SUMOylation, the attachment of SUMO  proteins to a target protein, regulates the HIF pathway, although the impact of SUMO on HIF activity remains controversial. Here, we examined the effects of SUMOylation on the expression pattern of HIF-1α in response to pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG) in intestinal epithelial cells. We evaluated the effects of SUMO-1, SUMO-2, and SUMO-3 overexpression and inhibition of SUMOylation using a novel selective inhibitor of the SUMO pathway, TAK-981, on the sensitivity of HIF-1α in Caco-2 intestinal epithelial cells. Our findings demonstrate that treatment with TAK-981 decreases global SUMO-1 and SUMO-2/3 modification and enhances HIF-1α protein levels, whereas SUMO-1 and SUMO-2/3 overexpression results in decreased HIF-1α protein levels in response to DMOG. Reporter assay analysis demonstrates reduced HIF-1α transcriptional activity in cells overexpressing SUMO-1 and SUMO-2/3, whereas pretreatment with TAK-981 increased HIF-1α transcriptional activity in response to DMOG. In addition, HIF-1α nuclear accumulation was decreased in cells overexpressing SUMO-1. Importantly, we showed that HIF-1α is not directly SUMOylated, but that SUMOylation affects HIF-1α stability and activity indirectly. Taken together, our results indicate that SUMOylation indirectly suppresses HIF-1α protein stability, transcriptional activity, and nuclear accumulation in intestinal epithelial cells.The hypoxia-inducible factor (HIF) is a master regulator of the cellular transcriptional response to hypoxia. While the oxygen-sensitive regulation of HIF-1α subunit stability  Hypoxia arises when the cellular oxygen demand exceeds the vascular supply. On a cellular level, tissue adaptation to hypoxia is primarily controlled by the hypoxia-inducible factor (HIF) signaling pathway. HIFs are heterodimeric transcription factors that regulate the cellular response to hypoxia by inducing the expression of over 400 hypoxia-dependent genes involved in adaptive processes, such as glycolysis, erythropoiesis, and cell survival, consequently, normalizing oxygen homeostasis in hypoxic tissues  and CREB-binding protein/p300, leading to increased transcriptional expression of HIF-dependent genes, thus driving the cellular transcriptional response to hypoxia  in intestinal epithelium. Previous work from our group has shown HIF-1α to be highly protective in models of inflammatory bowel disease , 18, 19.A). Downregulation of SUMO-1 and SUMO-2/3 modifications was a later event, with their expression fully inhibited at 4 h after treatment with 100 nM of TAK-981 . A dose of 100 nM of TAK-981 produced substantial inhibition of the SUMO pathway after 4 to 6 h, thus we selected this dosage and time points for further experiments.We first demonstrated that the novel pharmacological SUMO inhibitor TAK-981 inhibits the SUMO pathway in Caco-2 intestinal epithelial cells in a dose- and time-dependent manner by analyzing global SUMO-1 and SUMO-2/3 modifications . TAK-981Fig. 1A). Densitometric analysis of HIF-1α expression confirmed upregulation of HIF-1α protein levels in cells treated with a combination of TAK-981 and DMOG (B). Western blot together with densitometric analysis validate that pharmacologic inhibition of SUMOylation with TAK-981 enhances sensitivity of Caco-2 cells to DMOG by elevating HIF-1α protein levels. Together, our results indicate that SUMOylation is a negative regulator of HIF-1α stability in intestinal epithelial cells.Having demonstrated the inhibitory effects of TAK-981 on the SUMO pathway, we next investigated the impact of TAK-981 on HIF-1α protein levels. The pan-hydroxylase inhibitor DMOG was used to stabilize the HIF pathway in Caco-2 cells. To determine whether SUMOylation affects HIF-1α expression, cells were simultaneously treated with TAK-981 and DMOG for 4 h or pretreated with TAK-981 for 2 h prior to treatment with DMOG for 4 h. We found that inhibition of the SUMO pathway with TAK-981 enhanced HIF-1α protein expression in response to DMOG A. Densitand DMOG B. WesterA). Furthermore, SUMO-2 and SUMO-3 overexpression also promoted a decrease in HIF-1α protein levels; however, the effects of overexpressed SUMO-2/3 isoforms on global SUMO-2/3 modifications were less pronounced (B). Densitometric analysis of HIF-1α expression further confirmed reduced HIF-1α protein expression in cells overexpressing various SUMO isoforms (C and D). Western blot together with densitometric analysis validate that SUMO-1, SUMO-2, and SUMO-3 suppress DMOG-activated HIF-1α protein expression in Caco-2 cells.Having demonstrated that pharmacological inhibition of the SUMO pathway enhanced HIF-1α expression, we next evaluated the effects of SUMO-1, SUMO-2, and SUMO-3 overexpression on HIF-1α protein levels in response to DMOG. To determine whether SUMO isoforms affect HIF-1α expression, we overexpressed SUMO-1, SUMO-2, or SUMO-3 protein for 48 h and then treated cells with DMOG. SUMO-1 overexpression resulted in a decrease in HIF-1α protein expression in response to DMOG, which was correlated with an enhanced level of SUMO-1 modifications A. Furtheonounced B. Densitisoforms , C and DA). Interestingly, before DMOG administration to the cells (0 h time point), basal HIF-1α transcriptional activity was decreased because of SUMO-1 overexpression. In contrast, pretreatment with TAK-981 had no effect on HIF-1α transcriptional activity in cells stimulated with DMOG . Moreover, we observed similar result when cells were overexpressing SUMO-2 and SUMO-3 isoforms. HIF-1α transcriptional activity was significantly reduced at early time points (0–6 h) and after 24 h of DMOG treatment because of SUMO-2 and SUMO-3 overexpression (C and D). Altogether, these data indicate that SUMO-1, SUMO-2, and SUMO-3 reduce HIF-1α transcriptional activity in Caco-2 cells before stimulation and after stimulation with DMOG, whereas inhibition of the SUMO pathway with TAK-981 enhances HIF-1α transcriptional activity only after 24 h of DMOG treatment.Having demonstrated a negative effect of SUMOylation on HIF-1α protein expression, we next evaluated the impact of SUMO-1 and SUMO-2/3 overexpression and TAK-981 on HIF-1α activity at the transcriptional level in intestinal epithelium. Using an HRE Gaussia Luciferase reporter system, we demonstrated that stimulation of cells with DMOG resulted in time-dependent increase in HRE activity, which was dependent upon functional HIF-1α transcriptional activation . OverexpFig. 4A). We found that cells overexpressing SUMO-1 displayed lower nuclear levels of accumulated HIF-1α, whereas inhibition of the SUMO pathway with TAK-981 enhanced nuclear stabilization of HIF-1α (A). By analyzing the mean fluorescent intensity of DMOG-induced HIF-1α nuclear levels, it became evident that SUMO-1 overexpression significantly reduced it, whereas TAK-981 upregulated HIF-1α nuclear accumulation (B). We further investigated the effect of SUMOylation on nuclear and cytoplasmic protein levels of HIF-1α using subcellular fractionation. We found that SUMO-1 overexpression reduced nuclear protein levels of HIF-1α (C and D). However, pretreatment of cells with TAK-981 also reduced nuclear levels of HIF-1α. We also observed that global protein SUMOylation with SUMO-1 mainly occurs in the nucleus. Our results suggest that SUMO-1 controls DMOG-induced HIF-1α nucleocytoplasmic shuttling, whereas inhibition of the SUMO pathway with TAK-981 seems to have a more complex effect on HIF-1α localization in intestinal epithelium.Having established that SUMO-1 isoform promoted the strongest decrease in HIF-1α protein expression and transcriptional activity when compared with SUMO-2/3 isoforms, we next investigated whether the changes in HIF-1α in the presence of overexpressed SUMO-1 and TAK-981 were due to the effects of SUMOylation on nuclear accumulation of HIF-1α. By using confocal immunofluorescence microscopy, we demonstrated accumulation of stabilized HIF-1α in the nuclei in Caco-2 cells treated with DMOG when compared with those treated with dimethyl sulfoxide A. We fouf HIF-1α A. By anamulation B. We furf HIF-1α , C and DFinally, our previous findings demonstrate that SUMOylation significantly influences HIF-1α activity and stability in intestinal epithelial cells. Therefore, we investigated whether HIF-1α could be directly SUMOylated. To do this, we coimmunoprecipitated HIF-1α from whole-cell lysates of Caco-2 cells treated with DMOG for 4 h. The expression of HIF-1α, SUMO-1, and SUMO-2/3 proteins was determined in coimmunoprecipitation (co-IP) and input samples by Western blot. We did not detect direct SUMOylation of HIF-1α in our system. Upon analyzing the coimmunoprecipitated HIF-1α samples, we observed the presence of SUMO-1 and SUMO-2/3 modified proteins, as demonstrated by bands around 100 kDa . HoweverConsidering the significance of SUMOylation in regulating biological functions in various pathophysiological conditions, a number of small-molecule inhibitors targeting SUMOylation have been suggested as potential therapeutic opportunities for multiple diseases , 22. To via activation of protective transcriptional responses  were cultured in Dulbecco’s modified Eagle's medium (Lonza) containing 4.5 g/l glucose. Media were supplemented with 10% fetal bovine serum (Gibco), 1% penicillin/streptomycin antibiotic solution , and 1% MEM nonessential amino acid solution (Gibco). Cells were maintained in an incubator at 37 °C, 21% ODimethyl sulfoxide was used as vehicle control. The SUMOylation enzymatic cascade inhibitor subasumstat (TAK-981) was obtained from MedChemExpress (HY-111789). The pan-hydroxylase inhibitor DMOG was purchased from Cayman Chemical . Reagent doses and treatment durations are listed in the figure legends.Human SUMO-1, SUMO-2, and SUMO-3 complementary DNA (cDNA) plasmids were purchased from OriGene . HRE-Gaussia Luciferase reporter construct was provided by Dr Alex Cheong (Aston University). Caco-2 cells were grown to 30% confluency. Before transfection, the media were replaced with penicillin/streptomycin-free Dulbecco’s modified Eagle's medium containing 10% fetal bovine serum and 1% nonessential amino acid. Cells were transfected with 500 ng of SUMO-1, SUMO-2, or SUMO-3 plasmid and/or 750 ng of HRE-GLuc plasmid for 48 h using Lipofectamine 2000  and Opti-MEM (Gibco). After transfection, the cells were treated as desired.2, 10 mM KCl, 200 mM sucrose, 1% NP-40 supplemented with 1× protease inhibitor cocktail  and 40 mM N-ethylmaleimide , incubated on ice for 10 min, and centrifuged at 12,000 rpm for 5 min at 4 °C. Supernatant containing cytoplasmic proteins was collected, and the pellet was resuspended in nuclear lysis buffer containing 20 mM Hepes (pH 8), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 25% glycerol supplemented with 1× PIC and 40 mM NEM. Samples were incubated for 30 min on ice with gentle agitation and then centrifuged at 14,000 rpm for 5 min at 4 °C. The collected supernatant contained nuclear fraction.To isolate cytoplasmic and nuclear fractions, cell-culture media were removed, and cells were washed with ice-cold PBS. Cells were lysed using cytoplasmic lysis buffer containing 10 mM Hepes (pH 8), 1 mM MgClWhole-cell protein lysates were generated by lysing cells in radioimmunoprecipitation assay buffer (Merck) containing 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, and 10 μM EDTA and supplemented with 1× PIC and 40 mM NEM. Proteins were denatured by boiling samples at 95 °C for 5 min in 1× SDS loading buffer containing 5% mercaptoethanol, resolved in 8% or 10% SDS-polyacrylamide gels by SDS-PAGE and then transferred onto 0.45 μm nitrocellulose membranes by a wet transfer system (Bio-Rad). The membranes were blocked in 5% milk for 1 h and incubated with primary antibodies overnight at 4 °C. Western blot analysis was performed using the following antibodies: HIF-1α (D1S7W) XP rabbit monoclonal antibody #36169, SUMO-1 #4930, SUMO-2/3 (18H8) rabbit monoclonal antibody #4971, anti-rabbit immunoglobulin G (IgG) horseradish peroxidase–linked #7074 , anti-β-actin A5441 , and DyLight 680 Goat Antimouse IgG #35519 . Enhanced chemiluminescence WesternBright horseradish peroxidase substrate (Advansta) was used for signal immunodetection by Fusion FX Spectra Imager (Vilber Lourmat). Fluorescent signal was detected by Odyssey CLx Imager (Li-Cor Biosciences). Quantification of protein band intensity was analyzed using Image Studio Lite software (Li-Cor Biosciences) and normalized to the density of the β-actin band.2, to remove nonspecific interactors. For Western blotting, 1× NuPAGE LDS sample buffer  was added to samples, and they were then heated at 95 °C for 10 min with 300 rpm rotation to elute antibody-bound proteins. Samples were then centrifuged at 2000 rpm for 1 min at 4 °C to precipitate the beads. Supernatant was collected, and 100 mM DTT was added to each sample and boiled at 95 °C for 5 min. Co-IP protein samples were resolved by SDS-PAGE as previously described.Whole-cell protein lysates were generated as previously described and normalized to 1 mg of protein. Normalized protein lysates were incubated with a primary antibody against HIF-1α  overnight at 4 °C with end-to-end rotation. Input samples were normalized to 10% of the co-IP samples by adding 5× sample buffer, beta-mercaptoethanol, and distilled water, following by boiling at 95 °C for 5 min. To isolate the antibody-bound proteins, 20 μl of Protein A Agarose Beads  were added to each sample and incubated for 4 h at 4 °C with end-to-end rotation. Agarose beads were washed twice with IP wash buffer, containing 100 mM Tris–HCl, 300 mM NaCl, and 10 mM MgCld-lysine . After desired treatment, cells were fixed using 4% paraformaldehyde for 10 min and washed with Dulbecco’s PBS without Ca2+ and Mg . Cells were blocked in 5% donkey serum, 0.3% Triton X-100  in DPBS for 1 h, and then probed for HIF-1α  made up in 0.1% Triton X-100, 0.1 g bovine serum albumin  in DPBS overnight at 4 °C. The following day, cells were incubated with secondary donkey anti-rabbit IgG Alexa Fluor 568 antibody  made up in 0.1% Triton X-100 and 0.1 g bovine serum albumin in DPBS for 2 h at 4°C. Cells were then counterstained with 1 μg/ml of 4′,6-diamidino-2-phenylindole nucleic acid stain  in DPBS for 10 min at room temperature. Coverslips were inverted and then mounted onto glass slides using Fluoromount Mounting Media . Slides were imaged using Zeiss LSM800 laser-scanning confocal microscope with 63× oil-immersion objective. HIF-1α nuclear fluorescent intensity was quantified in ImageJ software; National Institutes of Health and the Laboratory for Optical and Computational Instrumentation (University of Wisconsin). “Freehand” selection tool was used to record mean fluorescence intensity by highlighting the nuclear regions in 2D-rendered confocal images acquired from five different fields of view for each biological replicate (n = 3). Nuclei stained with 4′,6-diamidino-2-phenylindole were used as a nuclear localization control. A mean fluorescence intensity was then calculated for each technical and biological replicate.Caco-2 cells were seeded onto sterilized coverslips coated with 50 μg/ml of poly-HRE-Gaussia Luciferase reporter system was used to measure HIF-1α transcriptional activity. Caco-2 cells were transfected with 750 ng of HRE-Gluc plasmid for 48 h as previously described. Secreted bioluminescence in the media was collected after each treatment with DMOG. Bioluminescence was quantified in a 96-well plate using the Pierce Gaussia Luciferase Glow Assay Kit  according to the manufacturer’s protocol. Luminescence was determined using a SpectraMax M3 Microplate Reader set to 140 nm.RNA was isolated from cells using Trizol TRI Reagent . cDNA was generated from 1 mg of total RNA using Moloney murine leukemia virus reverse transcriptase. Target cDNAs were amplified and quantified using SYBR Green  on the QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Samples were incubated for 1 h and 40 min with the thermal cycle set up to 10 min at 95 °C, 15 s at 95 °C, and 60 s at 60 °C repeated for 40 cycles. Relative fold expression of genes of interest relative to β-actin RNA was analyzed using the δ–δ Ct method. Forward and reverse sequences of SYBR Green oligonucleotide primers were as follows:HIF-1α_Forward: 5′-TTCCAGTTACGTTCCTCGATCA-3′,HIF-1α_Reverse: 5′-TTTGAGGACTTGCGCTTTCA-3′,β-actin_Forward: 5′-CGACAGGATGCAGAAGGAGA-3′,β-actin_Reverse: 5′-CATCTGCTGGAAGGTGGACA-3′.p value < 0.05 was considered to be statistically significant. Results are presented as the mean ± SD.Statistical analyses were performed using the GraphPad Prism 7 software . One-way ANOVA followed by Holm–Sidak’s test for multiple comparisons was applied when comparing three or more groups. For all experiments, All data are contained within the article and available from the corresponding author upon reasonable request.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Inflammation plays a significant role in various cancers, including lung cancer, where the inflammatory cytokine IL-1β is often elevated in the tumor microenvironment. Patients with lung adenocarcinoma show higher levels of serum IL-1β compared to healthy individual. Moreover, IL-1β blockade reduces the incidence and mortality of lung cancer. Our prior studies revealed that alveolar type-II cells, the precursors for lung adenocarcinoma, display an induction in the expression of the enzyme tryptophan 2,3-dioxygenase (TDO2) during normal lung development. This induction of TDO2 coincides with an increase in IL-1β levels and is likely caused by IL-1β. Given that cancer cells can co-opt developmentally regulated pathways, we hypothesized that IL-1β may exert its pro-tumoral function by stimulating TDO2 and indoleamine 2, 3-dioxygenase-1 (IDO1), parallel enzymes involved in the conversion of tryptophan (Trp) into the immune-suppressive oncometabolite kynurenine (Kyn). Our goal was to determine whether IL-1β is a common upstream regulator of immune checkpoint regulators.To determine whether IL-1β regulates IDO1, TDO2, PD-L1, and PD-L2, we measured mRNA and protein levels in lung adenocarcinoma cells lines  grown in 2D or 3D and in immortalized normal lung epithelial cells (HBEC3-KT and HSAEC1-KT). To determine the importance of the NFκB pathway in mediating IL-1β -regulated cellular effects, we used siRNA to knockdown RelA/p65 in IL-1β treated cells. The levels of Trp and Kyn in the IL-1β-treated cells and media were measured by mass spectrometry.Upon IL-1β stimulation, lung adenocarcinoma cells exhibited significant increases in IDO1 mRNA and protein levels, a response that depended on the NFκB pathway. Interestingly, this induction was more pronounced in 3D spheroid cultures compared to monolayer cultures and was not observed in normal immortalized lung epithelial cells. Furthermore, the conversion of Trp to Kyn increased in cells exposed to IL-1β, aligning with the heightened IDO1 expression. Remarkably, IL-1β also upregulated the expression of programmed death ligand-1 (PD-L1) and PD-L2 in multiple cell lines, indicating that IL-1β triggers parallel immune-suppressive mechanisms in lung adenocarcinoma cells.Our studies demonstrate that lung adenocarcinoma cells, but not normal immortalized lung epithelial cells, respond to IL-1β signaling by inducing the expression of parallel immune checkpoint proteins that have the potential to promote immune evasion.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01348-1. Lung cancer is the leading cause of cancer-related deaths in both men and women . ApproxiT- cells possess the ability to detect and eliminate cancer cells, but cancer cells can develop strategies to evade this immune response by activating immune-evading pathways. This underscores the significance of employing immune checkpoint blockade as a treatment approach for non-small cell lung cancers (NSCLCs) lacking oncogenic driver mutations like EGFR, BRAF, or EML4-ALK, which can be specifically targeted therapeutically . HoweverTwo crucial pathways in cancer immune evasion are: the increased expression of programmed cell death ligand-1 (PD-L1), and the upregulation of the Trp metabolite, Kyn , 9. PD-LThe inflammatory cytokine interleukin-1β (IL-1β) is commonly elevated in tumors from various origins, such as lung, breast, prostate, colon, and head and neck cancers, and its elevated levels are associated with unfavorable patient outcomes –21. In NGiven that cytokines have the capacity to stimulate the expression of enzymes such as IDO1 and TDO2, and taking into account that lung adenocarcinoma is thought to originate from type-II alveolar epithelial cells that are capable of producing IL-1β , 27, we 4 cells were plated per well in RPMI supplemented with 5% FBS. Spheroids formed spontaneously between 24 to 48 h and were collected for analysis on day 5 after seeding. To isolate protein and RNA, the cells were plated on 1% agarose-coated 6- or 10-cm dishes and collected after the same duration.Lung cancer cell lines, NCI-H1792, NCI-H1838, NCI-H2228, NCI-H2347, HCC364 and HCC827 and normal cells: human bronchial epithelial cells (HBEC3-KT) and human small airway epithelial cells (HSAEC1-KT) both immortalized with cyclin-dependent kinase 4 (CDK4) and human telomerase reverse transcriptase (hTERT) were obtained from the Hamon Center Repository . HBEC3-KTotal RNA from cells was extracted using RNeasy Mini Kit . RNA was incubated with DNase  to remove any contaminating DNA, and 1 μg of RNA was reverse transcribed using the iScript Reverse Transcription Supermix . For quantitative analysis of mRNA, a BioRad CFX384 Real‐Time PCR Detection System was used with iTaq Universal SYBR Green Supermix  for the detection of PCR products. The cycling conditions were 95 °C for 30 s, followed by 39 cycles of 95 °C for 15 s, and 60 °C for 30 s. Each sample was analyzed in triplicate, and the mean of Cq value was calculated. The relative fold changes were calculated using the comparative Ct method (2–ΔΔCt). Primers used in RT‐qPCR are listed in Table S2, 1X protease inhibitor, 1X phosphatase inhibitor). Protein concentration was measured using the Pierce BCA Protein Assay Kit . For Western blot analysis, 20–30 µg protein was separated using a 10% sodium dodecyl sulfate polyacrylamide gel . Proteins were transferred from the gel to 0.45 μm pore size nitrocellulose membrane and total protein visualized using Ponceau S. The membrane was blocked with 2.5% (wt/vol) BSA  in 1X tris-buffered saline with Tween 20 . Primary and secondary antibodies were diluted in 2.5% BSA in 1X TBST. Protein blot bands were visualized using SuperSignal West Femto Maximum Sensitivity Substrate . Primary antibodies included IDO1 , RelA/p65 , PD-L1 , GAPDH  and the secondary antibody was Peroxidase-linked anti-Rabbit IgG .Western blot analysis was done as previously described . BrieflyCells were incubated in their regular growth media  with vehicle control (PBS) or 5 ng/mL IL-1β for the denoted duration. After incubation, 500 µL of the media were collected, centrifuged at 14,000 rpm for 5 min to pellet cells and other debris. The supernatants were snap frozen in liquid nitrogen and stored at -80ºC until further processing. The cells were washed twice with 0.9% NaCl (Baxter) to remove any residual media and then collected in 80:20 acetonitrile:water (Optima LCMS grade) and flash frozen in liquid nitrogen and stored at -80ºC until further processing. Polar metabolites were extracted from cells by 3 rounds of freeze–thaw in liquid nitrogen and 37ºC water bath to lyse cells. Aliquots (50 µL) of the medium were added to 950 µL 80:20 acetonitrile:water (Optima LCMS grade). Samples were centrifuged at 14,000 rpm for 10 min and supernatant was transferred to a fresh tube. Extracts were subjected to BCA assay (Pierce) and normalized to 7 µg in 100 µL of 80:20 acetonitrile before LCMS analysis. Metabolite analysis used a Vanquish UHPLC coupled to a Thermo Scientific QExactive HF-X hybrid quadrupole orbitrap high-resolution mass spectrometer (HRMS) as performed previously , 32. Rel+ cells, first the DAPI stained blue nuclei were counted to obtain total cell number and the red fluorescent cells were counted to obtain number of IDO1+ cells. Results are represented as % IDO1 + cell/total cells in an entire section of the spheroid.Immunofluorescence was performed as previously described . Spheroi4 HCC827 or H1792 cells were plated per well in 1% agarose-coated 96-well plates. Spheroids formed spontaneously in 24–48 h and CellTiter Glo assay was performed according to manufacturer’s instructions on day 4 after seeding. The spheroids and 100 µL of the media were transferred to a White opaque 96-well microplate  and 50 µL of the CellTiter Glo 3D reagent was added, covered in aluminum foil and mixed by shaking for 5 min. The plate was then incubated for another 25 min at room temperature and the luminescence was measured using Cytation5 Imaging Reader.Cell viability assay was performed as previously described –35. For http://www.cbioportal.org) [IDO1, CD274 (PD-L1) and PDCD1LG2 (PD-L2) genes. Co-expression data was plotted on GraphPad Prism.Data containing the mRNA expression of 503 patients with NSCLC adenocarcinoma  was retrieved from The Cancer Genome Atlas using the cBioPortal  . We analRELA (p65) siRNA  using DharmaFECT 1 transfection reagent . Non-targeting siRNA duplex was used as a negative control . Knockdown was performed using 40 nM siRNA treatment 6 h before IL-1β treatment. RT-qPCR was used to confirm mRNA knockdown.Cells were transfected with siGENOME human P-value ≤ 0.05 was considered statistically significant. Graphs are shown as the average of a minimum of 3 biological replicates ± standard deviation (SD) and plotted using Prism Graphpad. All data points are shown on the graphs.All experiments were repeated a minimum of 3 times. Statistical significance was determined using unpaired student t-test. TDO2 mRNA was elevated in A549, H1792, H2347, and H2228 cells, while IDO1 and IDO2 were low in most cell lines , and response to IL-1β stimulation, which was measured as fold change in CXCL8 induction, were detected in immortalized normal cells and lung adenocarcinoma cells , and found that Ifnγ could induce IDO1 mRNA   [RelA mRNA in H1792 and HCC827 cells and then treated with vehicle or IL-1β (5 ng/mL) for 48 h. RT-qPCR and Western blotting showed that RelA mRNA  . To deteRNA Fig. A was effRNA Fig. B was alsRNA Fig. C and proRNA Fig. D in H179RNA Fig. E. Our reIDO1 mRNA and protein induction was maximal at 6 h after treatment, and the levels decreased until 24 h and then stayed constant until 48 h  and PD-L2 (PDCD1LG2 gene) mRNA were among top significant genes that were positively correlated with IDO1 mRNA in lung adenocarcinoma tumors  and PDCD1LG2 (PD-L2) mRNA  [While studies have shown myeloid cell–derived IL-1β to be present in syngeneic models of lung tumors using LLC-1 cells , whether9648419) , 56. PreCancer cells have evolved an innate program to respond to different cytokine stimuli  by upregulating both IDO1 and PD-L1 to promote cancer cell survival. Our study has identified IL-1β as an upstream regulator of IDO1/PD-L1 axis of immune suppression in lung adenocarcinoma cells, which is partly through NFκB-p65 activity. These studies indicate that lung adenocarcinoma cells, but not normal lung epithelial cells, are primed to respond to IL-1 signaling by inducing the expression of cytoprotective immune checkpoint proteins, which could promote tumor cell survival through immunosuppression.Additional file 1: Table S1. Primer Sequences.Additional file 2: Supplemental Fig. 1. (A) Heatmap showing the FPKM values of the IL-1 receptors, IL-1 ligands, immune checkpoint genes  Heatmap showing the FPKM values of TDO2, IDO1, IDO2, CXCL8 and IL-1α mRNA of the cell lines used in this study from the cancer cell line encyclopeAQ11dia (CCLE) database. Additional file 3: Supplemental Fig. 2. (A-B) HCC827 cells transfected with 40 nM of non-targeting or RELA/p65 siRNA for 24 h followed by treatment with vehicle (ctrl) or 5 ng/mL IL-1β for 48 h followed by (A) RT-qPCR for CD274 and PDCD1LG2 mRNA and (B) western blotting for PD-L1 protein. (C) Western blot of PD-L1 in HCC827 cells treated with 5 ng/ml IL-1β ± BAY11-7085 (p65/NFκB inhibitor) for 48 h. (D) HCC827 cells transfected with 40 nM of non-targeting or CEBPβ siRNA for 24 h followed by treatment with vehicle (ctrl) or 5 ng/mL IL-1β for 48 h followed by western blotting for PD-L1. (D) Western blot of p-STAT1 and p-STAT3 in HCC827 cells stimulated with IL-1β for 6 h. GAPDH is loading control.Additional file 4: Supplemental Fig. 3. (A – C) RT-qPCR of IDO1 (A), PD-L1 (B) and PD-L2 (C) mRNA in H1792 and HCC827 cells treated with 1, 10 or 100 ng/mL mouse-derived (mIL-1β) or 1 ng/mL human-derived IL-1β (hIL-1β) for 48 h. Note that P-value is represented by comparing all treatments to 1 ng/mL hIL-1β. (D) Western blot of IDO1 and PD-L1 protein in HCC827 cells treated with 5 ng/mL IL-1β. GAPDH is loading control. Error bars represent ± SD of 3 biological replicates: *P ≤ 0.05, ** ≤ 0.005, *** ≤ 0.0005. mRNA levels were normalized to 18s mRNA and represented as fold change over control treatment.


Scientific Reports 10.1038/s41598-023-29848-7, published online 13 February 2023In the original version of this Article Zsuzsanna Kövi was incorrectly affiliated with ‘Department of Personality and Health Psychology, Institute of Psychology, Károli Gáspár University of the Reformed Church, Bécsi Street 324, Budapest 1037, Hungary.’The correct affiliation is listed below:Centre of Specialist Postgraduate Programmes in Psychology, Institute of Psychology, Károli Gáspár University of the Reformed Church, Bécsi street 324, Budapest, H-1037, Hungary.The original Article has been corrected.


In vitro, we confirm that adhesion to integrin ligands and CCL25-dependent and independent migration of intraepithelial lymphocytes are Myo1f-dependent. Mechanistically, Myo1f deficiency prevents correct chemokine receptor and integrin polarization, leading to reduced tyrosine phosphorylation which could impact in signal transduction. Overall, we demonstrate that Myo1f has an essential role in the adhesion and migration in γδT intraepithelial lymphocytes.γδT intraepithelial lymphocyte represents up to 60% of the small intestine intraepithelial compartment. They are highly migrating cells and constantly interact with the epithelial cell layer and lamina propria cells. This migratory phenotype is related to the homeostasis of the small intestine, the control of bacterial and parasitic infections, and the epithelial shedding induced by LPS. Here, we demonstrate that Myo1f participates in the adhesion and migration of intraepithelial lymphocytes. Using long-tailed class I myosins KO mice, we identified the requirement of Myo1f for their migration to the small intestine intraepithelial compartment. The absence of Myo1f affects intraepithelial lymphocytes’ homing due to reduced CCR9 and α4β7 surface expression.  Salmonella and Toxoplasma gondii  domains, respectively . These mMyo1e participates in neutrophils and B cells integrin mediated-adhesion and migration , 24 as win vitro. Mechanistically, Myo1f was relevant for actin-mediated membrane protrusion formation during adhesion and migration. Its absence impacts CCR9 and integrin polarization and leads to tyrosine phosphorylation defects, which could impact in signal transduction.Here, we show that intraepithelial lymphocytes express Myo1e and Myo1f. Furthermore, the absence of Myo1e and Myo1f impacts the homing of intraepithelial γδT lymphocytes, but Myo1f shows a more significant impact. This deficiency was due to a reduction in the surface expression of intestinal homing receptors CCR9 and α4β7 integrin, having consequences on the 2D migration of γδT lymphocytes https://www.immgen.org/). Heat map reveals that among class I myosins, Myo1e and Myo1f are expressed mainly by intraepithelial γδT lymphocytes, both Vγ7+ and Vγ7-. Myo1f was also expressed in activated spleen Vγ4+ and Vγ4- γδT lymphocytes but did not in resting spleen γδT lymphocytes. Mature thymic Vγ5 γδT lymphocytes also express Myo1f. Whereas the rest of class I myosins remain less represented in γδT lymphocytes . As a first approach, mRNA class I myosins expression was analyzed in intraepithelial, thymus, and spleen γδT lymphocytes with data from The Immunological Genome Project  algorithm shows that Myo1e did not considerably affect the proportions of IEL. However, Myo1f deficiency negatively affected CD8αα γδT, CD8αβ, and CD8αα αβT IEL while positively affecting CD4 αβT cells and ILCs. In contrast, the absence of both myosins affected CD8αβ αβT negatively, and CD8αα ILCs positively. Unexpectedly, CD8αα γδT IEL proportion was positively affected in the absence of both myosins . However, the possibility that the difference between the antibodies employed may contributes to the differences observed between Myo1e and Myo1f expression can not be ruled out. Consistently to the data available in the Immunological Genome Project, neither thymic nor spleen γδT cells express Myo1e, but ~25% of cells express Myo1f. Additionally, activated γδT cells increased Myo1f expression compared to their naïve counterpart. These suggest that long-tailed class I myosins could be expressed after activation or differentiation. It has been previously demonstrated that monocytes acquire Myo1f expression upon macrophage differentiation . However, this needs to be experimentally confirmed. Despite this, dKO mice have no additive effects in the γδT cell proportion. It has been demonstrated that dKO macrophages showed increased actin waves and Arp2/3 recruitment during Fc-mediated phagocytosis , fibronectin, and collagen. Mechanically, Myo1f-/-deficiency results in fewer filopodia formation. This filopodia formation depends mainly on the actin cytoskeleton polymerization dependent on the activity of Cdc42 . In agreIntraepithelial lymphocyte homing depends on CCR9, and its deficiency reduces γδT proportion , 13. SimTypically, class I myosin localize in the plasma membrane and colocalizes with cortical actin. This localization has been related to membrane tension regulatory properties. In this regard, it has been reported that Myo1g controls plasma membrane tension in B cells . AdditioFinally, we demonstrated that altered CCR9 and α4β7 polarization reduce tyrosine phosphorylation compared to the WT counterpart. Together, this work provides evidence about the role of Myo1f in adhesion and migration in γδT intraepithelial lymphocytes, mediating the lipid raft dependent-CCR9 and α4β7 polarization that could affect cell signaling.tm1.1FlvMyo1e/J), Myo1f-/- (B6.129S6-tm1.1FlvMyo1f/J) and Myo1e-/-+Myo1f-/- (dKO) (generated in the CINVESTAV by crossing Myo1e-/- with Myo1f-/- during ten generations to obtain double homozygous knock out) mice were bred and maintained at CINVESTAV-UPEAL   facilities. Additionally, Myo1f-/- mice were maintained in the Johns Hopkins Bloomberg School of Public Health  facilities for some experiments. WT mice were purchased from Jackson Laboratories. Myo1e-/- and Myo1f-/- mice were kindly donated by Dr. Richard Flavell from Yale School of Medicine, New Haven, CT. Mice genotypification was carried out employing the primers described previously  C57BL/6 wild type, Myo1e-/- (B6.129S6(Cg)-eviously , 51.in vivo experiment) guide.All experiments were approved by the Animal Care and Use Committee at Cinvestav and carried out following ARRIVE , Myo1b (10354432), Myo1c (10378697), Myo1d (10389022), Myo1e (10586781), Myo1f (10443980), Myo1g (10384154) and Myo1h (10524515) γδT lymphocytes RMA (Robust Multiarray Averaging)-normalized mRNA expression value was manually obtained from microarray data using the Gene Skyline browser and graphed as a heat map using Prism Software  version 8 in order to show the relative expression.Class I myosin mRNA expression was obtained from The Immunological Genome Project in the portal +2 and Mg+2  for 30 min, twice. The cell suspension was filtered using a 70 μM cell strainer , transferred to 50 mL tubes, and centrifuged at 1500 rpm for 10 min. Pellets were washed twice with HBSS and then resuspended in discontinuous 40/70% Percoll®  gradient and centrifuged at 2500 rpm for 30 min   in HBSS  without Car 30 min . The intIEL were incubated with anti-CD16/32  antibody  and Live/Dead Aqua (dilution 1:1000)  for 30 min on ice. Then, IEL were washed and incubated with anti-CD45  , -CD3ε  (eBioscience), -TCRγδ  , -CD8α  (BD Biosciences), -CD8β  (Biolegend) and CD4  (BD Biosciences) for total IEL analysis. With anti-CD3ε (eBioscience), -TCRγδ (BD Bioscience), -TCRVγ1.1/1.2  (Biolegend), -TCRVγ2  (BD Biosciences) for Vγ specific γδT IEL analysis. Anti-CCR9  (eBioscience), -α4β7  , -αE  (eBioscience), -αLβ2  (Biolegend), -αM  (Biolegend), -β1  (Biolegend), were also employed. For intracellular staining, cells were fixed with 1% paraformaldehyde (PFA) for 5 min. Then the cells were permeabilized with saponin 0.1% for 15 min, next incubated with 10% goat serum  at 37°C, and further incubated with anti -Myo1e , -Myo1f , -CCR9  and α4β7  antibodies. For indirect staining, secondary anti-rabbit FITC , anti-rat AF647 , or anti-rabbit Pacific Blue (dilution 1:1000 Jackson ImmunoResearch Laboratories) were employed. Finally, cells were resuspended in PBS and analyzed in Cytoflex LX , LSR Fortessa , BD Influx  or Attune Next  cytometers. Data were analyzed using FlowJo Software (BD Biosciences) version 10.6.IEL were incubated with anti CD16/32 for 30 min at 4 °C. Then, cells were incubated with anti-TCRαβ biotin-coupled (H57-597) (Biolegend) for 30 min at 4°C. Next, cells were incubated with anti-biotin beads  for 15 min at 4°C. Finally, cells were washed, and γδT IEL were negatively selected by an LD MACS column (Miltenyi Biotec). γδT IEL were incubated with Live/Dead Scarlet (Thermo Fisher Scientific) and anti-TCRγδ-PE (Biolegend) antibody for 30 min at 4°C. Cell viability and purity were confirmed by flow cytometry. Cell viability was >95%, and purity was 90%. IEL were cultured as described previously . PurifieProteins from total extracts (30 μg) were resolved in 10% SDS-PAGE gels. Proteins were transferred to a nitrocellulose membrane , and protein transfer was confirmed with Ponceau red. First, membranes were blocked with 5% milk in 0.05% TBS-Tween 20 buffer for 1 h at room temperature. Next, anti-Myo1e  and -Myo1f  antibodies were incubated in 2% milk in TBS-T overnight at 4°C on independent membranes; next, membranes were washed and incubated with anti-rabbit HRP antibody  for 1 h at room temperature. The same membranes were then incubated with anti-β actin , in 2% milk in TBS-T overnight at 4°C; next, membranes were washed and incubated with anti-mouse HRP antibody  for 1 h at room temperature. Finally, membranes were washed and revealed with a super signal western blot system  in C-digit equipment . Densitometric analysis was performed using Image J software  version 1.52.Duodenum sections were obtained from WT and Myo1f-/- mice. First, tissue fractions were pulled in optimal cutting temperature media  and frozen in liquid nitrogen. Next, tissue sections (5 μm thick) were placed in poly-L-lysine coated slides, fixed with absolute acetone for 20 min at -20°C, and then blocked with 5% BSA for 1 h at room temperature. Next, tissues were incubated with purified anti-TCRγδ  and anti-E-cadherin  antibodies for 1 h a room temperature. Afterward, tissues were stained with anti-hamster-AF555  and anti-rat-FITC  secondary antibodies, respectively. Finally, tissues were incubated with DAPI  for 10 min and mounted with 10% glycerol. Tissues were analyzed immediately in an Olympus X microscope . γδT cell count was performed with Image J software (NIH), version 1.52.5 γδT IELs or PMA activated-γδT IEL were seeded and incubated at 37°C for 1 h. After that, non-adhered cells were removed, and plates were washed once with PBS. Then cells were fixed with PFA 4% for 10 min at room temperature. Next, plates were washed and stained with crystal violet for 30 min; plates were washed 5 times and then incubated with 10% SDS for 30 minutes. Finally, plates were read in a 680-microplate reader  equipment.Plates (96-wells) were pre-coated with murine recombinant MadCAM-1 , fibronectin, collagen, or 5% BSA overnight at 4°C. Then, plates were blocked with 5% BSA in PBS for 1 h a 37°C. Next, 2 x 105) were seeded in 1.5 mm coverslips previously coated with 5 μg/mL of Collagen I . Cells were incubated for 1 h at 37°C with 5% CO2. Next, cells were fixed with 4% formaldehyde for 30 minutes at room temperature and then permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 15 minutes. After that, cells were stained with phalloidin-CF555®  for 1 h at 37°C and mounted with ProlongGold with DAPI (Thermo Fisher Scientific). Cells were observed in a GE DeltaVision OMX-SR microscope . Images were analyzed using Fiji Software  version 2.3.0.24-hours cultured γδT IEL (1 x 105) were seeded in an μ-Slide Chemotaxis® chamber coated with collagen IV  for 30 min at 37°C. Then, non-adhered cells were removed, and the chamber was filled with a complete RPMI-1640 medium. Murine recombinant CCL25  (100 ng/ml) was added in one slide plug. Slides were immediately placed on the stage of a DeltaVision Elite microscope . Time-lapse videos were taken every 30 sec for 30 min. Distance and velocity analysis was made with Fiji software (Fiji Software (NIH), version 2.3.0, with the TrackMate plugin  (Biolegend) or anti α4β7 antibody for 20 min at 4°C. Cells were washed with PBS and incubated with the anti-rat-AF594  antibody for 30 min at 37°C to induce cross-linking. A parallel cold incubation was employed as negative control of capping. Cells were washed and then fixed with 4% PFA. For intracellular staining, cells were permeabilized with 0.1% Triton X-100 for 15 min, then incubated with 5% BSA for 1 h, and then incubated with anti- Myo1e , anti- Myo1f  (Santa Cruz Biotechnology) and anti-phospho-tyrosine  (Biolegend) antibodies. Then, cells were incubated with anti-mouse FITC  and anti-rabbit AF647 . After staining, cells were mounted in poly-L-lysine (Sigma-Aldrich) pre-coated coverslips with ProlongGold with DAPI. In some experiments, cells were pre-incubated with 5 mM MβCD (Sigma-Aldrich) for 15 min at 37°C. CD3ε (clone 145-2C11) stimulation was employed as positive phosphotyrosine control. For the phosphotyrosine detection, cells were treated with 1 mM vanadate . To define capping, we employed an index between the cap length and the cell circumference as previously reported . Cells wResults shown are mean with +/- SD. The normality of the data was analyzed by Anderson-Darling, D’Agoston & Pearson, Shapiro-Wilk or Kolmogorov test. The specific statistical test, p values and the number of samples or cells (n) used are mentioned in each figure legend.The original contributions presented in the study are included in the article/Conceptualization: IM-V, LS-A, PTR; Methodology: IM-V, MS-B, CM-R, FH-C; Validation: IM-V, LS-A, PTR; Investigation: IM-V, MS-B, CM-R, FH-C; Resources: LS-A, PTR; Writing : IM-V; Review and Editing: IM-V, LS-A, PTR; Visualization: IM-V, LS-A, PTR; Supervision: LS-A, PTR; Funding: LS-A, PTR. All authors contributed to the article and approved the submitted version.


Rb2CoCl2(C2O4) consists of chains of Co2+ cations connected via oxalate bridging ligands. In K2Li2Cu(C2O4)3·2H2O, the metal cations are interconnected by oxalate ligands forming a novel three-periodic network.Rb 2[Co(C2O4)2(H2O)2]·2H2O, (I), catena-poly[dirubidium [[dichloridocobalt(II)]-μ-oxalato]] or dirubidium cobalt(II) dichloride oxalate, {Rb2[CoCl2(C2O4)]}n, (II), and poly[dipotassium [tri-μ-oxalato-copper(II)dilithium] dihydrate] or dipotassium dilithium copper(II) tris dihydrate, {K2[Li2Cu(C2O4)3]·2H2O}n, (III), have been grown under hydrothermal conditions and their crystal structures determined using single-crystal X-ray diffraction. The structure of (I) exhibits isolated octahedral [Co(C2O4)2(H2O)2] units, whereas (II) consists of trans chains of Co2+ ions bridged by bidentate oxalato ligands and (III) displays a novel tri-periodic network of Li+ and Cu2+ ions linked by oxalato bridging ligands.Single crystals of three novel transition-metal oxalates, dirubidium diaquadioxalatocobalt(II) dihydrate or dirubidium cobalt(II) bis tetrahydrate, Rb Their magnetic properties are in part due to the oxalato ligand, which is known to facilitate magnetic exchange between transition-metal cations, and the compounds are known to exhibit both ferro- and antiferromagnetic interactions 2·4H2O (I) consists of isolated [Co(C2O4)2(H2O)2] octahedra. The Co2+ cation lies on the 2c Wyckoff position with a site symmetry of trans disposition of the bidentate oxalato and aqua ligands 2(H2O)2] octahedra are interlinked by hydrogen bonding of both types of water molecules, as shown in Fig. 2trans coordinating water molecules  form hydrogen bonds with the oxalate ligands of the neighbouring [Co(C2O4)2(H2O)2] octahedra, whilst the second type of water molecule  forms hydrogen bonds (in part bifurcated) with the oxalate ligands of two separate [Co(C2O4)2(H2O)2] octahedra. Numerical data for the hydrogen-bonding interactions are given in Table 1Rbds Fig. 1. The ave2CoCl2(C2O4) (II) consists of octahedrally coordinated Co2+ cations. They are linked by bis-bidentate oxalate ligands to form chains extending parallel to the a axis, as shown in Fig. 3trans to one another whilst the Cl− anions cap each side of the octahedron. Co—O bond lengths are 2.0616 (17) Å and longer for the Co—Cl bond at 2.4863 (9) Å, with a calculated bond-valence sum of 2.03 valence units for Co. The Rb+ cation has a coordination number of eight and lies between the layers formed by the Co2+ chains , Co2+ , Cl− , O  and C . The presence of the oxalate-bridged Co2+ chain could allow for magnetic exchange 3·2H2O (III) are shown in Fig. 5d9 Cu2+ cations display classic Jahn–Teller distortion with elongation of the axial Cu—O bonds. The equatorial Cu—O bond lengths are 1.938 (3) (O2) and 1.942 (3) (O1) Å whilst the axial bonds are significantly longer at 2.473 (4) Å (O6). The Cu2+ ion lies on a special position with Wyckoff position and site symmetry of 6b and 2+ coordination environment consists of four oxalate ligands, two of which act as bidentate bridging ligands and two of which are axially oriented and bind to four metal cations with a tricoordinate oxygen atom. The Li+ cation is tetrahedrally coordinated by three oxalate molecules, one of which is bidentate whilst the other two are monodentate. The Cu2+ and Li+-centred polyhedra are interconnected into a tri-periodic network, as shown in Fig. 6+ cation lies within this network and consists of eight oxygen atoms from the oxalate ligands and two water molecules. These water molecules exhibit disorder of the O7 atom, which is split into two positions. The interatomic distances between the water molecules is ∼3.7 Å, which is too far apart to facilitate hydrogen bonding.The Cu3.et al., 2016et al., 2013+ rather Rb+ cations, but is not isostructural. For (II), there are several compounds containing transition-metal oxalate chains with the same binding environment, although with quite different cations involved. For example BEJHOQ {catena-dithiolodithiin-1-ium) bistetrachlorodiiron(III) dichloromethane solvate]} and EYALIB {catena-diselenolodithiin-1-ium) bistetrachlorodiiron(III)]; Zhang, 2016trans monodentate oxalate ligands, containing a tricoordinating oxygen atom. The search revealed evidence of only two similar compounds, viz. ADAJUL [octaammonium hexakisbisdiaquatetracopper(II) tetrahydrate] and ASOXOV {bisbisdiaquabisdicopper(II) tetrahydrate; Kadir et al., 2006et al., 2004Database surveys were carried out using the Cambridge Structural Database  and (II) were synthesized as by-products from the reaction of rubidium carbonate, sodium carbonate, cobalt chloride hexahydrate and oxalic acid dihydrate in molar ratios of 2:2:1:1.5 and 1:1.5:1:1.5 at 433 and 463 K, respectively. Compound (III) was synthesized by the reaction of potassium carbonate, lithium carbonate, copper chloride dihydrate and oxalic acid dihydrate (1:3:1:3) at 463 K. Single crystals were isolated from a mixture of products for further analysis. The resulting crystals were filtered and dried overnight at 323 K prior to analysis by X-ray diffraction.The samples were synthesized 5.Uijs were refined independently.Crystal data and refinement details of the three compounds are summarized in Table 210.1107/S2056989023001822/wm5668sup1.cifCrystal structure: contains datablock(s) I, II, III, global. DOI: 10.1107/S2056989023001822/wm5668Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989023001822/wm5668IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989023001822/wm5668IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 2245039, 2245038, 2245037CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Trimethylphosphine oxide and water molecules form 16-membered hydrogen-bonded rings interconnected into layers. 3PO·2H2O, crystallizes in the orthorhombic space group Pbca with eight formula units per unit cell. The extended structure displays O—H⋯O hydrogen bonding, with Me3PO molecules as acceptors and water molecules acting as donors and acceptors of hydrogen bonds, forming hydrogen-bonded layers, which propagate in the ac plane.The title hydrate, Me R3P=O , are good hydrogen-bond acceptors and have been employed for co-crystallization and stabilization of hydrogen-bond donor species such as hydrogen peroxide 3C6H2]3PO·H2O 3C6H2]3PO·2H2O  and diethyl ether-d10 (0.3 ml) in an NMR tube that was capped and cooled to −20 °C in an ethanol cooling bath. The Dewar flask containing the bath and sample was sealed and placed in a freezer at −80 °C. Crystals of Me3PO·2H2O grew within 3 days. The single crystals were examined, selected, and transferred to the diffractometer employing a previously described low-temperature crystal-mounting procedure  was dissolved in a mixture of acetone-et al., 2010Crystal data, data collection, and structure refinement details are summarized in Table 210.1107/S2414314623003140/hb4428sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314623003140/hb4428Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623003140/hb4428Isup3.cmlSupporting information file. DOI: 2254008CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


One of the Zn atoms displays a tetrahedral coordination environment, while the other Zn atom adopts a five-coordinate geometry intermediate between square pyramidal and trigonal bipyramidal. Additionally, O—H⋯O hydrogen-bonding patterns involving the water molecules of crystallization serve as a structure-stabilizing element by aggregating the double-strand motifs.In the title compound, {[Zn Previously our group had isolated a zinc tBuip coordination polymer featuring bis(4-pyridylmethyl)piperazine coligands; this phase manifested a twofold interpenetrated pcu 3-D network structure 2} dimeric clusters with a Zn⋯Zn distance of 3.969 (1) Å. The full span of the tBuip ligands connect the dimeric clusters into [Zn2(tbuip)2]n coordination polymer strands oriented along the a axis 2]n strand motifs are connected into [Zn2(tbuip)2(dpu)]n coordination polymer double strands by tethering dpu ligands that span a Zn⋯Zn distance of 14.394 (3) Å  short water-molecule chains and C(4) cyclic water molecule tetramers 2.6H2O , 5-tert-butylisophthalic acid (tBuipH2) , 1,3-di(pyridin-4-yl)urea (dpu) , and 0.75 ml of a 1.0 M NaOH solution were placed into 10 ml of distilled H2O in a Teflon-lined acid digestion bomb. The bomb was sealed and heated in an oven at 393 K for 48 h, and then cooled slowly to 273 K. Colorless crystals of the title complex were obtained in 55% yield.Zn I, 1R. DOI: 10.1107/S2414314623006594/im4021Isup2.hklStructure factors: contains datablock(s) I. DOI: 1959995CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Cytokines of the common γ chain (γc) family are critical for the development, differentiation, and survival of T lineage cells. Cytokines play key roles in immunodeficiencies, autoimmune diseases, allergies, and cancer. Although γc is considered an assistant receptor to transmit cytokine signals and is an indispensable receptor in the immune system, its regulatory mechanism is not yet well understood.This study focused on the molecular mechanisms that γc expression in T cells is regulated under T cell receptor (TCR) stimulation.The γc expression in TCR-stimulated T cells was determined by flow cytometry, western blot and quantitative RT-PCR. The regulatory mechanism of γc expression in activated T cells was examined by promoter-luciferase assay and chromatin immunoprecipitation assays. NFAT1 and NFκB deficient cells generated using CRISPR-Cas9 and specific inhibitors were used to examine their role in regulation of γc expression. Specific binding motif was confirmed by γc promotor mutant cells generated using CRISPR-Cas9. IL-7TgγcTg mice were used to examine regulatory role of γc in cytokine signaling.We found that activated T cells significantly upregulated γc expression, wherein NFAT1 and NFκB were key in transcriptional upregulation via T cell receptor stimulation. Also, we identified the functional binding site of the γc promoter and the synergistic effect of NFAT1 and NFκB in the regulation of γc expression. Increased γc expression inhibited IL-7 signaling and rescued lymphoproliferative disorder in an IL-7Tg animal model, providing novel insights into T cell homeostasis.Our results indicate functional cooperation between NFAT1 and NFκB in upregulating γc expression in activated T cells. As γc expression also regulates γc cytokine responsiveness, our study suggests that γc expression should be considered as one of the regulators in γc cytokine signaling and the development of T cell immunotherapies.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01326-7. This in2+-dependent nuclear factor of activated T cells (NFAT) and the PKCθ-dependent nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) are transcription factors (TFs) that mediate immune response in T lymphocytes [The Caphocytes –9. NFκB phocytes . Upon T phocytes . Consequphocytes , 12. Thephocytes  in T celphocytes . They haphocytes , 15. In phocytes . In the phocytes , 9.Although it is known that γc expression is regulated in thymocyte developmental stages –18 and tnull (γcKO) were obtained from The Jackson Laboratory , and BALB/c mice from Charles River Laboratories . The generation of γc transgenic (Tg) mice has been previously described [C57BL/6 (B6) mice and IL-2Rγescribed . We then+ T cells, total LN cells were incubated with α-CD4 (GK1.5) for 30 min on ice and negatively selected using the BioMag goat α-mouse IgG (Qiagen) and BioMag goat α-rabbit IgG beads (Qiagen) for 40 min on ice. The purity of the isolated total LN T and LN CD8+ T cells was confirmed using FACSCanto II  and Attune NxT Flow cytometry .LNs were collected from mice and minced to isolate LN cells. Total LN cells were incubated with BioMag goat α-mouse IgG beads  for 40 min on ice to isolate LN T cells via negative selection . To isol+ T cells, and EL4 cells were cultured in RPMI 1640 medium , whereas HEK293 T cells were cultured in Dulbecco’s Modified Eagle Medium (Welgene) containing 10% fetal bovine serum , L-glutamine, 100,000 U/ml penicillin plus 100 mg/ml streptomycin , non-essential amino acids , sodium pyruvate (Sigma-Aldrich), and β-mercaptoethanol (Gibco). LN T cells and LN CD8+ T cells were stimulated with plates bound with α-TCRβ /α-CD28  antibodies . LN CD8+ T cells were stimulated with recombinant human IL-2 (10 ng/ml), IL-7 (10 ng/ml), IL-15 (100 ng/ml), or interferon (IFN)-γ (10 ng/ml)  or with plates bound with α-TCR in the presence of α-IL-2 antibody 10 µg/ml (PeproTech Inc.) to block IL-2 signaling. EL4 cells were stimulated with phorbol 12-myristate 13-acetate (12.5 ng/ml)  and ionomycin (1 µM) . To identify the mechanisms involved in regulating γc expression, LN CD8+ T cells stimulated with plates bound with α-TCR/α-CD28 in the presence of PD98059 (10 µM) or wortmannin (1 µM), NFAT1 inhibitor , and/or NFκB inhibitor  . EL4 cells were pretreated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole , cycloheximide , MG132 (500 nM), cyclosporine A , INCA6, and/or Bay11-7802 (Bay11) for 2 hr and then stimulated with PMA/Iono for 16 hr.LN T, LN CD8To induce acute polyclonal T cell stimulation in vivo, BALB/c mice were injected intraperitoneally with 10 μg anti-CD3 antibodies  or the appropriate isotype control IgG. Anti-CD3 and isotype control antibodies were purchased from BioXCell . After 16–18 hr, LNs were harvested from overnight α-CD3 antibody- or isotype control IgG-injected mice, and T cell surface marker expression was assessed.Escherichia coli (New England Biolabs) cells were incubated at 37 °C on an LB plate containing ampicillin. An ExprepTM Plasmid SV kit  was used to extract the gRNA plasmids for transfection. For transfection, 2 × 106 EL4 murine lymphoma cells provided by Dr. Se-Ho Park  were cultured in RPMI 1640 medium supplemented with 5% antibiotics and 10% fetal bovine serum in 24-well plates. One day after seeding, Cas9 expression plasmids (750 ng) and gRNA plasmids (250 ng) were transfected using Lipofectamine™ 2000 . After 3 days, the genomic DNA was purified using Proteinase K extraction buffer . The insertion and deletion (indel) ratios and patterns of each genomic DNA sample were analyzed using next-generation sequencing (NGS) on an Illumina Mini-Seq platform  and Cas-Analyzer (www.rgenome.net) [Guide RNA (gRNA) vectors targeting NFAT1, p65, and cRel were produced, starting with an empty expression vector. The vector was cleaved using BsaI New England  and ligated to each targeted annealed oligonucleotide. The ligation products were transformed into DH5α chemically competent cells , and transformed DH5α ome.net) . To obtaOrgans were processed into single-cell suspensions, stained, and analyzed using FACSAria or FACSCanto II (Becton Dickinson) and Attune NxT Flow cytometry (Thermo Fisher Scientific). Dead cells were excluded by forward light-scatter gating and propidium iodide staining. Data were analyzed using FlowJo version 10.3 (TreeStar). Antibodies with the indicated specificities were used for staining: CD69 (H1.2F3), γc (4G3), and human CD3 (Leu4) ; IL-7Rα (A7R34), IL-2Rα (CD25), Foxp3 (FJK-16s), and CD4 (GK1.5) (eBioscience); and TCRβ (H57-597), CD8α (53-6-7), and phosphorylated STAT5 (Tyr694) . An anti-mouse CD16/32 antibody  was used to block the Fc receptor. pSTAT5 expression was determined in cytokine-stimulated cells (30 min) by methanol/acetone fixation and intracellular staining , 23. IntRpl13 in the same sample.Total RNA was extracted from cultured cells using Ribospin (GeneAll). RNA was reverse-transcribed to cDNA via oligo(dT) priming using a QuantiTect Reverse transcription kit (GeneAll). RT-qPCR was conducted using a LightCycler 96 Real-Time PCR System  and a SYBR Green detection system . The following primer sequences were used: mγc , sγc , IL-7Rα , and RPL13 . The thermal cycling conditions were as follows: 40 cycles of denaturation at 95 °C for 10 s, annealing at 59 °C for 30 s, and extension at 72 °C for 30 s. Gene expression values were normalized to those of EL4 cells were stimulated with PMA 12.5 ng/ml) /Iono (1 µM) for 16hr and culture supernatant was collected. The level of sγc was detected using a sandwich enzyme-linked immunosorbent assay with γc-specific polyclonal antibodies  as capture antibodies and biotin-conjugated γc-specific monoclonal antibodies  as detection antibodies, as previously described  ng/ml /I. RecombiCell lysates were obtained from stimulated LN T, EL4, and EL4 KO, EL4 mutant cell lines, resolved using sodium dodecyl sulphate–polyacrylamide gel electrophoresis  under reducing conditions, and transferred to polyvinylidene difluoride membranes . Blots were incubated with biotinylated anti-mouse IL-2Rγ , p65 (F-6), cRel (C), NFATc2 (4G6-G5) , and GAPDH, phospho p65(ser536), phospho AKT (ser473), phospho MEK1/2 (Ser217/221)  antibodies, followed by horseradish peroxidase (HRP)-conjugated streptavidin  or HRP-conjugated anti-rabbit or anti-mouse IgG  and HRP-conjugated anti-mouse β-actin antibodies . The membranes were then incubated with enhanced chemiluminescence to determine the reagents  and exposed using the LAS-3000 Imaging system  and Imager 680 (Amersham™). The summary graph indicates the densitometric analysis of western blots (relative intensity) with an arbitrary number of the intensity ratio of γc to β-actin or GAPDH using ImageJ software.The deletion versions of γc promoter regions were produced by serially eliminating the predicted NFAT1 binding sites and were inserted into the pGL4 reporter vector using XhoI and HindIII restriction enzymes (New England Biolabs). To generate NFAT1-binding site-deleted constructs, the different forward primers and equal reverse primers in the first exon of the γc gene were used. The primer sequences used to obtain different combinations of the construct were as follows: forward, 1397 bp, 5'-CACTAACACTCTCTCCCCCAGA-3'; 910 bp, 5'-CCAGTTTGTGGGTTACGGGA-3'; 743 bp, 5'-TGAGGTTTCAAGTCGGGCAG-3'; 513 bp, 5'-GTACCCAC ATGAATCATGTCAG-3'; 278 bp, 5'-TCTCCCTGGGGACTTAGCTT-3'; 194 bp, 5'-CCGGAAGCTACGACAAAAGG-3'; 167 bp, 5'-GGAGAGTGGTTCAGGGTTCT-3'; and reverse, 5'-TTCGCACTGGACATGAGGAC-3'. The sequences of all the constructs were confirmed using sequencing. To generate NFκB-binding site-deleted constructs, different forward primers and equal reverse primers in the first exon of the γc gene were used. The primer sequences used to obtain different combinations of the construct were as follows: forward, 1397 bp, 5'- CACTAACACTCTCTCCCC CAGA-3'; 910 bp, 5'- CCAGTTTGTGGGTTACGGGA-3'; 672 bp, 5 '-TTTGCAGGGAGCTAGGAAGT-3'; 514 bp, 5'-AGTACCCACATGAATCA-3 '; 278 bp, 5'-TCTCCCTGGGGACTTAGCTT-3'; 167 bp, 5'- GGAGAGTGGTTCAGGGTTCT -3', and reverse, 5'-TTCGCACTGGACATGAGGAC-3'. The sequences of all the constructs were confirmed using sequencing. The two NFAT1-binding sites at positions -737 and -111 were subjected to site-directed mutagenesis . The three NFκB-binding sites at positions -440, -180, and -114 were subjected to site-directed mutagenesis . The sequences of the cloned DNA fragments were confirmed using DNA sequencing.Renilla luciferase activity, was also transfected to normalize the firefly luciferase activity of the pGL4 vector. After 16 hr, the HEK293 T cells were stimulated with PMA/Iono for 6 h and then lysed using a dual-luciferase assay system . Firefly and Renilla luciferase activities were measured using a Victor 3 multi-well plate reader .The human embryonic kidney cell line HEK293 T, received from Dr. Sang-Mo Kwon , was transiently co-transfected with full-length, NFAT1, or NFκB-binding site-deleted γc promoter constructs containing a reporter vector, together with NFAT1 or NFκB-expressing vectors using Lipofectamine™ 2000 (Invitrogen) for 16 h. The hRluc vector, which expresses T cells were stimulated with α-TCR/α-CD28 for 16 hr, crosslinked for 10 min at 37 °C using 1% formaldehyde, and lysed. The crosslinking reaction was arrested by exposure to glycine in phosphate-buffered saline, and the cell layer was collected in phosphate-buffered saline containing protease and phosphatase inhibitors. The chromatin was sheared by sonication on ice. Lysates were incubated with antibodies against NFAT1 and NFκB (p65 and cRel), or without antibodies, overnight at 4 °C, and then, protein was digested using proteinase K (0.4 mg/ml). The ChIP-enriched DNA was subjected to qPCR. Each experiment was performed two or three independent times. ChIP and non-antibody signals were normalized to the total input. The primer pairs used for detecting γc promoter were NFAT1 #1 , NFAT1 #2 , NFAT1 #3 , NFAT1 #4 , NFκB #1 , NFκB #2 , NFκB #3 , and NFκB #4 .5 EL4 cells were collected and resuspended in electroporation solution (Buffer R), and ssODNs  and Cas9 /sgRNA  were added to the resuspended cells. Electroporation was conducted using a Neon Transfection System (Invitrogen). Electroporated cells were slowly loaded into a growth medium with RS-1  and cultured at 37 °C under 5% CO2 conditions; 48 hr after transfection, genomic DNA was purified using Proteinase K extraction buffer. The indel ratio, homology-directed repair frequency, and genomic DNA patterns were analyzed using Illumina Mini-Seq and Cas-Analyzer, as mentioned above [To generate γc promoter -111 (TC→GA) mutant cells, 1.5 × 10ed above . t-tests or one-way ANOVA with GraphPad Prism (GraphPad Software). p values of < 0.05 were considered statistically significant. *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.The data are shown as the mean ± SEM. Statistical differences between groups were examined using Student’s two-tailed+, CD8+ and regulatory T cells was assessed upon TCR stimulation. We found that increase of γc expression upon TCR stimulation was consistent across these different T cell subsets  and IFN-γ. Unlike the effect of IL-7 and IL-15 stimulation, which was not significant to γc expression, IL-2 slightly enhanced the expression level  using EL4 cells, which are murine lymphoma T cells. Prior to this, we first examined whether γc expression in EL4 cells was regulated by TCR stimulation, similar to that in LN T cells. We found that γc expression was upregulated in a time-dependent manner at the protein and mRNA levels with stimulated PMA/Iono Fig. A. Next, To predict the potential regulatory elements in the upstream region of the γc gene, several potential regulatory elements that have highly conserved binding sites for NFAT1 and NFκB between humans and mice were determined using rVISTA 2.0 and TRANSFAC database analysis Figure SA and B. To further confirm the vital binding sites of NFAT1 and NFκΒ, the -106 to -105 region of the γc promoter in EL4 cells was mutated from TC to GA (mut111) using CRISPR/Cas9-mediated gene editing  LN T cell treated with α-TCR/α-CD28 in the presence of PD980 and wortmannin specifically inhibited MEK and AKT phosphorylation by western blot analysis. (B) Upregulation of γc expression is not inhibited by inhibitors for AKT and ERK pathway. CD8+ T cells were cultured O/N on α-TCR/α-CD28 coated plates in presence of PD980 and Wortmannin. Surface expression level of γc and CD69 was assessed by flow cytometry. (C) Viability of LN T cells under α-TCR/α-CD28 treated with Bay11 or INCA6 dose dependent. Activated T cells were treated with NFAT1 and NFκB inhibitors  in vitro. After 16 hr of incubation, cell viability was assessed using Propidium Iodide (PI) staining. (D) Western blot analysis showed that LNT cells under α-TCR/α-CD28 treated with Bay11 (3 µM) or INCA6 (5 µM) or Bay11 (2.5 µM)/INCA6 (2.5 µM) inhibited NFAT1 and p65 phosphorylation. (E) LN CD4+ T cells were cultured O/N on α-TCR/α-CD28 coated plates in presence of Bay11 (3 µM) and INCA6 (5 µM). Surface expression level of γc (left), CD69 (middle), and TCRβ (right) was assessed by FACS. The bar graph shows the relative surface expression γc (left), CD69 (middle), and TCRβ (right). The relative γc, CD69, and TCRβ expression were calculated as the ΔMFI associated with the inhibitor-treated group over the ΔMFI associated with the DMSO control group. Data are means ± SEM of three independent experiments. * P < 0.05, ** P < 0.01, PD; PD980, wort; wortmannin, Bay; Bay11, INCA; INCA6. Figure S2. NFAT1 and NFκB pathways are related to the regulation of γc expression. (A) Expression of γc protein is increased time-dependently upon PMA (12.5 ng/ml)/Iono (1 µM) stimulation. EL4 cells were stimulated with PMA/Iono for indicated time point. The γc and CD69 expression levels were determined by flow cytometry. Results are the summary of three independent experiments. (B) γc expression is specifically inhibited by treatment of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Cell surface γc staining of EL4 cells incubated with DRB (50 µM) and cyclohexamide  in presence of PMA/Iono. Surface γc and CD69 staining was assessed as mean fluorescence intensity (MFI) in EL4 cells. Data are summary of two independent experiments. (C and D) EL4 cells were cultured O/N with PMA/Ionomycin in presence of (C) MG132 (500 nM) or (D) CsA (1 ug/ml) for indicated time point. The γc expression level was measured by flow cytometry (left) and mRNA level was measured by quantitative RT-PCR analysis (right). DMSO was used as vehicle control. (E) Upregulation of γc expression is significantly inhibited by Bay11 (3 µM) or INCA6 (5 µM) and synergistically blocked by both Bay11 (2.5 µM) and INCA6 (2.5 µM). EL4 cells were stimulated with PMA/Iono in the presence or absence of the Bay11 and INCA6 as specific inhibitors for NFκB and NFAT1, respectively. The γc protein expression is analyzed by FACS and mRNA expression was determined by RT-qPCR. Fold induction of γc content was calculated to that of DMSO. DMSO was used as vehicle control. Data are means ± SEM of three independent experiments. *p < 0.05, ** p < 0.01 and *** p < 0.001. Figure S3. Targeted disruption of NFAT1 and NFκB gene by CRISPR/Cas9 system in EL4 cells. NFAT1, p65, cRel, NFκB KO , and NFAT1/NFκB KO targeted by using CRISPR/Cas9 technology in EL4 cells. (A) Western blot analysis of γc, NFAT1, cRel expression in NFAT1 (#21 and #24) and NFκB and NFAT1/NFκB (#7 and #10) KO EL4 cells with PMA/Iono stimulation for 16hr. GAPDH was used as the loading control. Data are representative of three independent experiments. (B) The γc and CD69 expression levels in NFAT1 KO (#21 and #24), NFκB KO and NFAT1/NFκB KO (#7 and #10) EL4 cells treated with PMA/Iono for 16hr were determined by flow cytometry. (C) NFAT1, p65, and cRel mRNA levels in EL4 and EL4KO cell line by RT-qPCR analysis. Relative gene expression was calculated with Rpl13 housekeeping gene. (D) NFAT1, p65, cRel protein levels in EL4 KO cell by western blot (left). Bar graphs show a summary of western blot data from three independent experiments. Relative gene expression was calculated with β-actin (right). Data are means ± SEM of three independent experiments. *p < 0.05, ** p < 0.01 and *** p < 0.001. Figure S4. NFAT1 and NFκB predicted binding regions on upstream of γc gene. ECR browser analysis of the mouse and human γc loci is shown. (A) six NFAT1 binding sites of γc upstream region motifs were predicted. (B) Eight NFκB binding sites of γc upstream region motifs were predicted. (C) HEK293 T cells were transfected with pGL4-NFAT1-luc plasmid that carry whole γc promoter region and then were stimulated with PMA/Iono for indicated time dependent. Luciferase activity in the lysates of transfected cells was determined by a dual luciferase assay. Data are means ± SEM of three independent experiments. * p < 0.05. Figure S5. Regulation of γc expression in mutant NFAT1/NFκB binding site in γc promoter region. The mutant EL4 cell lines  were stimulated with PMA/Iono for 16 hr. (A) Western blot analysis of γc expression in mut111 (#120 and #126) and control EL4 cells. GAPDH was used as the loading control. Data are representative of three independent experiments. (B) The γc and CD69 expression level in mut111 (#120) and WT EL4 cell lines were analyzed using FACS. γc and CD69 expression (open histogram) were assessed in activated mut111 (#120) and control cell lines and overlaid with control antibody staining (shaded histogram). Bar graph is summary of three independent experiments. *** p < 0.001.Additional file 3. NGS-seq data of mutant cell lines (mut111).Additional file 4. Full immunoblot images.


N-ethyl-N-n-propyltryptamine is reported as its salt with a fumarate dianion and a fumaric acid adduct.The structure of the synthetic psychedelic prodrug 4-hydroxy- H-indol-3-yl)ethyl](ethyl)propylazanium} but-2-enedioate–(E)-butenedioic acid (1/1)), 2C17H25N2O2+·C4H2O42−·C4H4O4, was determined by single-crystal X-ray diffraction. The asymmetric unit consists of a singly protonated tryptammonium cation, one half of a fumarate dianion and one half of a fumaric acid molecule. In the crystal, the ions and molecules are linked together in infinite chains propagating along [001] through a series of N—H⋯O and O—H⋯O hydrogen bonds.The solid-state structure of the title salt/adduct (systemic name: bis{[2-(4-acetyloxy-1 N,N-dimethyltryptamine) is a natural product found in many species of mushrooms. It functions as a prodrug of psilocin  via enzymatic hydrolysis of the phosphoryloxy group to generate the active psychedelic. Psilocin is an agonist at serotonin  receptors, most notably the serotonin 2A (5-HT2A) receptor, which is primarily responsible for the psychoactive and therapeutic effects of the molecule. Psilocybin has shown promise in the treatment of pervasive human disorders, including depression  which, like psilocybin, also functions as a prodrug of psilocin. Our recent in vivo studies in mice demonstrate that psilacetin is more efficient than psilocybin at delivering psilocin, resulting in increased potency at equimolar amounts. This is supported by background hydrolysis rates which show psilacetin to hydrolyze forty times faster than psilocybin, but also demonstrate that the hydrolysis of either prodrug in the body must be enzymatic  is a putative prodrug of the synthetic psychedelic, and psilocin analogue, 4-hydroxy-N-ethyl-N-n-propyltryptamine (4-HO-EPT). When competitive binding assays are compared, binding is observed for 4-HO-EPT across many more receptors than 4-AcO-EPT, and significantly stronger binding is observed at most receptors where 4-AcO-EPT is also competitive  fumarate–fumaric acid.One thing that is not clear from the N-ethyl-N-n-propyltryptammonium (C17H25N2O2+) cation, one half of a fumarate (C2HO2−) dianion, and one half of a fumaric acid (C2H2O2) molecule. The indole ring system of the cation is near planar with a r.m.s. deviation from planarity of 0.018 Å. The ethylamino arm is disordered over two orientations in a 0.895 (7):0.105 (7) ratio. The major component of the ethylamino arm is nearly co-planar with the indole ring, showing a C7—C8—C9—C10 torsion angle of −176.3 (2)°. The ethyl and n-propyl groups are disordered over two sets of sites with a 0.741 (6):0.259 (6) ratio, with the methylene carbon atoms of the n-propyl groups being in close proximity to the two ethyl group C atoms. The complete fumarate dianion is generated through crystallographic inversion symmetry, and is near planar with an r.m.s. deviation from planarity of 0.009 Å. The complete fumaric acid dianion in generated similarly and only slightly less planar with an r.m.s. deviation of 0.070 Å.The molecular structure of 4-AcO-EPT fumarate–fumaric acid is shown in Fig. 1N-ethyl-N-n-propyltryptammonium cations, fumarate dianions and neutral fumaric acid molecules are linked together in infinite one-dimensional chain propagating along [001]. The tryptammonium cations are linked to the fumarate dianions through N2—H2⋯O5 hydrogen bonds, and the fumaric acid molecules are linked to the fumarate dianions through O4—H4A⋯O6 hydrogen bonds  fumarate–fumaric acid were grown from the slow evaporation of an aqueous solution of ‘4-AcO-EPT fumarate’ obtained from ChemLogix.Crystals of bis Å and 1.20 Ueq of parent N atoms, and O—H distances of 0.88 (1) Å and 1.50 Ueq of parent O atoms. All other hydrogen atoms were placed in calculated positions with appropriate riding parameters. Ethyl and propyl groups showed overlap disorder with respect to each other and were treated using SADI C—C distance restrains, DELU rigid body restraints, and ISOR isotropic restraints.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314623007794/hb4447sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314623007794/hb4447Isup2.hklStructure factors: contains datablock(s) I. DOI: 2293403CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


I)–(III), the molecular structures of which differ in the location of the benzene fragment relative to the cytisine core. Intermolecular C—H⋯O hydrogen bonds cross-link the molecules into infinite chains.Arylsulfonation of cytisine with three types of substituted arylsulfonyl chlorides produced products -N-[(4-ethylphenyl)sulfonyl]cytisine, C19H22N2O3S (I) {systematic name: -3-[(4-ethylphenyl)sulfonyl]-1,2,3,4,5,6-hexahydro-8H-1,5-methanopyridodiazocin-8-one}, -N-[(4-chlorophenyl)sulfonyl]cytisine, C17H17ClN2O3S (II) {systematic name: -3-[(4-chlorophenyl)sulfonyl]-1,2,3,4,5,6-hexahydro-8H-1,5-methanopyridodiazocin-8-one} and -N-[(3-nitrophenyl)sulfonyl]cytisine, C17H17N3O5S (III) {systematic name: -3-[(3-nitrophenyl)sulfonyl]-1,2,3,4,5,6-hexahydro-8H-1,5-methanopyridodiazocin-8-one}. The crystal structures of the compounds were determined on the basis of single-crystal X-ray diffraction data. The crystal structures of (I)–(III) are distinguished by the arrangement of two fragments of the molecule around the sulfonyl site. For all structures, weak C—H⋯O hydrogen bonds are developed. Hirshfeld surface analysis shows that H⋯H (for I and II) and H⋯O/O⋯H (for III) interactions make the most important contribution to the crystal packing.By arylsulfonylation of cytisine in the presence of triethylamine, three new compounds have been obtained in good yields: (7 To this day, other sources of cytisine have been found . Cytisine is a quinolizidine alkaloid, which is found in different sources by different names: 1,2,3,4,5,6-hexahydro-1,5-methano-8H-pyrido-diazocin-8-one -cytisine -cytisine.Cytisine was first isolated by Huzeman and Marme from the seeds of et al., 2018et al., 2011et al., 2000et al., 2012et al., 2010et al., 2010et al., 2000et al., 2007et al., 2019et al., 2020Various studies report modern methods for the synthesis of cytisine -N-[(4-ethylphenyl)sulfonyl]cytisine (I), -N-[(4-chlorophenyl)sulfonyl]cytisine (II) and -N-[(3-nitrophenyl)sulfonyl]cytisine (III).This communication describes the synthesis and crystal structures of three 2.I)–(III) are virtually identical and also do not differ from that of the cytisine molecule itself –(III) obtained by arylsulfonation of cytisine, the configurations are 7R, 9R in each case.The conformations of the cytisine cores in structures (I) consists of one molecule of -N-[(4-ethylphenyl)sulfonyl]cytisine  of the ethyl group bound to the phenyl ring is disordered over two sets of sites. In the crystal structures of (II) and (III), both asymmetric units likewise comprise one molecule of -N-[(4-chlorophenyl)sulfonyl]cytisine and -N-[(3-nitrophenyl)sulfonyl]cytisine, respectively –(III) are almost superimposable in the three molecules . Corresponding torsion angles C1′—S1—N12—C11 and N12—S1—C1′—C2′ are listed in Tables 1The asymmetric unit of –(III), weak intermolecular hydrogen bonds of the type C—H⋯O(C) are developed. In the crystal structures of (I) and (II), C—H⋯O1 hydrogen bonds link molecules into chains directed parallel to [100]  interactions (Tables 4III), the C—H⋯O1 interactions link the molecules into a chain running along -8-oxo-1,3,4,5,6,8-hexahydro-2H-1,5-methanopyridodiazocin-3-ium perchlorate  and 10 ml of acetone were placed in a two-necked flask with a volume of 50 ml. After cooling, a previously prepared solution (1.9 g (0.01 mol) of cytisine and 0.01 mol of triethylamine in 15 ml of acetone) was added under stirring through a separatory funnel. The reaction mixture was stirred at room temperature for 10 h. The reaction mixture was then left in the open air overnight to produce a dry mass. The mass was treated with 15 ml of distilled water and the remaining solid filtered off and dried in air. The reaction scheme is shown in Fig. 11-N-[(4-ethylphenyl)sulfonyl]cytisine (I)Rf = 0.59 [5:1 (v/v) benzene–ethanol].Yield 64% (2.29 g), m.p. 456–458 K, -N-[(4-chlorophenyl)sulfonyl]cytisine (II)Rf = 0.71 [5:1 (v/v) benzene–ethanol].Yield 76% (2.77 g), m.p. 488–490 K, -N-[(3-nitrophenyl)sulfonyl]cytisine (III)Rf = 0.50 [5:1 (v/v) benzene–ethanol].Yield 72% (2.71 g), m.p. 524–526 K, I)–(III) suitable for X-ray analysis were obtained by slow evaporation of an ethanol solution.Colourless crystals of (6.I), the methyl C8′ atom is disordered over two positions . The site occupancy factors of the disordered fragment were refined with a free variable to a ratio of 0.55 (2):0.45 (2). Hydrogen atoms bonded to C atoms were placed geometrically  and included in the refinement in a riding motion approximation with Uiso(H) = 1.2Ueq(C) or Uiso = 1.5Ueq(C) for methyl H atoms.Crystal data, data collection and structure refinement details are summarized in Table 710.1107/S2056989023001950/wm5674sup1.cifCrystal structure: contains datablock(s) I, II, III, Global. DOI: 10.1107/S2056989023001950/wm5674Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989023001950/wm5674IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989023001950/wm5674IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 2245793, 2245792, 2245791CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Nature 10.1038/s41586-023-06726-w Published online 15 November 2023Correction to: y-axis label now reading “GAL-3 area/cell area” originally said “G3BP1 area/cell area”. In Fig. 4h, two labels now reading “ZNFX1” said “ZNFZ1”. The errors have been corrected in the HTML and PDF versions of the article.In the version of the article initially published, there was an error in Fig. 1c where the