The results of the [3H]uridine uptake study showed that the replacement of the naphthalene moiety with the benzene moiety could abolish the inhibitory effects on ENT1 and ENT2. The addition of chloride to the meta position of this benzene moiety could restore only the inhibitory effect on ENT1 but had no effect on ENT2. However, the addition of the methyl group to the meta position or the ethyl or oxymethyl group to the para position of this benzene moiety could regain the inhibitory activity on both ENT1 and ENT2. The presence of a halogen substitute, regardless of the position, in the fluorophenyl moiety next to the piperazine ring was essential for the inhibitory effects on ENT1 and ENT2. Among the analogues tested, compound 3c was the most potent inhibitor. Compound 3c reduced Vmax of [3H]uridine uptake in ENT1 and ENT2 without affecting Km. The inhibitory effect of compound 3c could not be washed out. Compound 3c did not affect cell viability, protein expression and internalization of ENT1 and ENT2. Therefore, similar to FPMINT, compound 3c was an irreversible and non-competitive inhibitor. Molecular docking analysis also showed that the binding site of compound 3c in ENT1 may be different from that of other conventional inhibitors. It is expected that structural modification may further improve its potency and selectivity and lead to the development of useful pharmacological agents.Equilibrative nucleoside transporters (ENTs) play a vital role in nucleotide synthesis, regulation of adenosine function and chemotherapy. Current inhibitors of ENTs are mostly ENT1-selective. Our previous study has demonstrated that 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N--1,3,5-triazin-2-amine (FPMINT) is a novel inhibitor of ENTs, which is more selective to ENT2 than to ENT1. The present study aimed to screen a series of FPMINT analogues and study their structure-activity relationship. Nucleoside transporter-deficient cells transfected with cloned human ENT1 and ENT2 were used as  FPMINT and its analogues were purchased from TopScience . Other chemicals were obtained from Sigma-Aldrich .Culture media and supplements were purchased from Thermo Fisher Scientific . Antibodies were obtained from Alomone Labs . Sulfo-NHS-SS-biotin was purchased from Thermo Fisher Scientific  uridine  for 1 min. Passive uridine uptake was determined by incubating the cells in HEPES-buffered Ringer’s solution containing [3H]uridine in the presence of 0.5 mM NBMPR. The cells were rapidly washed five times with ice-cold phosphate-buffered saline (PBS). After air-drying, the cells were solubilised by overnight incubation in 500 μl of 5% Triton-X100. Next, 300 μl of the cell lysates were mixed with 2 ml of scintillation liquid. The radioactivity of each sample was quantified by a scintillation counter. The protein content in each well was determined spectrophotometrically using a commercial bicinchoninic acid assay . Uptake values were expressed as picomoles of [3H]uridine per milligram of cellular protein per minute.Confluent monolayers of PK15NTD/ENT1 and PK15NTD/ENT2 cells in 24-well plates were washed thrice with HEPES-buffered Ringer’s solution containing (mM): 135 NaCl, 10 glucose, 5 KCl, 5 HEPES, 3.33 NaH3c or an equivalent volume of a vehicle (0.5% dimethyl sulfoxide (DMSO), as a control) in a serum-free medium for another 24 or 48 h. For measuring cell membrane integrity, the lactate dehydrogenase (LDH) released into the culture medium was measured with a detection kit  according to the manufacturer’s instructions. The absorbance at 490 nm was measured with a microplate absorbance reader. For measuring cell viability, 0.5 mg/ml methylthiazolyldiphenyl-tetrazolium bromide (MTT) was added to each well. The cells were further incubated for 4 h at 37°C to allow the formation of MTT formazan crystals. Finally, 100 μl of DMSO was added to lyse the cells and solubilise the crystals. The absorbance was measured at 560 nm, with the absorbance at 655 nm serving as the background.PK15NTD/ENT1 and PK15NTD/ENT2 cells were seeded onto 96-well plates until they reached 80–90% confluent. The cells were then treated with different concentrations of compound 3c or an equivalent volume of a vehicle (0.5% DMSO) for 24 h or 48 h. The cells were washed thrice with ice-cold PBS. Total protein was extracted by cell lysis in a lysis buffer  with a protease inhibitor cocktail . The protein content was measured by bicinchoninic acid assay. Protein samples were separated by 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies  (1:200 [v/v] dilution in a blocking solution) at 4°C overnight. After washing thrice with PBS containing 0.02% (v/v) Triton X-100, the membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies  at room temperature for 2 h. The membranes were then again washed, and the bound secondary antibodies were detected using an enhanced chemiluminescence kit . The antibodies recognized ENT1 as a protein of 40 kDa, and ENT2 as proteins of 50 and 47 kDa , but internalized transporters were protected and still contained biotin. Subsequently, 50 mM iodoacetamide in PBS was used to neutralize glutathione. Cells were then immediately solubilized in extraction buffer . After centrifugation at 13,000 x g for 5 min, supernatants with equal amounts of total proteins were agitated with streptavidin-agarose beads at 4°C overnight. Finally, the biotinylated transporters in the pull-down complexes, which should represent internalized transporters, were eluted by Laemmli buffer and resolved by SDS-PAGE as described above. Antibody against ENT1 or ENT2 was used to detect internalized transporters in the pull-down complexes and total transporter expressions in cell extracts.To label all surface proteins, after washing with ice-cold PBS, PK15NTD/ENT1 and PK15NTD/ENT2 cells were incubated with sulfo-NHS-SS-biotin (300 μg/ml) for 30 min at 4°C to biotinylate cell surface proteins. The unbound biotin was washed away by PBS containing 0.1% BSA at 4°C. The biotinylated cells were then incubated with the original growth media with or without 50 µM compound http://www.rcsb.org/pdb) (PDB ID: 6OB7). Two-dimensional structures of compound 3c and draflazine were downloaded from the PubChem database (http://pubchem.ncbi.nlm.nih.gov)  and was transformed into three-dimensional structure using Chem3D 19.0 software. The structures of ENT1, compound 3c and draflazine were pretreated and standardized with the Protein Preparation Wizard module of Schrödinger Maestro suite 2015 and the LigPreg module of Schrödinger LigPrep suite 2015, respectively. Autodock Vina software and Lamarckian Genetic Algorithm program were used to analyse the molecular interactions between ENT1 and drugs with default parameters. ENT2 was not studied because its crystal structure is not available.The X-ray crystal structure of human ENT1 was obtained from the Protein Data Bank . Data are expressed as mean ± standard error (SE) of three experiments performed in triplicate. The IC3H]uridine uptake in PK15NTD/ENT1 and PK15NTD/ENT2 cells were 18.42 ± 2.93 pmol/mg protein/min and 61.35 ± 6.67 pmol/mg protein/min, respectively. The dose-response curves for each FPMINT analogue against ENT1 and ENT2 are shown in 50 values for ENT1 and ENT2 were shown in PK15NTD/ENT1 and PK15NTD/ENT2 cells were used to screen a series of FPMINT analogues listed in 1a, 1b, 1c, 1d and 1e are the analogues of FPMINT, with modification in the N-naphthalene moiety. Compound 1a and 1e showed negligible effects on both ENT1 and ENT2. Compound 1b inhibited ENT1 with an IC50 value of 1.82 µM, but it had no effect on ENT2. Similar to FPMINT, compound 1c was more selective to ENT2 than to ENT1. The IC50 values of compound 1c against ENT1 and ENT2 were 171.11 µM and 36.82 µM, respectively. Although the potencies of compound 1c against ENT1 and ENT2 were lower than that of FPMINT, the selectivities of FPMINT and compound 1c were comparable . Unlike compound 1c, compound 1d was more selective to ENT1 than to ENT2 with IC50 values of 0.59 µM and 77.12 µM, respectively.Compounds 2a, 2b, 3a, 3b and 3c are the analogues of FPMINT, with changes in the N-naphthalene moiety and/or the fluorophenyl moiety. Compound 3a had no effect on both ENT1 and ENT2. Compounds 2a and 3b inhibited ENT1 with IC50 values of 104.92 µM and 1.65 µM, respectively. However, they did not inhibit ENT2. Compounds 2b and 3c inhibited both ENT1 and ENT2, but they were relatively more selective to ENT2. The IC50 values of compounds 2b and 3c against ENT1 were 12.68 µM and 2.38 µM, respectively, whereas their IC50 values against ENT2 were 2.95 µM and 0.57 µM, respectively. Compared to FPMINT, compound 3c showed a stronger potency, but compound 2b showed a weaker potency. However, their selectivities were close to that of FPMINT .Compounds 3c was selected for further investigation. The kinetic study of [3H]uridine transport was performed by measuring the concentration-dependent uptake of [3H]uridine. This study could provide clue about the mode of action of compound 3c. The uptake of [3H]uridine by ENT1 and ENT2 was saturable and conformed to Michaelis-Menten kinetics. Compound 3c decreased the Vmax of ENT1 but had no effect on Km. Specifically, the apparent Vmax values were 2236 ± 56.32, 2083 ± 36.80, 1867 ± 27.63, 1377 ± 37.81 and 732.9 ± 12.02 (pmol/mg protein/min) and the Km values were 0.143 ± 0.018, 0.142 ± 0.012, 0.143 ± 0.010, 0.144 ± 0.019 and 0.142 ± 0.011 (µM) in the presence of 0, 0.01, 0.1, 1 and 10 µM compound 3c, respectively  was 2.79 μM  and the Km values were 0.220 ± 0.035, 0.220 ± 0.041, 0.228 ± 0.059, 0.229 ± 0.050 and 0.226 ± 0.051 (µM) in the presence of 0, 0.01, 0.1, 1 and 10 µM compound 3c, respectively . The cells were then washed five times with buffer. After wash-out for every single time [3H]uridine uptake was measured. The inhibitory effects of compound 3c on both ENT1 and ENT2 were only partially reversed by washing  was much stronger than the analogue with an ethyl substitute . Moreover, the analogue with a chloride substitute at the meta position of this benzene ring  was slightly stronger than the analogue with a methyl substitute . The reason for this phenomenon is not known, but a common characteristic of the chloride and oxymethyl substitutes is that they possess lone pairs of electrons, which may increase the interaction with certain functional groups in the binding site of ENT1 through hydrogen bonds.First, the change from the naphthalene moiety to the benzene moiety abolished the effect on ENT1. This finding was confirmed based on the data of compounds 2a) reduced but not abolished the inhibitory effect on ENT1. Replacing the fluoride substitute at the ortho position with the chloride substitute at the meta position  did not significantly affect the inhibitory effect on ENT1. Moreover, comparison of the results of compounds 1b and 3b showed that changing the fluoride substitute from the ortho position to the para position did not alter the inhibitory effect on ENT1. It is apparent that the presence of a halogen substitute, regardless of its position on the phenyl moiety, was critical to the inhibitory effect on ENT1, probably through the electrostatic interaction with the binding site.Another determinant for the inhibitory effect on ENT1 is related to the fluorophenyl moiety next to the piperazine ring. The removal of the fluoride substitute from the fluorophenyl moiety  not only decreased but also completely eliminated the inhibitory effect on ENT2. Similar to that for ENT1, the change of the fluoride substitute at the ortho position to the chloride substitute at the meta position  restored the inhibitory effect on ENT2. Modification of the N-naphthalene moiety also greatly affected the inhibitory effect on ENT2. The replacement of the naphthalene moiety with the benzene moiety  not only abolished the inhibitory effect on ENT1 but also on ENT2. In contrast to that for ENT1, the addition to a chloride substitute to the meta position of the benzene ring could not restore the inhibitory effect on ENT2. This assumption was clarified when we compared compounds 1a against 1b, and compounds 3a against 3b. However, the addition of the ethyl or oxymethyl group to the para position of this benzene ring  enabled the compounds to regain the inhibitory activity, although the inhibitory effect was smaller than that of FPMINT. It remains unclear whether the presence of a para-substitute facilitates the binding of the compounds to ENT2. However, we believe that the methyl group in this benzene moiety may also be of paramount importance. The reason is that the change of the chloride substitute at the meta position of this benzene moiety  to the methyl substitute  could drastically make the compound, which was originally insensitive to ENT2, to become highly sensitive to ENT2.The presence of a halogen substitute on the fluorophenyl moiety played an even more critical role in the inhibitory effect on ENT2 because the absence of fluoride , which occupies the central cavity of ENT1 . Kinetic dilazep . Moreove dilazep . Interes of ENT1 , which i3c, they are not cytotoxic to PK15NTD cells. Nevertheless, further experiments are still required to completely reveal their safety profile. It is believed that the optimisation of the chemical structures of FPMINT and compound 3c may further improve the selectivity to ENT2. Development of a selective ENT2 inhibitor will be a useful research tool or even a potential therapeutic agent. ENT2 is particularly abundant in skeletal muscle and adrenal gland (It has been reported that certain piperazine derivatives may have cytotoxic effects on hepatocytes and cancer cell lines . Althougal gland  but its al gland . Moreoveal gland . These eIn conclusion, our study has provided evidence that the modification of the N-naphthalene moiety and the fluorophenyl moiety in FPMINT can change the inhibitory potency and selectivity for ENT1 and ENT2. The insights obtained from the structure-activity relationship may be useful in designing and developing more potent and selective ENT2 inhibitors.


Candida albicans filamentation, the ability to convert from oval yeast cells to elongated hyphal cells, is a key factor in its pathogenesis. Previous work has shown that the integral membrane protein Dfi1 is required for filamentation in cells grown in contact with a semisolid surface. Investigations into the downstream targets of the Dfi1 pathway revealed potential links to two transcription factors, Sef1 and Czf1. Sef1 regulates iron uptake and iron utilization genes under low-iron conditions, leading us to hypothesize that there exists a link between iron availability and contact-dependent invasive filamentation. In this study, we showed that Sef1 was not required for contact-dependent filamentation, but it was required for wild-type (WT) expression levels of a number of genes during growth under contact conditions. Czf1 is required for contact-dependent filamentation and for WT levels of expression of several genes. Constitutive expression and activation of either Sef1 or Czf1 individually in a dfi1 null strain resulted in a complete rescue of the dfi1 null filamentation defect. Because Sef1 is normally activated in low-iron environments, we embedded WT and dfi1 null cells in iron-free agar medium supplemented with various concentrations of ferrous ammonium sulfate (FAS). dfi1 null cells embedded in media with a low concentration of iron (20 μM FAS) showed increased filamentation in comparison to mutant cells embedded in higher concentrations of iron (50 to 500 μM). WT cells produced filamentous colonies in all concentrations. Together, the data indicate that Dfi1, Czf1, Sef1, and environmental iron regulate C. albicans contact-dependent filamentation.IMPORTANCECandida albicans is an opportunistic pathogen responsible for a larger proportion of candidiasis and candidemia cases than any other Candida species. The ability of C. albicans cells to invade and cause disease is linked to their ability to filament. Despite this, there are gaps in our knowledge of the environmental cues and intracellular signaling that triggers the switch from commensal organism to filamentous pathogen. In this study, we identified a link between contact-dependent filamentation and iron availability. Over the course of tissue invasion, C. albicans cells encounter a number of different iron microenvironments, from the iron-rich gut to iron-poor tissues. Increased expression of Sef1-dependent iron uptake genes as a result of contact-dependent signaling will promote the adaptation of C. albicans cells to a low-iron-availability environment. Candida albicans is a human commensal organism that commonly resides in the gastrointestinal (GI) tract  tract , 2. Whildidiasis . The abial forms –6. This l forms ––11.DFI1 results in a filamentation defect in cells grown on or embedded in agar medium. It has been shown that a deletion of DFI1 also results in a reduction in lethality of C. albicans in the intravenously inoculated mouse model of systemic candidiasis . Numerous members of the Sef1 regulon were identified as differentially expressed in the presence or absence of Dfi1p. Further investigations revealed that Sef1 activation is able to bypass the invasive filamentation defect of the Dfi1 null mutant and promote contact-dependent filamentation. Taken together, the results demonstrate a role for Sef1 in the induction of C. albicans on a semisolid surface  using Bowtie, and differential gene expression was analyzed using Cuffdiff. A total of 6,264 genes were analyzed for each treatment group.Therefore, Caeviously . Briefly2+/A23187, gene expression in WT cells treated with Ca2+/A23187 versus gene expression in dfi1 null cells treated with Ca2+/A23187, and gene expression in untreated WT cells versus gene expression in untreated dfi1 null cells , were not detected, indicating that transcription of these genes was not altered by the absence of Dfi1 under the conditions of these experiments.Interestingly, of these 93 genes, 13 were members of the Sef1 regulon. These genes are represented in bold in sef1 null mutant strain  Bright-field images (4× objective) of WT and sef1 null cells grown in liquid YPS (rich high-iron) medium (left) or grown embedded in YPS (rich high-iron) agar medium for 4 days at 25°C (right). (B) Growth curve of WT and sef1 null cells in YPS media at 25°C. Black solid line, WT; gray shading, SD. Blue dotted line, sef1 null; blue shading, SD. (C) Filamentation of WT and sef1 null colonies grown embedded in YPS agar media on days 3 and 4 postembedding at 25°C. Each point represents 1 biological replicate. Results from 3 experiments with 3 biological replicates per experiment are shown. Bar shows the means; error bars indicate SD (D) Images of invading cells left on YPS plates after washing away cells from the agar surface after 4 days of growth. Images were taken using 2.5× and 10× objectives. Download FIG S1, TIF file, 1.1 MB.Sef1 is not required for contact-dependent filamentation. WT and Copyright © 2022 Junier et al.2022Junier et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00779-21.2FIG S2sef1 null cells were grown overnight in YPD medium at 30°C and then either back diluted to an OD of 0.1 in NIMS (noniron media plus 2% sucrose) supplemented with 20 μM ferrous ammonium sulfate (FAS) and grown over a day at 30°C or embedded in the same media with agarose and allowed to grow for 4 days at 25°C. (A) Bright-field images (4× objective) of WT and sef1 null cells grown in liquid NIMS supplemented with 20 μM FAS (left) and embedded in NIMS agarose supplemented with 20 μM FAS (right). (B) Growth curve of WT and sef1 null cells in NIMS plus 20 μM FAS media at 25°C. Black solid line, WT; gray shading, SD. Blue dotted line, sef1 null; blue shading, SD. (C) Filamentation of WT and sef1 null cells grown embedded in NIMS plus 20 μM FAS agarose media on days 3 and 4 postembedding. Each point represents 1 biological replicate. Results from 3 experiments with 3 biological replicates per experiment are shown. Download FIG S2, TIF file, 0.5 MB.Sef1 is not required for contact-dependent filamentation in low-iron media. WT and Copyright © 2022 Junier et al.2022Junier et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the sef1 null mutant strain to produce filamentous colonies under embedded conditions. The WT and sef1 null strains were embedded in yeast extract-peptone-sucrose (YPS) media and grown as described in Materials and Methods. Representative images of embedded colonies are shown in sef1 null strains exhibited about 90% filamentous colonies , members of the Sef1 regulon that were not RNA-seq hits , and genes that are regulated by low iron (FET33 and FTR2). To investigate whether expression of these genes was altered in the sef1 null mutant, we harvested cells grown on the surface of YPS agar medium, as described in Materials and Methods. After harvesting of the cells from the surface of the plates, the presence of invading cells was used to demonstrate the invasiveness of the strain during growth on the agar. Visual inspection, using 2.5× and 10× objectives, of the invading cells left behind after removing the colonies from the plates showed no discernible difference in invasive filamentation between WT and sef1 null mutant strains . Despite there being no difference in filamentation, a significant decrease in expression was observed for 5 of the genes examined: CFL5, CSA1, SOD4, CFL2, and FTR1  Gene expression of WT, czf1 null, czf1+CZF1, and czf1+SEF1/GAD1 cells that were grown overnight in YPD medium at 30°C and then plated on the surface of YPS agar plates and allowed to grow for 3 days at 25°C. Each point represents 1 biological replicate. Results from three experiments with 2 or 3 biological replicates per experiment are shown. Results are normalized to average WT expression for each experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, two-way ANOVA with post hoc Dunnett’s test for multiple comparisons on log transformed data. Download FIG S3, TIF file, 0.4 MB.Constitutive activation of Sef1 and Czf1 rescue gene expression defect. (A) Gene expression of WT, Copyright © 2022 Junier et al.2022Junier et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the sef1 null cells grown in liquid, rich high-iron media were harvested during either log phase or post-exponential phase (4 days at 25°C). Under these conditions, we observed no difference in expression of CFL5, CSA1, SOD4, CFL2, or FTR1 between the WT and the sef1 null mutant  scanned for Czf1p binding sites. Of these 200 randomly selected genes, only 27% contained a Czf1p binding site in their promoter. This represents a significant enrichment of Czf1 binding sites among Sef1 regulon genes , leading us to hypothesize that Czf1 may regulate genes in the Sef1 regulon. Analysis of transcripts by RT-qPCR of the 13 genes listed above from WT and czf1 null cells plated on rich high-iron media showed a significant decrease in expression of 10 genes: CFL5, BMT9, CSA1, OPT1, SOD4, FET31, FTR1, GDH3, MRS4, and FET33 , Czf1 only , or both Sef1 and Czf1  for WT levels of expression during growth under contact conditions.Czf1 is a zinc cluster DNA binding protein that is required for WT filamentation in cells grown in contact with agar but not under liquid growth conditions , 22. Rece points , consiste points . Of the WRSCGCCG ) in theind FET33 . Interes10.1128/mSphere.00779-21.4FIG S4czf1 null cells were grown overnight in YPD medium at 30°C and then either back diluted to an OD of 0.1 in YPS media and grown overnight at 30°C or embedded in YPS agar media and allowed to grow for 4 days at 25°C (A) Bright-field images (4× objective) of WT and czf1 null cells grown in liquid or grown embedded in YPS (rich high-iron) agar media. (B) Filamentation of WT and czf1 null cells grown embedded in YPS agar media on days 3 and 4 postembedding. Each point represents 1 biological replicate. Results from 3 experiments with 3 biological replicates per experiment are shown. Results are normalized to average WT expression for each experiment. ***, P < 0.001, t test. Download FIG S4, TIF file, 0.6 MB.Czf1 is required for contact-dependent filamentation. WT and Copyright © 2022 Junier et al.2022Junier et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the CFL5, CSA1, SOD4, and FTR1 expression in cells growing on the surface of agar. However, despite 5 genes demonstrating Sef1-dependent expression during plated growth, none of these genes required Sef1 for WT levels of expression during liquid growth, indicating a potential role for Sef1 in contact-dependent filamentation.In summary, Sef1 is not required for contact-dependent filamentation, while WT levels of embedded filamentation require Czf1. Both transcription factors are required for WT levels of dfi1 null mutant. An activated Sef1 fusion  was used. The activated allele encodes a fusion of a Gal4 activation domain (GAD) to the C terminus of Sef1, causing the protein to be constitutively activated. The activated SEF1 allele is constitutively expressed under the control of the ADH1 promoter  Bright-field images (4× objective) of WT and dfi1 strains alone, with SEF1/GAD, or with CZF1/GAD alleles grown in liquid YPS. (B) Bright-field images (4× objective) of WT and dfi1 strains alone, with SEF1/GAD, or with CZF1/GAD alleles grown embedded in YPS agar. Download FIG S5, TIF file, 2.1 MB.Constitutive activation of Sef1 and Czf1 in Copyright © 2022 Junier et al.2022Junier et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the dfi1, WT/SEF1-GAD, and dfi1/SEF1-GAD strains were embedded in rich, high-iron media as described in Materials and Methods. On day 4, the WT strain exhibited filamentous colonies. The dfi1 null mutant strain yielded only 25% as many filamentous colonies as the WT , confirming the filamentation defect for the dfi1 mutant that has been previously reported . Activated Sef1p was thus sufficient to rescue the filamentation defect seen in the dfi1 null mutant strain . Thus, activation of Sef1 was sufficient to bypass the filamentation defect caused by the lack of Dfi1. Sef1 activation, however, did not lead to filamentation under liquid growth conditions.WT, reported   were taken at various times. Download FIG S6, TIF file, 1.4 MB.Time course of constitutive activation of Sef1 in Copyright © 2022 Junier et al.2022Junier et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the dfi1 null mutant did not exhibit a consistent defect in filamentation and the SEF1-GAD allele did not increase filamentation. Most likely, alternative filamentation regulatory pathways are activated at 37°C and these pathways mask the defect of the dfi1 null mutant and prevent the detection of bypass by the SEF1-GAD allele (data not shown).Embedded filamentation by these strains was also analyzed at 37°C. Under these conditions, the FTR1, CFL5, CFL2, OPT1, and CFL1. These genes were selected because of their various degrees of dependence on Sef1 and Czf1 as shown in CFL5, FTR1, and CFL2 required Sef1 for WT levels of expression in plated cells grown on rich, high-iron media. We observed that the WT/SEF1-GAD strain exhibited a 10-fold increase in expression of FTR1, CFL5, CFL2, and CFL1 relative to the WT  , but only FTR1, CFL5, and OPT1 required Czf1 for WT levels of expression under plated conditions  when constitutively expressed and activated Czf1 was present  . Similarnditions . Analysi present . Constitth model .czf1 null strain with activated Sef1 (czf1 null+SEF1/GAD1) and a czf1 null strain with ectopically expressed CZF1. Both strains exhibited normal morphology when grown in liquid YPS media . CFL2 expression was not defective in the czf1 null strain. Introduction of constitutively activated Sef1 to czf1 null cells increased expression of FTR1, CFL5, and CFL2 above the level observed in the null strain alone  Bright-field images (10× objective) of WT, czf1 null, czf1+CZF1, and czf1+SEF1/GAD1 cells grown overnight in YPD medium at 30°C and then back diluted to an OD of 0.1 in YPS media and grown overnight at 30°C. (B) Bright-field images (10× objective) of WT, czf1 null, czf1+CZF1, and czf1+SEF1/GAD1 cells grown embedded in YPS agar or NIMS agarose media supplemented with 20 μM FAS for 2 days at 25°C. Download FIG S7, TIF file, 1.7 MB.Complementation, constitutive activation of Sef1, and low-iron media rescue Copyright © 2022 Junier et al.2022Junier et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the dfi1 mutant. To test this hypothesis, we grew cells in YNB media containing different levels of iron . Cells dfi1 mutant exhibited lower levels of filamentous colonies , we observed that the colonies  and B, ccolonies  and B, ientation . At veryentation . Upon cl10.1128/mSphere.00779-21.8FIG S8dfi1 null, or dfi1+DFI1 cells were grown overnight in YPD medium at 30°C and then back diluted to an OD of 0.1 in liquid YPS or NIMS supplemented with 20 μM or 200 μM FAS and grown overnight at 30°C. Bright-field images (10× objective) of WT, dfi1 null, and dfi1+DFI1 cells grown in liquid rich high-iron or minimal low-iron media are shown. Download FIG S8, TIF file, 1.4 MB.Liquid low-iron medium does not induce filamentation. WT, Copyright © 2022 Junier et al.2022Junier et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the czf1 null strain to these conditions. As shown above  under contact conditions  tract is an iron-replete environment . ContactCFL1, have been shown to have functions in filamentation, with deletions leading to impaired filamentous growth and altered cell wall architecture under liquid conditions. Recently, Luo et al. showed that iron acquisition was required for sustained hyphal development but not hyphal initiation   medium at 30°C. For specific studies, cells were grown in complete minimal medium minus uridine (CM-U) or yeast extract-peptone-sucrose (YPS) . Noniron medium (NIM) was adapted from Hsu et al.  at 100 μM was used to remove any residual iron. Ferrous ammonium sulfate (FAS) was added at 0 to 500 μM concentrations; 2% sucrose was also added (noniron media plus 2% sucrose [NIMS]). To create embedded conditions, 0.8% agarose was added and cells were embedded as described above. Cells were routinely cultured at 30°C or 25°C. Some mutants were obtained as part of a deletion collection . After 30 min, 10 mL of cells was collected for RNA extraction and analysis. Cells for RNA were washed and frozen in RNAlater at −80°C.Artificial activation of the Dfi1p pathway in liquid medium was done as previously described by Davis et al. . BrieflyC. albicans cells frozen in RNAlater using the Qiagen RNeasy kit with the following modifications. Cells were broken by bead beating with 0.5-mm silica zirconia beads on a Mini-Beadbeater-24 machine (Biospec) with 3 rounds of 1-min bead beating and 5 min on ice between. RNA was extracted from the bead beating supernatant as described by Qiagen, including on-column DNase treatment, and eluted with 30 μL of water. RNA was stored at −80°C.RNA was extracted from Candida albicans SC5314  and annotation GFF file were used as the reference genome. A bowtie2 index was created using bowtie2-build (v2.2.1) from the fna sequence file, while the downloaded GFF was converted to GTF using gffread for downstream use. The raw sequencing reads for each sample were mapped to the bowtie2 index using bowtie2 (v2.2.1) with default parameters and then sorted and converted to bam format using samtools v1.9 (sort function). The resulting sorted bam files were used as input for Cuffdiff (Cufflinks v2.1.1) with the triplicates grouped. Genes with a fold change of over 2 were considered.RNA was sent to the Tufts University Core Facility for library preparation and sequencing using the Illumina TruSeq RNA library preparation kit and the HiSeq 2500 instrument. Sequencing data were analyzed using the Tuxedo Suite as previously described . NCBI geThe growth of colonies under embedded conditions was performed as previously described by Zucchi et al. . BrieflyCells were grown as previously described . BrieflycDNA was synthesized by reverse transcription of 10 μg of total RNA using SSIII (Invitrogen) following the manufacturer’s protocol. Resulting cDNA was diluted 1:20 and used for gene expression analysis via qPCR. qPCRs were set up using SYBR green master mix (Applied Biosystems). qPCRs were run on the Applied Biosystems StepOnePlus RT-qPCR system using standard reaction parameters. Primers used for qPCR are listed in 10.1128/mSphere.00779-21.10TABLE S2Table S2, DOCX file, 0.02 MB.List of primers used in this study. Download Copyright © 2022 Junier et al.2022Junier et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the GSE193641.RNA-seq data generated by this project are available in the GEO database under accession no.


Reg3A expression are negatively correlated with TNM classification (P < 0.001), lymph node (P < 0.001) in GC. Reg3A significantly suppresses cell proliferation in GC (P < 0.05). Bioinformatic analysis and experimental results confirmed that Reg3A positively regulates the expression of deleted in malignant brain tumor 1 . Besides, Reg3A and DMBT1 all prolong the overall survival , post-progression survival , and first progression survival . The function of Reg3A inhibiting cell proliferation is abolished by DMBT1 siRNA in GC (P < 0.05). In conclusion, Reg3A may act as a novel tumor suppressor by promoting DMBT1 expression, which may be a potential therapeutic target in patients with GC.Regenerating family member 3 alpha (Reg3A) encodes a pancreatic secretory protein that may be involved in cell proliferation or differentiation. However, the function and downstream regulatory mechanism of Reg3A in gastric cancer (GC) remains elusive. This study aimed to clarify the function and mechanism of Reg3A regulating cell proliferation in GC. The expression levels of Reg3A were confirmed in GC patients and cells using qRT-PCR and western blotting. TCGA datasets and clinical samples were used to explore the correlation between Reg3A and clinicopathologic features in GC. Cell viability, colony formation, and xenograft tumorigenesis assays were performed to detect the function of Reg3A on cell proliferation. Besides, we predicted the correlated genes of Reg3A by analyzing TCGA datasets, and further investigated the downstream regulatory mechanism of Reg3A in GC. Our results demonstrated that Reg3A is down-regulated in  GC waRegenerating family member 3 alpha (Reg3A) encodes a pancreatic secretory protein that may be involved in cell proliferation or differentiation . RecentlDeleted in malignant brain tumor 1 (DMBT1) is a member of the scavenger receptor cysteine-rich superfamily, which mediates cell-extracellular matrix interactions . DMBT1 iIn the present study, we aimed to clarify the function and the regulatory mechanisms of Reg3A in GC. We explored the expression of Reg3A in GC and analyzed the correlation of Reg3A with clinical characteristics, such as TNM classification, lymph node, grade of differentiation, and survival time. Besides, We illuminated the functions and correlations of Reg3A and DMBT1 in GC. Considering the clues above, we hypothesized that Reg3A acts as a tumor suppressor by targeting DMBT1 in GC. So, we further explored the regulatory mechanism between Reg3A and DMBT1 in GC.This study is the first to report the regulatory relationship between Reg3A and DMBT1. We hope that these data may provide novel biomarker candidates for the diagnosis and treatment of GC.2.2.12.The human GC cells  and human gastric epithelial cells (GES-1) were obtained from the Cell Bank of Shanghai Academy of Chinese Sciences. Forty paired clinical specimens (tumor and adjacent tissues) were provided by patients with GC in Hebei General Hospital (from June 2018 to June 2020). Inclusion benchmarks were: (1) newly histologically diagnosed stomach adenocarcinoma; (2) received no prior therapy before surgery; (3) underwent gastrectomy; (4) provided informed consent of using specimens. Exclusion benchmarks were: (1) tumor volume was insufficient for the analysis; (2) additional surgery after endoscopic resection; (3) no histories of other cancer. Ethical approval was obtained from Hebei General Hospital. AGS cell line was cultured in F12K medium. Other cell lines were cultured in RPMI 1640 medium. All mediums were supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were cultured at 37°C under 95% air, 5% CO2.2−ΔΔCT method. The parameters were used: 95°C for 60 sec, followed by 40 cycles of 95°C for 15 sec, 55°C for 15 sec, and 72°C for 20 sec. qRT-PCR primers were as follows: Reg3A forward: 5ʹ- AGCTACTCATACGTCTGGATTGG-3ʹ, Reg3A reverse: 5ʹ-CACCTCAGAAATGCTGTGCTT-3ʹ; DMBT1 forward: 5ʹ-CACCTCAGAAATGCTGTGCTT-3ʹ, DMBT1 reverse: 5ʹ-GCCACCAATCTGTCGTAGTAGAA-3ʹ.Total RNAs were extracted using Trizol reagent  by the manufacturer’s instructions. For complementary DNA synthesis, 1 μg total RNA was reverse transcribed using the Primescript RT reagent kit . Relative expression levels were detected using SYBR Green PCR Kit  and calculated using the 22.3Reg3A siRNA, Reg3A overexpression vector, DMBT1 siRNA, and DMBT1 overexpression vector were synthesized by GenePharm , which sequences are as follows: Reg3A siRNA, 5ʹ- GCUGCUGUCUCAGGUUCAAGG-3ʹ(sense), 5ʹ-UUGAACCUGAGACAGCAGCAU-3ʹ (antisense); DMBT1 siRNA, 5ʹ-GGUGGAUGUGUAUUAUUAAGA-3ʹ (sense), 5ʹ-UUAAUAAUACACAUCCACCUU-3ʹ (antisense). The overexpression vector is GV492. Lipofectamine 2000 reagent  was used to transfect AGS or SGC-7901cells.2.4Cell viability was measured with Cell Counting Kit-8 . In brief, SGC-7901 and AGS cells were seeded in 96-well plates and incubated for 24 h, 48 h, or 72 h, respectively. Then, 10 µl CCK-8 solution was added to 100 µl medium and incubated for 1 h at 37°C. A microplate reader detected the optical density at 450 nm.2.52 after transfection. About two weeks later, cells were fixed with 95% alcohol for 30 min at room temperature. Cells were stained with crystal violet (0.1%). Plaques were calculated with Image J software. Each assay was repeated three times.For colony formation assay, GC cells were seeded at 200 cells per well in 12-wells plates and were incubated at 37°C under 5% CO2.6https://chat.lionproject.net/) [http://gepia.cancer-pku.cn/) [https://kmplot.com/analysis/) [http://www.linkedomics.org/login.php) [Cancer Hallmarks Analytics Tool   was usedpku.cn/)  was perfalysis/)  was usedgin.php)  was used2.7Western blot assays were performed to detect the protein expression of Reg3A, Ki67, PCNA, and β-actin . Cells were lysed with Lysis Buffer  on ice for 30 min, which was centrifuged at 14,000 rpm for 10 min at 4°C. The protein samples were normalized using a BCA assay kit . Equal amounts of protein lysates were electrophoresed on SDS-PAGE gels and then transferred onto nitrocellulose membranes. After blocked 1 h at room temperature, membranes were incubated with primary antibody overnight at 4°C. Membranes were then washed in PBST (PBS with 0.1% Tween) and incubated with horseradish peroxidase-labeled secondary antibody for 1 h at room temperature. After being washed in PBST three times, membranes were visualized by enhanced chemiluminescence reagents .2.8ad libitum access to food and water) for 1 week [6/mouse) were resuspended in 100 µl PBS and then subcutaneously injected into each mouse. The tumor volume was measured with a caliper every three days. After thirty days, the mice were euthanized by cervical dislocation in deep anesthesia (isoflurane), and the tumors were surgically isolated and fixed for immunohistochemistry.Nude mice  were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. The care and breeding of animals were performed in compliance with national guidelines. Mice were acclimatized to laboratory conditions . Immunohistochemistry assays were performed using a one-step Immunohistochemistry kit . Paraffin sections were dewaxed in xylene and rehydrated in graded ethanol. Then, 3% hydrogen peroxide was used to inactivate endogenous enzymes. Tissue sections were immersed in citrate buffer and heated for antigen retrieval. After being blocked using 5% bull serum albumin, tissue sections were incubated with primary antibody, biotin–peroxidase secondary antibody, DAB reagents. Finally, tissue sections were counterstained with hematoxylin and photographed with a microscope.2.10P values <0.05 were deemed statistically significant.Data management and analysis were performed with IBM SPSS 21.0 software. The data were normalized using mean± standard deviation for three independent experiments. Significance levels of measurement data were estimated using the student t-test or one-way ANOVA. 3.3.1P < 0.01). We verified the Reg3A mRNA expression levels in GC patients and cell lines. These results indicated that Reg3A is downregulated in GC patients  but not with the M stage (P < 0.01), PPS (P < 0.001), and FP (P < 0.001) of patients with GC. However, Reg3A did not prolong the RFS of patients with GC , and overexpression of Reg3A significantly decreases colony formation rate in AGS and SGC-7901 cells , PPS (P < 0.05), and FP (P < 0.01, CHAT revealed that DMBT1 is mainly correlated with genome instability and mutation, tumor-promoting inflammation, immune destruction, and evading growth suppressors . RT-PCR 3.6P < 0.01, P < 0.05, P < 0.05, P < 0.05, P < 0.05, P < 0.05, DMBT1 was significantly knocked down and overexpressed in GC cells (4.The effect of Reg3A on a variety of human cancers has been gradually recognized in recent years. Wang et al. found that Reg3A functions as a tumor promoter in pancreatic carcinoma, which is involved in the Reg3A-JAK2/STAT3 positive feedback loop . HoweverThis study confirmed that Reg3A is a tumor suppressor in GC, which is consistent with that of Qiu et al. who revealed that Reg3A suppresses cell invasion, proliferation, and promotes apoptosis . HoweverBesides, we explored the potential correlate genes of Reg3A in the TCGA dataset . We chosRemarkably, we found that Reg3A is relatively high expression in SGC-7901 cell, and is relatively low expression in AGS cell. So, we performed transfection experiments using AGS and SGC-7901 cells. Reg3A overexpression vector and DMBT1 siRNA were co-transfected into AGS cells. Reg3A siRNA and DMBT1 overexpression vectors were co-transfected into SGC-7901 cells. Our findings revealed that the function of Reg3A inhibiting cell growth is reversed by DMBT1 siRNA in AGS cells, and the function of Reg3A siRNA promoting cell growth was reversed by DMBT1 in SGC-7901 cells. These results revealed that Reg3A can target DMBT1 to regulate cell proliferation.Based on the above results, this study first suggested that Reg3A can target DMBT1 to regulate cell proliferation in GC cells. Future studies will further determinate the regulatory mechanisms of Reg3A and DMBT1. A large-scale clinical trial will be conduct to verify the effect of Reg3A and DMBT1 on patients with GC.5.GC contains various pathological classifications, such as adenocarcinoma, signet ring cell carcinoma, and undifferentiated carcinoma. Different types of GC have distinct incidence, clinicopathological features, treatments, and prognoses ,35. More6.DMBT1 acts as a novel target of Reg3A suppressing GC proliferation. Reg3A and DMBT1 may be potential biomarkers of diagnosis and prognosis in patients with GC.


Arabidopsis thaliana to decouple its nucleus- and chloroplast-associated functions and found hypomorphic mutants pointing at essential amino acids. While the origin of the PAP8 gene remained elusive, we have found in its sequence a micro-homologous domain located within a large structural homology with a rhinoviral RNA-dependent RNA polymerase, highlighting potential RNA recognition motifs in PAP8. PAP8 in vitro RNA binding activity suggests that this domain is functional. Hence, we propose that the acquisition of PAPs may have occurred during evolution by different routes, including lateral gene transfer.Chloroplast biogenesis depends on a complex transcriptional program involving coordinated expression of plastid and nuclear genes. In particular, photosynthesis-associated plastid genes are expressed by the plastid-encoded polymerase (PEP) that undergoes a structural rearrangement during chloroplast formation. The prokaryotic-type core enzyme is rebuilt into a larger complex by the addition of nuclear-encoded PEP-associated proteins (PAP1 to PAP12). Among the PAPs, some have been detected in the nucleus (PAP5 and PAP8), where they could serve a nuclear function required for efficient chloroplast biogenesis. Here, we detected PAP8 in a large nuclear subcomplex that may include other subunits of the plastid-encoded RNA polymerase. We have made use of PAP8 recombinant proteins in  Chloroplasts are of endosymbiotic origin, and as a remnant of their cyanobacterial ancestry, they have retained a small but highly conserved genome. Plastid genomes are composed of nearly 120 genes encoding housekeeping and photosynthesis genes found in multiple copies of circular DNA . HoweverArabidopsis [In the germinating seedling of angiosperms, ignition of chloroplast biogenesis requires light exposure (reviewed in the work of ), leadinbidopsis . On the According to their amino acid sequence and domain predictions, the PAPs have been classified into four functional groups  on the recombinant protein predicted a chloroplast transit peptide with a likelihood of 0.9941 and a processing site (PS) at position 54–55 (VVK-VD), confirming that the translational fusion did not alter its predicted localization. However, the novel algorithm consistently shortens the cTP by 5 amino acids as compared to the obsolete ChloroP algorithm (PS at position 59-60 VDDVD-AD) previously used for the cloning designs [To follow the dually localized PAP8, PAP8-GFP C-terminal translational fusions were cloned but failed to complement the pap8-1 mutant . Here, a designs .PAP8 promoter sequence (pP8) in the pap8-1 mutant. The double heterozygous PCR-screened primary transformants  restores most of the PAP8 function in the pap8-1 mutant. A minor defect corresponding to a pale green phenotype in the center of the rosette occurred, which did not affect the life cycle of the plant or the production of seeds. The expression of the translational fusion GFP-PAP8 gave rise to similar fluorescent patterns as that of PAP8-GFP [327F328 amino acids. On the other hand, the cTP8-GFP-PAP8 provides a functional framework allowing for other interesting tags or domains to be fused to PAP8.Functional complementation was tested with such GFP fusion under the control of the 1kb-formants B were teformants C. Four ttg−/tg−” D,E. HencPAP8-GFP . Under tm5) , where the NLS sequence of PAP8 was re-introduced in the permissive context (see pLC23) behind the cleavage site of the NLSm5 variant  and a construction carrying a mutated nuclear localization signal replacing the five positively charged lysine and arginine residues by glycine  generated 1/4 of the delayed greening phenotype  produced a progeny of 1/4 seedling with a delayed greening  produced ¼ of albino seeds showing no interference of the transgene with the phenotype of pap8-1  simultaneously would have provided definitive proof only if they were totally independent. Instead, the phenotypic analysis revealed that the uncoupled nuclear function carried by ΔcTP does not provide any benefit to a plant carrying the NLSm5 recombinant gene. This suggests that the nuclear function of PAP8 is entirely masked by the inability of PAP8 to properly work during chloroplast biogenesis. In that context, it would be of interest to test whether uncoupling the genomes in a gun1 mutant background, for example, would affect the phenotypes of plants carrying NLSm5 alone or in combination with ΔcTP. The recombinant gene BB647 (cTP8-N8-NLSm5) is an even stronger hypomorphic allele since the 5-amino acid substitution can potentially affect both nuclear and plastid functions. Future work could test other dually localized PAPs for which the NLS removal does not affect the molecular function of the protein.The presence of PAP8 in different compartments triggers questions of functionality. Does PAP8 play the same molecular function, whether it is in the nucleus or in the chloroplast, and are these compartmentalized functions dependent on each other. The uncoupling of these functions by using two distinct carriers )—PAP8/pTAC6: At1g21600; PAP5/HMR/pTAC12: At2g34640; PAP7/pTAC14: At4g20130; PAP12/pTAC7: At5g24314; PAP10/TrxZ: At3g06730; EF1α: At5g60390.TAIR , and Col-0: SALK_6000, Ai15 (PAP8ΔcTP) and Ai10 (PAP8NLSm5) described in the work of [E. coli DH5α strain (lacZ-ΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17(rK-mK+) supE44 thi-1 gyrA96 relA1) was used for cloning. Agrobacterium tumefaciens strain C58C1 pMP90 was used for transgenesis. Rosetta™2 (DE3)  cells were used for protein production with pAG21d (H6-PAP8ΔcTP). Mustard seeds  were grown for 7 days on soil under a photoperiod of 16 h of white light/8 h dark at 20 °C in a growth chamber. Cotyledons were harvested at the end of the night to avoid starch granules and immediately used for the preparation of organelles in a cold room. Arabidopsis plants were grown on ½-strength MS media, sucrose 1% pH5.7 and 0.8% agar. Seeds were imbibed and stratified for 2 days at 4 °C, before growth at 21 °C for 3 days in darkness. Plants were then transferred to white light (30 µmol m−2 s−1). work of . E. coliSinapis alba cotyledons (2 kg) were homogenized in ice-cold isolation buffer using a Waring blender and filtered through one layer of 56 µm mesh nylon; detailed protocol and buffers as in the work of [g for 5 min at 4 °C; the supernatant corresponded to sample “T”. Nuclear fraction 25 mL in lysis buffer; centrifugation 750× g for 10 min; the pellet was resuspended in 10 mL lysis, triton 0.5%, 10 min on ice plus stirring bar, then centrifugation at 750× g for 10 min. Pellet was resuspended in 10 mL lysis buffer; repeated as long as it was green. Resuspend the nuclei in equal pellet volume with washing buffer + glycerol to 50% (2 mL). Nuclei were sonicated with the following parameters (1 s/2 s relapse) in 1 min cycles × 3 . Samples were centrifugated at 16,000× g 15 min; the supernatant corresponded to sample “N”. Nanodrop (N6× protein 2 mg/mL and DNA 270 ng/µL). Loadings for blue native gels: 50 µL (45 µL + 5 µL Coomassie blue). Run for 16 h at 4 °C; then Coomassie blue running buffer exchanged with clear running buffer for 6 additional hours. Gels were scanned on a ChemidocXRSTM imaging system . Gels were transferred to a nylon membrane (BioRad), then blocked in TBS, Tween 0.1%, non-fat dry milk 5% w/v. The membrane was probed in TBS-Tween 0.1%, with the primary antibody against PAP8 [ work of . In shorTM High-Fidelity DNA Polymerase . Final constructions were introduced in derivatives of pART27 binary vectors [Minipreps were performed using Qiagen kits and DNA in-gel purification using GeneClean III kit . PCR cloning was generated using Phusion vectors . pBB61: pap8-1 was used as the progeny of a heterozygous plant; transgenic plants were then selected to carry the segregating mutant allele pap8-1, yielding albino plants in the progeny and to carry the selection marker using the corresponding antibiotic or the GFP signal in mature seeds when using the “alligator” system [Electro-competent Agrobacterium were transformed with binary plasmids containing our transgenes see  selected” system .7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS). After 5 min at 10,000× g, 400 µL of supernatant was added to 400 µL isopropanol. After 10 min at 10,000× g, the pellet was washed with 750 µL of EtOH 80% and then dried. DNA was then suspended in 50 µL of water. The PCR was performed with indicated primers. PAP8 (op8_rtp_F + op8i2_R) pap8-1 (oPAP8_rtp_F + oLBb1.3) BB647 transgene (op8F257S + oP8E3_R) HPTII (ohpt1 et ohpt2). oPAP8_rtp_F, tggtggtgatggagatatcg; oPAP8_rtp_R, tttgagacactgaagtctcg; op8i2_R, aaggaagtctcagaacaacgc; oLBb1.3, attttgccgatttcggaac; oP8E3_R, tagtcactcattgcacatcg; EF1α: F, caggctgattgtgctgttcttatcat; R, cttgtagacatcctgaagtggaaga. oHPT1, ttcgatgtaggagggcgtgg; oHPT2, ggtcaagaccaatgcggagc.gDNA preparation: leaf tissues were ground in 1.5 mL reaction tubes, then homogenized in 400 µL of EB buffer  with α set to 0.05 or to retrieve 2) was conducted using the Biolistic PDS 1000/He Particle Delivery System (BioRad)  with DNA onto 1 µm gold particles  following instructions. After 16 to 40 h in the dark at 24 °C, the epidermis was peeled and observed by fluorescence microscopy with a Nikon AxioScope equipped with FITC filters and an AxioCam MRc camera. Pictures acquired with Nikon’s Zen system. Confocal microscopy was performed on a Leica TCS SP2 or a Zeiss LSM800 using standard settings. Protein localization of stably transformed plants was examined on cotyledons.Transient expression in onion cells (bulb sliced to ~16 cm8. Binding mix: rdr32 (probe) 100 nM, buffer first-strand 5×, tRNA (0.2 mg/mL), DTT 1 mM, glycerol 2%, RNasin (10 U/µL), PAP8 (from 0 to 8 µM). Polyacrylamide gel was polymerized with APS and Temed at room temperature. Wells were washed with a syringe filled with running buffer. Pre-run at 4 °C in running buffer for 1 h at 90 volts. Meanwhile, the binding mix was prepared on ice. The probe is denatured using heat (95 °C for 30 s) and then directly stored on ice to keep it single-stranded . The run took about 75 min, at 90 volts, before in-gel cy5 detection using AmershamTM ImageQuant .Polyacrylamide gel 5%: acrylamide/bisacrylamide (29:1) 40%, TBE 1×, APS 10%, Temed 1%. Running buffer: TBE 10×: Tris base, boric acid, EDTA pHg, for 25 min, at 4 °C. The cell pellet was resuspended and sonicated in 30 mL of lysis buffer: 50 mM Tris HCl, pH8, 0.5 M NaCl, 10 mM β-mercaptoethanol, 20 mM imidazole pH8 with a CompleteTM Protease inhibitor Cocktail tablet . The lysate was centrifuged at 15,000× g, for 40 min, at 4 °C. The purification was performed at 20 °C. After filtration, the supernatant was applied onto a NiNTA column in 50 mM Tris HCl, pH8, 0.5 M NaCl, 20 mM imidazole pH8. After elution, PAP8 was loaded on a Superdex 200 10/30 and eluted with 10 mM Tris HCl pH8, 50 mM NaCl, 5 mM DTT.Rosetta2 cells were grown overnight in 50 mL LB with 100 µg/mL of carbenicillin and 34 µg/mL of chloramphenicol at 37 °C. A total of 1 L of LB + antibiotics was then inoculated and cultivated at 37 °C to 0.1 OD600. At 0.6 OD600, the temperature was decreased to 16 °C, and 0.5 mM of isopropyl β-D-1-thiogalactopyranoside was added. After an overnight induction, cells were harvested by centrifugation at 5500×


The curative effect of high-efficiency progesterone and other therapeutic drugs for endometrioid adenocarcinoma patients with preservation of reproductive capacity has not been satisfactory so far. Novel therapeutic drugs need to be explored.LMTK3 and ESR1 in 1647 UCEC samples.We investigated the cytoplastic and nuclear expression levels of LMTK3 between endometrioid adenocarcinoma tissues and adjacent endometrial tissues by immunohistochemistry. We detected the effects of LMTK3 on cell viability of Ishikawa cells by CCK-8. We detected the effects of LMTK3 on cell cycle and apoptosis of Ishikawa cells by flow cytometry. We also detected the effects of LMTK3 knockdown on mRNA and protein levels of ERα by qRT-PCR and western blotting, respectively. We also used the cBioPortal online database to analyze the coexpression of P < 0.05). LMTK3 knockdown decreased the proliferation of Ishikawa cells through decreasing cell viability (P < 0.01), increasing G1 (P < 0.001) arrest, and promoting apoptosis (P < 0.01). There was a positive correlation between the mRNA expression levels of LMTK3 and ESR1 . Knockdown of LMTK3 also reduced the mRNA (P < 0.001) and protein (P < 0.001) levels of ERα.We used TMAs to identify that LMTK3 was mainly detected in the cytoplasm of endometrioid tissues, and cytoplasmic LMTK3 expression in endometrioid tissues was higher than that in adjacent endometrial tissues (Inhibitors of LMTK3 may be a possible future treatment for ERα and LMTK3 highly expressed endometrioid adenocarcinoma following appropriate studies. Uterine corpus endometrial carcinoma (UCEC) is the second most common malignant tumor of the female reproductive system in China  and the Lemur tyrosine kinase 3  is an oncogenic kinase associated with proliferation in different types of cancer [breast , lung 11, stomac2. The following antibodies were used: ERα rabbit monoclonal  and LMTK3 mouse monoclonal .Ishikawa, well-differentiated endometrioid adenocarcinoma cells, obtained from Shanghai Cell Bank of Chinese Academy of Sciences, which were cultured in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin, were incubated at 37°C with 5% COhttp://www.cbioportal.org/)  , 21, an TMAs consisting of 34 human EEC tissues were purchased from Outdo Biotech Co. Ltd. Immunohistochemical staining was performed using Biotin-Streptavidin HRP Detection Systems (Zhongshan Golden Bridge) according to the manufacturer’s instructions. After deparaffinization and rehydration, TMA was pretreated using heat-mediated antigen retrieval with sodium citrate buffer (pH6.0) for 20 min. Then TMA was incubated with 10 µg/ml anti-LMTK3 rabbit polyclonal antibody at room temperature for 15 min. A goat antirabbit secondary antibody was used to detect the primary. All the tissue information on the TMA was scanned and imaged by an automatic digital slide scanner  to form a document. This document was opened by pannoramic viewer software to magnify 1–400 times for observation and image capture and then was analyzed to obtain nuclear and cytoplasmic staining. LMTK3 immunoreactivity was mainly detected in the cytoplasm of endometrial carcinoma and adjacent tissues epithelial cells, and little in the nucleus to a variable degree. Cytoplasmic staining was scored based on intensity ranging from 0 to +3, where 0 = null, +1 = low, +2 = intermediate, and +3 = high level of staining intensity. The positive rate of staining was 0–100%. Cytoplasmic H-scores = intensity score x staining positive rate, which were as follows: low (1–80) and high (81–200).4/well and then was transfected with a control siRNA, siLMTK3-1, siLMTK3-2, and siLMTK3-3 plasmids respectively generated by GeneChem Co. Ltd. Proliferation assay was performed using a CCK-8 reagent (Beyotime). The absorbance at 450 nm was detected after 24- and 72-h transfection. The experiment was repeated three times independently.Cells were seeded in 96-well plates at 1×10After transfection for 48 h with a control siRNA and siLMTK3-3 plasmids, respectively, the cells were digested with 0.25% trypsin without EDTA. After digestion, the cells were collected and centrifuged at 1,500 rpm for 5 min. The supernatant was removed and resuspended with PBS. The cells were washed twice with PBS at 1,500 rpm for 5 min. Apoptosis detection kit (Beyotime) was used for detection. The cells were resuspended with 195 μl annexin V-FITC binding buffer, then added with 5 µl annexin V-FITC, and after being mixed well, 10 ul PI was added; the reaction was at room temperature without light at 20 min and then detected by flow cytometry .After transfection for 48 h with a control siRNA and siLMTK3-3 plasmids, respectively, the cells were digested with 0.25% trypsin without EDTA. After digestion, the cells were collected and centrifuged at 1,000 rpm for 5 min. The supernatant was removed and resuspended with PBS. The cells were washed twice with PBS at 1,000 rpm for 5 min. The cells were resuspended with 100 µl PBS. About 700 µl of precooled 80% ethanol was added slowly to make the final concentration of ethanol of 70%. The cells were fixed at 4 °C for more than 4 h. The cells were washed twice with precooled PBS at 1,000 rpm for 5 min. About 100 µl RNase (50 µg/ml) was added at 37 °C for 30 min; 400 µl PI (50 µg/ml) was added and dyed at 4 °C without light for 30 min and was then detected by flow cytometry .LMTK3 are as follows—forward: 5’-CAAGTGCTGTGGTTGTGTAATG-3’; reverse: 5’-CAGGCATCTTGTCGAGGATGG-3’. The primers for ESR1 are as follows—forward: 5’-GGGAAGTATGGCTATGGAATCTG-3’; reverse: 5’-TGGCTGGACACATATAGTCGTT-3’.Total RNA was isolated using RNAiso Plus . Reverse transcription was performed using a PrimeScript™ RT reagent kit with a gDNA eraser . qRT-PCR analysis was performed on a QuantGene 9600 (Bioer) using primers for LMTK3 and ERα purchased from Sangon Biotech. The primers for Whole cell lysates were prepared using NP40 lysis buffer  glycerol, 1% NP40, 5 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM EGTA, 50 µM leupeptin, and 30 µg/ml aprotinin). These extracts were then centrifuged at 15,000 rpm for 15 min at 4°C. The bicinchoninic acid (BCA) protein assay (Pierce) was used to determine the protein concentration. Lysates were incubated in 6x sodium dodecyl sulfate (SDS) sample buffer , subjected to 8% or 10% SDS-PAGE, and blotted on a nitrocellulose membrane . The membranes were then blocked in TBS containing 0.1% (v/v) Tween20 and 5% (w/v) non-fat milk for 1 h and incubated overnight with different antibodies. After being washed with TBS/Tween20 clearly, the membranes were incubated with goat antirabbit IgG or goat antimouse IgG  for 60 min. The immunocomplexes were detected using enhanced chemiluminescence (ECL) detection. The intensity of the bands was quantified using the Image J software .All statistical analysis was performed using the software packages SPSS 22.0 and GraphPad Prism 5.0. Our data were expressed as x ± SEM; the appropriate t test was applied to analyze the statistical significance between two groups. P < 0.05 was considered to have statistical significance.We used TMAs to explore the LMTK3 expression between EEC and adjacent endometrial tissues, and the level of LMTK3 immunostaining analysis of TMA section revealed that LMTK3 was predominantly detected in the cytoplasm of endometrioid tissues with little in the nucleus of endometrioid tissues (P < 0.01). After 72-h transfection with a control siRNA, siLMTK3-1, siLMTK3-2, or siLMTK3-3 plasmid, the A450 of the siRNA control group was 2.00 ± 0.07, the absorbance at 450 nm of the three siLMTK3 groups were 1.28 ± 0.04, 1.02 ± 0.05, and 0.82 ± 0.02, respectively, and the cell viability of the three siLMTK3 groups was also lower compared with the siRNA control group (P < 0.001)  Figure 2P<0.001) (P < 0.05)  (P<0.05) (P<0.01). All above made it clear that LMTK3 knockdown increased both early and late apoptosis of Ishikawa cells compared with the transfection with a control siRNA plasmid.Because increasing apoptosis is another factor that has an inhibition effect on cell growth, after 48-h transfection with a control siRNA or siLMTK3 plasmid, we detected the apoptosis of Ishikawa cells. The results indicated that the early apoptosis rate of Ishikawa cells transfected with a control siRNA plasmid was only 4.31 ± 0.60%, and that of Ishikawa cells transfected with plasmid siLMTK3 was increased to 7.08 ± 0.61%    (P < 0.001)   Figure 5LMTK3 and ESR1 in 1,647 UCEC samples, and finally we found that LMTK3 knockdown really reduced the mRNA and protein levels of ERα. In short, knockdown of LMTK3 inhibiting cell growth and ERα expression in a well-differentiated ERα-positive endometrioid adenocarcinoma Ishikawa cell line has been first identified. As Hu G et al. had reported that knockdown of ERα could inhibit the cell viability of Ishikawa cells at 48 h and increase G1 arrest, and the type, source, and culture environment of cells were consistent with us , the National Natural Science Foundation of China (No.81872125), and Outstanding Scientific Fund of Shengjing Hospital (NO.201704).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Acompeting off-cycle process leading to vinyl phosphine formation isdetailed for the hydrosilylation of benzophenone for which an inverseorder in [silane] is observed. Experimentally, the formation of sideproducts, including off-cycle vinyl phosphine, is favored by electron-donatingsubstituents on the phosphirenium cation, while catalytic hydrosilylationis promoted by electron-withdrawing substituents. These observationsare rationalized in parallel computational studies.Theutilization of phosphirenium ions is presented; optimized andbroadened three-membered ring construction is described together withthe use of these ions as efficient pre-catalysts for metal-free carbonylreduction with silanes. Full characterization of the phosphireniumions is presented, and initial experimental and computational mechanisticstudies indicate that these act as a “masked phosphenium”source that is accessed via ring opening. Catalysis proceeds via associativetransfer of {Ph The first exampleof a stabilized three-membered phosphirene–tungsten ring wasreported by Marinetti and Mathey in 19823  2 in CDCl3. 3·OTf does not form when using triflate salts, such as silver or irontriflates (entries 2 and 3), or by Brønsted acid activation withtriflic acid16 (entry 4) or activated triflicacid (entry 5). Acetic anhydride does activate 2, withthe formation of Ph2P(OAc)17 and Ph2P(O)PPh2.18 However, these species do not react further with 1 (entry6). Di-tert-butyl dicarbonate (Boc2O)is not effective for this transformation, and even at higher temperatures, 1 and 2 are recovered (entry 7). Following abrief solvent and temperature screening,19 the optimal conditions were determined to be CH3CN at60 °C for 30 min, which give full conversion to 3·OTf (entry 8).Due to the practicality of the reaction, Hirano andMiura’s procedure (where the phosphirenium ion was detectedbut not isolated)ArF tothe optimizedreaction mixture of 1, 2, and Tf2O in CD3CN forms 3·BArF, which is isolated in 66% yield  and 1.746(3)Å, and the C=C bond distance is 1.340(4) Å; bothare similar to reports from Mathey, Wild, and Cowley.21 Thealternating single and double bonds show that the structure depictedis the dominant resonance form of the phosphirenium ion in the solidstate. This species represents the first isolated phosphirenium ionwith a borate counterion.22Adding 1 equiv of NaB6% yield 1. A smal5·OTf, 6·OTf, 7·OTf, 8·OTf, and 10·OTf proved particularly challenging; see the Supporting Information for spectral data). The use of different substitutedSPOs, bearing bearing aryl or alkyl groups, is well tolerated. Forexample, using Cy2P(O)H, 4·OTf and 4·BArF are isolated in high yields . These speciesdisplay downfield 31P{1H} NMR signals comparedto 3, with a singlet at −85.4 and −86.5ppm, respectively, due to the increased electron donation of the alkylsubstituents. There is only a very modest difference in the 31P chemical shift observed between the ions bearing different anions, 3·OTf versus 3·BArF Δδ = 0.8 and 4·OTf versus 4·BArF Δδ = 1.1 ppm, which is as expected giventhe non-coordinating nature of the counterions.With the optimized conditions in hand, the generality of the methodologywas explored 2. Some otBuPhP(O)H and CyPhP(O)H, are also well tolerated with full conversionafter 1 h to form 5·OTfHlead to full conversion to 7·OTf with a phosphorussignal at −80.2 ppm and a downfield signal for the terminalC–H at 8.4 ppm. For all phosphirenium species presented, thereare deshielded signals in the proton and carbon NMR spectra, whichcan be explained by the resonance structure of the phosphirenium ion.26 1-Phenyl-1-propyne reacts well with 2 to form 8·OTf with a 31P signal at −103.6 ppm.Further functionalization of the alkyne is tolerated with 9·OTf (38%) and 10·OTf (64%) isolated using the optimizedconditions. The phosphorus signals for 9·OTf and 10·OTf appear at δ −104.3 and −110.9ppm, respectively, versus −108.3 ppm for 3·OTf; thus, by this measure, the alkyne substituents only have a minorimpact on P+ electronics, but as we show below, the samesubstituents have a dramatic effect on catalytic reactivity. Crystallizationis not limited to BArF counterions, and X-raysingle-crystal analysis of 10·OTf confirms the three-memberedstructure of this phosphirenium species  as 28% 13a and 16% 14a. By increasingthe temperature to 80 °C (entry 2), full conversion of the startingmaterial is observed with 84% formation of 13a, while16% 14a persists. Using 10 mol % isolated 4·BArF, lower conversions(73%) are observed after 18 h (entry 3), with 58% 13a. Isolated pre-catalyst 3·BArF shows higher reactivity, with increasedconversion to 14a versus 13a at 10 mol %loading (entry 5). When phosphirenium triflate species are used, suchas 3·OTf or 4·OTf, only deoxygenationoccurs with full conversion into 14a; this is due tohydrogen release from silane reacting with co-catalytic TfOH (formedfrom Tf2O).28 Thus, in linewith Miura’s recent studies on phosphirenium-derived heterocycles,11 we also note that the use of a base is crucial to quench TfOH releasedduring pre-catalyst formation and therefore increase the selectivitytoward 13a. See Table S2 in the Supporting Information where comprehensive benchmarking and optimizationwere undertaken demonstrating that not only does TfOH lead exclusivelyto the deoxygenation product 14a but also that superiorhydrosilylation catalysis takes place using a phosphirenium pre-catalystcompared to the equivalent reaction using TfOPPh2. A totalof 5 mol % 2,6-lutidine was added to 5 mol % in situ-prepared pre-catalyst 4·BArF prior to addition of substrates 11 and 12, and the selective formation of 13a is observed . Changing to 3·BArF, with 5 mol %2,6-lutidine, gives good reactivity at lower temperature (entry 6),and further improvement is seen with the electron-poor phosphireniumspecies 9·BArF, for which reaction with 5 mol % 2,6-lutidine leadsto full conversion into 13a at 60 °C after only1 h (entry 7). In contrast, electron-rich 10·BArF reacted at a slowerrate with full conversion to the hydrosilylation product requiring58 h at 80 °C (entry 8). Clearly the electronics of the alkyneused to prepare the phosphirenium pre-catalyst play a substantialrole in catalytic competency. Screening of the base with 10 mol % 3·BArF as the catalyst of choice shows lower conversion to 13a when using NEt3 and pyridine (entry 9 and 10) with 59and 52%, respectively, and recovery of the starting materials withK2CO3 (entry 11) after 18 h at 80 °C. Theseresults show that a slightly bulkier, aromatic organic base like 2,6-lutidineis most effective.29Initial studies were conducted with 10 mol % phosphirenium species  at r.t. 2. Initia2.33·BArF, by NMR spectroscopy  and product  in the 29Si{1H} NMR spectrum.However, a plethora of species are observed in the 31P{1H} NMR spectrum, with the major species being 3·BArF at −108.5ppm, along with P2Ph4 at −17.8 ppm, (E)-diphenylphosphine at 7.1 ppm,9 smaller amounts of (Z)-diphenylphosphineat −7.4 ppm,30 and benzhydryl diphenylphosphinateat 36.5 ppm.31 While phosphines are knownto facilitate hydrofunctionalization of carbonyls,32 noteworthy is the lack of catalytic activity observed using10 mol % HPPh2 or P2Ph4.We initiatedour mechanistic investigations by analyzing the catalytic reactionmixture, using in situ-prepared troscopy 2a. After3·BArF with subsequent additionof 2,6-lutidine and 12 results in two new species by 29Si{1H} NMR spectroscopy. The major species isidentified as Me2SiHOTf at 23 ppm, formed by the facilecleavage of the silane phenyl group.33 Analysisof the mixture by 31P{1H} NMR spectroscopy shows 3·BArF, P2Ph4, and vinyl phosphine as per the catalyticreaction, along with HPPh2 with its characteristic peakat −40 ppm .33 The latter mixture was also analyzed by 31P{1H} NMR spectroscopy, which shows the presence of 3·BArF, vinyl phosphine,and P2Ph4, but no traces of HPPh2 are visible at any point of the analysis -phenyl)vinyl)diphenylphosphine] insitu. Hydrosilylation to form 13a using 10 mol % 10·BArF pre-catalyst and 2,6-lutidine gives a complex array of peaks inthe 31P NMR spectrum, making identification difficult althoughlikely including vinyl phosphines.When performing hydrosilylationto form E)-diphenylphosphineand 20 mol % 2,6-lutidine in a catalytic reaction of 11 and 12 does not give an appreciable quantity of 13a after 18 h at 80 C, indicating that the vinyl phosphineis not an active catalyst in the hydrosilylation reaction, and thisis further supported by computational studies.34The use of 20 mol % . Therefore, the negative order in silane can be attributedto silane involvement in side-product formation or catalyst degradation.Comparable data are obtained when using isolated pre-catalyst 3·BArF in the absence of 2,6-lutidine, further supporting the assertionthat the role of 2,6-lutidine is simply to mop up TfOH released onformation of 3·BArF from reaction of Tf2O with alkyne, SPO,and NaBArF.26 Itis important to note that the kinetic data found for this transformationare in contrast with Piers’ hydrosilylation-type activationvia Lewis acids ,36 where the increase in ketone concentration slowsdown the reaction. Indeed, interrogating the 11B and 19F NMR spectra from catalytic reactions also shows that onlyborate is present in solution; no borane signals are observed, andso we feel that borane-mediated hydrosilylation is unlikely in thiscase.Next, we undertook kinetic studies using NMR spectroscopicmonitoring.By pre-forming the catalyst in situ, we have analyzed the order insubstrates ed for 12 Observation of vinylphosphine bothduring catalysis and in stoichiometric studies indicates that phosphireniumring opening could be involved, either as a catalyst deactivationprocess or on-cycle ring-opening/ring-closure sequence;(ii)The inverse order in silane indicatesthat there is a catalytically inactive pathway dominating at highersilane loadings.(iii)9·BArF > 3·BArF ≫ 10·BArF), but these changes in electronicsonly have a modest impact on the phosphorus center . We therefore believe that therole of the phosphirenium pre-catalyst is not merely to act as a simpleLewis acid.37Phosphirenium ions with differentelectronic substituents have very different capabilities in hydrosilylation with acetone and Me2PhSiH. Initial studies consideredthe direct nucleophilic attack of either the ketone or silane at thephosphirenium ring;29 however, the characterizationof these processes indicated that a ring-opening step occurs firstto form an ethenylium intermediate II at +22.0 kcal/mol.From II, catalysis is initiated via the nucleophilicattack of the ketone at the P center (see TS(II–III)P at +28.6 kcal/mol and results in both P–Obond formation and displacement of the alkyne to form a phosphenium-ketoneadduct IIIP. Catalysis now proceeds(TS(III–IV)P, +27.9 kcal/mol] to form IVP, in which partial hydride transfer hasoccurred . Silyl group migration then readily occurs to form VP at −7.8 kcal/mol, featuringa silylalkyloxonium adduct of the {Ph2P} moiety. Ketoneattack at VP then displaces thehydrosilylation product and generates IIIP that can then enter a second cycle with further reaction withsilane.38With these experimental results in hand, we undertook a computationalstudy of the reactions of the phosphirenium cation, nter see 4a. This proceeds4b via SiTS(II–III)P at 28.6kcal/mol to form IIIP, from whichcatalysis can then proceed with an energy span of only 10.8 kcal/mol.Catalysis from IIIP should thereforebe very efficient; however, the situation is complicated by at leasttwo possible competing reactions. The first of these is vinyl phosphineformation (II and proceeds via TS(II–III)H at +30.7 kcal/mol with net insertion into the Si–Hbond to give silylalkylphosphonium ion IIIH,Z at −9.0 kcal/mol, featuring a short P–Cbond (1.76 Å). Assuming facile rotation about the C–Cbond leads to IIIH,E , which can react with ketone with displacement of a silylethercation to form E-vinyl phosphine IVH,E .39 In principle, IVH,E could re-enter catalysis via hydride transfer to the silylethercation, generating the hydrosilylation product and intermediate II. However, this process has a computed barrier of 48.3 kcal/mol,and so the formation of vinyl phosphine represents an unproductiveoff-cycle process. The second competing process P at +3.3 kcal/mol with the release of thehydrosilylation product and formation of II. II can then rapidly undergo ring closure to reform I.Overall, the calculations imply a relatively slow initiation processvia ormation 4c. This  process 4d involvTS(II–III)H and TS(II–III)P, where the latter is more stable and ΔΔG1 = +2.1 kcal/mol. The productive onward reaction to form IIIP should therefore dominate; however,the small energy difference suggests that vinyl phosphine formationmay be significant at higher silane concentrations, and this is consistentwith the experimental identification of an inverse first-order reactionwith respect to silane. For the competing reactions of VP with either the ketone or the alkyne, thekey transition states are TS(III–IV)P and TS(V–II)P, respectively. In this case, TS(V–II)P is only marginally disfavored with ΔΔG2 = +0.4 kcal/mol, and this small energy difference suggestsreaction with alkyne and therefore the reformation of I will be competitive. However, unlike the vinyl phosphine IVE, I remains a competent catalystprecursor, albeit one that reacts with a relatively high overall barrierof 28.6 kcal/mol. There is therefore an ensemble of processes at play,in which efficient catalysis via IIIP is balanced by catalyst loss through vinyl phosphine formationand a less efficient cycle based on phosphirenium precursor I. The net effect of these various processes is qualitativelyconsistent with a reaction that requires several hours at 60 °Cto reach full conversion.The significance of these two competing processes is captured inthe free energy differences (ΔΔG) between the rate-limitingtransition states. For vinyl phosphine formation, these are 2.3.19+ and 10+ were similar to those for theparent species 3+ (see the Supporting Information). However, subtle changesin the relative energies of the key competing transition states arecomputed that have a significant impact on the behavior of these systems times to full conversion seen experimentally with 9+. In contrast, the electron-donating p-OMe substituents switch the preference toward vinyl phosphineformation from II  and favoring the reaction of VP with alkyne that leads to the formation of I overthe more direct catalytic reaction with ketone. Both factors shouldrender catalysis less efficient, consistent with the very sluggishcatalytic reaction that is observed with 10+ (58 h to full conversion). The calculations indicatethat while the p-OMe substituents in 10+ promote ring opening , they also disfavor the subsequent{Ph2P+} transfer via TS(II–III)P, and this results in hydride transfer via TS(II–III)H dominating.Reaction profilescomputed for cations temssee 5. For 9+2P+} moiety through reaction with silane, in accordancewith inverse order kinetics. Para-substituents on the phosphireniumaryl groups can significantly impact the behavior; electron-donating p-OMe substituents undermine catalysis by favoring the formationof side products.These combined experimental and computational studies allow usto propose a reaction mechanism for the hydrosilylation of carbonylscatalyzed by phosphirenium cations. These species act as a maskedphosphenium source that is accessed via ring opening. Productive turnoverthen involves associative phosphenium transfer to the ketone, Si–Haddition over the C=O bond, and associative displacement ofthe hydrosilylation product with further ketone. Spectroscopic andcomputational insights show that the formation of vinyl phosphineis an irreversible off-cycle process that will sequester the {Ph2 and P2Ph4 by in situ reaction monitoringalso supports the presenceof the ring-opened form in the catalytic reaction. Given the capriciousnature of unstabilized phosphenium ions, we do not observe free {Ph2P+} in the catalytic reaction. Calculations indicatethat loss of {Ph2P+} from the parent phosphireniumis uphill by 34 kcal/mol; thus, observation of the free species isunlikely, lending further support to it being transferred directlybetween nucleophiles during the cycle. Catalytic reactions do showthe presence of a phosphinate peak in the 31P NMR spectrum, which gives tentative support for the presenceof phosphenium adducts.Experimentally, the observation of HPPh2.413a can be isolated in 73% yield, after 5 h at 60 °Cusing 2.5 mol % 3·BArF . Alkyne-containing carbonyl substrates are notsuitable for this transformation, possibly due to alkyne scramblingof the propargyl group.41 Cyclohexanoneand acetone react well using 10 mol % 3·BArF, giving 13g and 13h in 87 and 62% yields, respectively, while acetophenonereacts sluggishly, and even with 10 mol % catalyst loading, only 23%of 13i is obtained. The use of other tertiary silaneswas found to be suitable, and 13j is obtained in fullconversion and high yield (85%). However, triethoxy silane and triphenylsilane do not convert, but the phosphirenium pre-catalyst is unaffectedand is still observed in the reaction mixture. Secondary or primarysilanes do not react with benzophenone 11 under the reactionconditions, and decomposition of the phosphirenium is observed byNMR spectroscopy.In this procedure, the highertemperature does not affect the reactionoutcome with hydrosilylation of diaryl ketones occurring selectivelyin 5 h. Unsymmetrical ketones with an aryl substituent and C13k–13p. Cinnamaldehyde reacts smoothly to yield 13q in 75%yield. The reaction also works well with 4-methylbenzaldehyde, furnishing 13r in 89% isolated yield, but is quite limited with otherelectron-rich aldehydes. 4-Dimethylamino benzaldehyde shows low conversioninto 13s (22%) after 18h with 10 mol % 3·BArF; 4-methoxybenzaldehydedoes not show any conversion after 24 h, which suggests a potentialdeactivation of the carbonyl or catalyst poisoning. Electron-withdrawinggroups in the ortho- and meta-positionare detrimental, giving low conversion and yield of 13t, 13u, and 13v. Tiglic aldehyde reactsusing 10 mol % catalyst at 80 °C with full conversion of silaneto 13w in only 40% spectroscopic yield, with traces ofthe product derived from the reduction of the double bond observedin situ. Alkyl aldehydes such as pentanal, heptanal, and dodecanalgive 13x, 13y, and 13z in 72–88%isolated yields, respectively, and cyclopropane aldehyde reacts togive 13aa in 77% isolated yield with no ring openingobserved, highlighting that radicals are not involved in the reaction.Several aldehydes were subjected to the optimizedreaction conditions,and the results are good to excellent when using electron-poor arylaldehydes, with 60–98% isolated yields for 3The ease of synthesis and thenewly discovered reactivity of phosphireniumions allow us to conclude that they are effective organocatalystsfor the hydrosilylation of ketones and aldehydes; to the best of ourknowledge, this is the first report of a phosphirenium ion being usedas a pre-catalyst. Full characterization of the phosphirenium ionsbearing triflate or borate counterions is reported, and further detailson their synthesis were explored. The practical pre-catalyst synthesisfrom typically cheap and readily available starting materials suchas phosphine oxides and alkynes allows a straightforward catalyticprocedure that is easily scalable. Our mechanistic investigationsgive insights into the principles governing their reactivity; thisis underpinned by DFT studies and experimentally determined kineticsand stoichiometric studies. The ability of these phosphirenium ionsto undergo ring opening paves the way for several reaction pathways,with the dominant and most favorable pathway being the release ofthe active catalyst, which is a phosphenium ion adduct. Increasingthe electron-donating ability of the alkyne used to prepare the pre-catalystslows catalysis, allowing an off-cycle deactivation process to dominate.These elusive phosphenium species are highly reactive and challengingto isolate and characterize; therefore, by disguising a phospheniumcatalyst as a more stable and manageable phosphirenium adduct, weopen up a multitude of opportunities in organic synthesis. Work isstill ongoing in our laboratory to shed light on diverse reactivityand the activation modes of phosphirenium rings and the broader applicabilityof masked phosphenium catalysts.44.1ArF (1 equiv.) were stirred in CD3CNor CDCl3 at 60 °C for 30 min. The reaction was monitoredby 31P{1H} NMR spectroscopy until complete disappearanceof the phosphine oxide peak. After the reaction was completed, themixture was concentrated under vacuum. The product was washed withpentane and recrystallized at −20 °C.In a J-Young NMR tube or a Schlenk tube, alkyne (0.25mmol), SPO (0.25 mmol), triflic anhydride (1 equiv.), and NaB4.2–3 mmol,2.7 mg) or diphenyl phosphine oxide , diphenylacetylene, triflicanhydride ,and NaBArF  were stirred in CH3CN (3 mL) for 30 minat 60 °C. After formation of pre-catalyst 3·BArF, the reactionmixture was cooled down, and 2,6-lutidine , benzophenone , and dimethylphenylsilane  were added,and the reaction was heated at 80 or 60 °C for 18 and 5 h, respectively.After drying the volatiles, the pure product was obtained by purificationby flash chromatography (9:1 petroleum ether/EtOAc) or vacuum distillation.To a J-Young NMR tube, dicyclohexylphosphine oxide /def2-TZVP level, which has been shown to be a reliableapproach for main group kinetics, thermodynamics, and non-covalentinteractions.42 These corrected energieswere then combined with the thermodynamic corrections from the gas-phasecalculations to give the free energies quoted in the text. Full detailsand references are supplied in the Supporting Information.Gaussian 09 (RevisionD.01) was used with geometry optimizations carried out at the TPSS/def2-SVPlevel and all stationary points characterized with analytical frequencycalculations. Electronic energies were recomputed at the M052X-D3(CH


Background: The prognosis of breast cancer varies according to the molecular subtype. Transmembrane 4 L six family 1 (TM4SF1) exhibits different expression patterns among the molecular subtypes of breast cancer. However, the expression profile of TM4SF1 in hormone receptor HR+HER2- breast cancer remains unclear.Methods: TM4SF1 mRNA levels were examined in major subclasses of breast cancer by analyzing The Cancer Genome Atlas (TCGA) datasets. In addition, TM4SF1 protein and mRNA levels in HR+HER2- breast cancer tissue samples were determined by immunohistochemistry and Western blot assay. The effect of TM4SF1 on cell proliferation was evaluated using MTT, colony formation, 3D organoid, and xenograft models, following the TM4SF1 overexpression or knockdown.Results: TCGA database analysis demonstrated that TM4SF1 was downregulated in breast cancer compared with the healthy adjacent breast tissue. In addition, the expression of TM4SF1 in basal-like one and the mesenchymal TNBC tissue was higher than that of the healthy adjacent breast tissue. Other types, including the luminal androgen receptor–positive TNBC tissue, expressed lower levels of TM4SF1. Immunohistochemistry and real-time quantitative PCR assays demonstrated that the TM4SF1 protein and mRNA levels were downregulated in the HR+HER2- breast cancer tissue compared with the healthy adjacent tissue. Moreover, the TM4SF1 overexpression reduced the viability of MCF-7 and ZR-75-1 breast cancer cells, whilst reducing the number of colonies and 3D-organoids formed by these cell lines. By contrast, TM4SF1 knockdown led to an increased MCF-7 cell proliferation. However, in the TNBC cell line, MDA-MB-231, TM4SF1 silencing reduced cell proliferation. In vivo, the TM4SF1 overexpression inhibited MCF-7 xenograft growth in a nude mouse model, which was associated with the downregulation of the Ki-67 expression, apoptosis induction, and inhibition of the mTOR pathway.Conclusion: TM4SF1 is downregulated in HR + HER2-breast cancer, and the overexpression of TM4SF1 suppresses cell proliferation in this cancer subtype. TM4SF1 r kinase . TM4SF1 r kinase , ovarianr kinase , glioma r kinase , colorecr kinase , liver  database was used to characterize TM4SF1 expression profiles between different types of breast cancer. TM4SF1 expression profiles in HR+HER2- breast cancer tissues were tested by immunohistochemistry and qRT-PCR assay; its roles in HR+HER2- breast cancer development were investigated upon TM4SF1 overexpression or knockdown.Hormone receptor (HR)+HER2-breast cancer contributes to most breast cancer deaths . Clarify2.MCF-7 was cultured in the MEM supplemented with 10% FBS and antibiotics. ZR-75-1 cells were cultured in the DMEM supplemented with 10% FBS and antibiotics. MDA-MB-231 cells were cultured in the RPMI-1640 medium supplemented with 10% FBS and antibiotics. 293T was cultured in the DMEM supplemented with 10% FBS and antibiotics. All cells were purchased from the Chinese Academy of Sciences Type Culture Collection. Cells were maintained at 37°C in a humidified atmosphere with 5% CO+HER2- breast cancer tissue samples were obtained from Taizhou Central Hospital with the consent of all patients. The tissue samples were fixed in formalin and embedded in paraffin. Each slide containing both healthy adjacent breast tissue and tumor breast tissue samples was used for immunohistochemistry. The present study was approved by the Medical Ethics Committee of Taizhou Central Hospital .Human hormone receptor (HR)Tissue sections were rehydrated using xylene and graded concentrations of ethanol, incubated in sodium citrate  for 10 min, and then cooled down to room temperature. Endogenous peroxidase activity was then blocked with 3% hydrogen peroxide for 30 min at room temperature. The sections were permeabilized with 0.1% Triton and blocked in 10% goat serum for 30 min. The following primary antibodies were used: anti-TM4SF1 , anti–Ki-67 , and anti-FLIP . All primary antibodies were diluted in 5% goat serum and added to the sections at 4°C overnight. The sections were then incubated with a biotinylated goat anti-rabbit antibody . For detection, streptavidin–horseradish peroxidase and a DAB substrate kit  were used. Counterstaining was carried out using hematoxylin.The pLenti-EF1a-EGFP-P2A-Puro-CMV-TM4SF13-Flag plasmid was designed and synthesized by OBiO Biotechnology Corp., Ltd. The GenBank accession number for the TM4SF1 gene is NM_014220.3. The pMDG2.G and psPAX2 plasmids for the lentivirus assembly were obtained from Addgene, Inc. The 293T cells stably expressing the TM4SF1 plasmid and a control vector were prepared by lentiviral transduction. Briefly, co-transfection was performed by combining the lentiviral plasmid (2.5 μg) with the packaging plasmids (0.75 μg pMD2. and 1.90 μg psPAX2). The 293T cells were then infected with viral supernatants containing 8 μg/ml polybrene (MedChemExpress). Transduced cells were selected using 1 μg/ml puromycin.Small interfering RNA (siRNA) transfection. Synthetic siRNAs targeting TM4SF1 were purchased from Shanghai GenePharma Co., Ltd. The sequences were as follows: siRNA-1, 5′-GCACGATGCATCGGACATTCT-3′; siRNA-2, 5′- GCTATGGGAAGTGTGCACGAT-3′; and control-siRNA, 5′-UUCUCCGAACGUGUCACGUTT-3′. siRNA was transfected using Lipofectamine®-RNAiMAX .® and then reverse-transcribed with the Prime Script™ RT reagent kit . The cDNA templates were amplified by qPCR using the PowerUp™ SYBR™ Green Master Mix . The primer sequences were as follows: TM4SF1 forward, 5′-TGCAGGATCTGGCTACTGTG-3′ and reverse, 5′-CAGAAGGTACTGGCCCTCAG-3′; and GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-GCCTTCTCCATGGTGGTGAA-3′. The thermocycling conditions were as follows: 1) 50°C for 120 s, 2) 95°C for 120 s, 3) 40 cycles of 95°C for 15 s and 60°C for 60 s, 4) 95°C for 15 s, 5) 60°C for 60 s, and 6) 95°C for 15 s. Gene expression levels were calculated using the ΔCq method. The mRNA levels of TM4SF1 were normalized to those of GAPDH mRNA.Total RNA was extracted using Trizolβ-actin   were obtained. Antibodies against Bax (BA0315), Bcl-2 (BA0412), caspase 3 (BM4340), caspase 9 (BM4619), and FLIP  were obtained from Boster Biological Technology . Antibodies against GAPDH , LC3 , and Beclin-1  from Santa Cruz Biotechnology  were used. All primary antibodies were polyclonal. Proteins were visualized using a peroxidase-conjugated secondary antibody  using ECL solution  for detection.Immunoblotting was conducted using standard procedures. Briefly, the cells were resuspended in the RIPA lysis buffer (Beyotime Institute of Biotechnology). The sample was transferred to a 1.5-ml centrifuge tube and then centrifuged at 10,000 x g for 5 min at 4°C to pellet the cell and tissue debris and collect the protein lysates. Equal masses of protein (50 μg) were analyzed by SDS-PAGE. Antibodies against TM4SF1 , AKT , phosphorylated AKT , and 3 cells were plated in a 96-well plate using a complete culture medium. After 4, 24, 48, or 72 h, MTT was added to each well at a final concentration of 0.5 mg/ml. The reaction was allowed to proceed for 4 h at 37°C. The formazan crystals were then dissolved in 100 μL DMSO. The plates were incubated in an orbital shaker at room temperature for 15 min, and the absorbance was then read at 490 nm. All assays were performed in triplicates.Cell proliferation was evaluated using MTT assays. Briefly, 1 × 103 cells were gently mixed with 30 μL Matrigel and plated as a droplet containing the single cells onto a prewarmed (in a 37°C incubator) 24-well ultra-low attachment plate. The droplets were allowed to solidify at 37°C for 15 min, then covered with 500 μL complete cell culture medium, and incubated at 37°C for 15 days 3D organoid images were examined under a microscope.Briefly , a total3 cells were seeded into 6-well plates and incubated for 10 days at 37°C with 5% CO2. The colonies were fixed in 100% methanol, then stained for 30 min in crystal violet, and washed in ddH2O. The clones were counted under a microscope.For colony formation assays, 1 × 10Cell apoptosis was analyzed by flow cytometry. Following different treatments, cells were trypsinized and washed with PBS twice. The cells were then stained with Annexin V-FITC/propidium iodide (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. Apoptosis was analyzed by MultiCycle software.ad libitum. A total of 107 parallel-controlled or TM4SF1-overexpressing MCF-7 cells were injected subcutaneously into the flank of the mice, respectively. Tumor dimensions were measured with calipers twice a week, and the volumes were calculated as (width)2 x length/2. After 12 weeks, the animals were sacrificed by cervical dissociation, and the tumors were removed and weighed. The largest tumor diameter was 1.434 cm, and none of the animals developed multiple tumors.All animal experiments were approved by the Medical Ethics Committee of the Taizhou University College of Medicine . A total of 24 male BALB/C-nu/nu mice  were obtained from Changzhou Cavens Animal Co., Ltd. The mice were housed in sterile cages under laminar airflow hoods at 20°C in a specific pathogen-free environment with a 12-h light/dark cycle and provided with autoclaved chow and water t-test or one way ANOVA by GraphPad Prism software (GraphPad Software). A p-value < 0.05 was considered significant.The data are presented as the mean ± standard deviation and were analyzed using Student’s unpaired n = 1,097) and normal breast (n = 114) TCGA data. TM4SF1 mRNA was expressed at lower levels in breast cancer than in normal tissue samples .As the repopulation of residual tumor cells can lead to cancer recurrence, which depends on the ability of cells to divide , the effin vivo and provide more accurate data about cell-to-cell interactions, tumor characteristics, drug discovery, metabolic profiling, stem cell research, and other types of diseases  versus normal breast epithelium revealed that the expression of TM4SF1 is significantly downregulated in DCIS (+PR+HER2- cell lines) were used to investigate the role of TM4SF1 in HR+HER2- breast cancer. The results demonstrated that the overexpression of TM4SF1 in these cell lines resulted in a decrease in their viability, 3D organoid formation, and colony formation. Conversely, TM4SF1 siRNA transfection increased the MCF-7 cell proliferation. By contrast, in the MDA-MB-231 TNBC cell line where TM4SF1 is overexpressed, TM4SF1 siRNA decreased the MDA-MB-231 cell proliferation. In vitro, TM4SF1-overexpressing MCF-7 xenografts grew at a slower rate than the control group. In tumor xenograft tissue, the frequency of Ki-67+ cells was significantly reduced following the TM4SF1 overexpression, whereas TUNEL staining was increased. Consistent with these findings, cleaved caspase 3/9 and Bax protein levels were upregulated, while those of BCL-2 and FLIP were downregulated following the TM4SF1 overexpression. These results suggested that the TM4SF1 overexpression could inhibit cell proliferation and induces apoptosis.The previous studies about TM4SF1 in breast cancer progression and development were also controversial cause utilizing different cell lines randomly . A compa in DCIS . In the  in DCIS . However+HER2- breast cancer development via the mTOR pathway.Moreover, it was also shown that the phosphorylation of AKT and p70S6K1 levels was reduced following the TM4SF1 overexpression. By contrast, the levels of the autophagy markers Beclin one and LC3 were increased . Excessi+HER2- breast cancer compared with noncancerous tissue samples. The overexpression of TM4SF1 inhibited breast cancer growth in vivo, as well as breast cancer cell viability and proliferation in vitro. Thus, TM4SF1 suppresses the HR+HER2- breast cancer cell proliferation, but the exact role of this molecule remains unclear and deserves further investigation.In conclusion, TM4SF1 mRNA and protein levels were downregulated in HR