Recent evidence indicates that the HIV-1 Integrase (IN) binds the viral genomic RNA (gRNA), playing a critical role in the morphogenesis of the viral particle and in the stability of the gRNA once in the host cell. By combining biophysical, molecular biology, and biochemical approaches, we found that the 18-residues flexible C-terminal tail of IN acts as a sensor of the peculiar apical structure of the trans-activation response element RNA (TAR), interacting with its hexaloop. We show that the binding of the whole IN C-terminal domain modifies TAR structure, exposing critical nucleotides. These modifications favour the subsequent binding of the HIV transcriptional trans-activator Tat to TAR, finally displacing IN from TAR. Based on these results, we propose that IN assists the binding of Tat to TAR RNA. This working model provides a mechanistic sketch accounting for the emerging role of IN in the early stages of proviral transcription and could help in the design of anti-HIV-1 therapeutics against this new target of the viral infectious cycle. Protein–nucleic acid interactions can occur through different types of protein binding domains and are responsible for a variety of essential molecular and cellular mechanisms and their regulation. This binding diversity has been well described by recent RNA interactome screenings revealing that the term RNA-Binding Domain (RBD) is no longer synonymous of a well-structured domain, but it also includes intrinsically disordered regions (IDR) endowed with non-canonical RNA-binding properties ,2,3. Mor2+ cofactors and folds similarly to nucleotidyl-transferases and nucleases -1-propanesulfonate), and 1 tablet of Protease Inhibitor Cocktail . The preparation was sonicated for 120 s on ice, then the resulting lysate was subjected to centrifugation at 11,000× g for 1h. The recovered supernatant was then applied to a HisTrapTM Fast Flow Crude column  and purified by an AKTA pure system . The sample was first abundantly washed with a lysis buffer containing 100 mM imidazole and 2M NaCl, then the protein was eluted using a gradient up to 500 mM imidazole in 10 column volumes. A second step of purification was carried out using a Superdex 75 10/300 GL column  with an isocratic elution carried out with a storing buffer . Mammalian FlagIN-FL (IN-FLm) was expressed in Baby Hamster Kidney suspension cells  using a vaccinia virus expression system as previously described [Construction of plasmid pET15b encoding N-terminal 6XHis tagged IN-CTD (aa 220-270) and IN-CTD-ΔCT (aa 220-288) were previously reported . The IN-escribed . IN-CT pescribed ,66. Thisescribed .32P-ATP  for 1 h at 37 °C then purified on denaturing 10% (w/v) polyacrylamide gel (29:1) as previously described [2, 2 mM DTT) for 3 min at 95 °C followed by 40 min of slow controlled cooling to room temperature and finally placed on ice.We produced several RNAs as listed in escribed . Before 2, 0.2 µg BSA, and 8% (v/v) PEG8000. The samples were incubated at 37 °C for 30 min before being resolved by native 6% polyacrylamide (19:1) gel electrophoresis in a 0.5x TAE (Tris acetate EDTA) buffer. Results were analysed by phosphorimaging using ImageQuant software. IN-FLm was incubated for 2 h at 4 °C with the RNA substrate. For the dose-dependent competition assay shown in D. Then, we added increasing concentrations of Tat.The electrophoretic mobility shift assays (EMSA) were performed as described in . SamplesEnzymatic treatments were performed in 10 µL of the reaction mix containing 0.5 pmol of 5′ radiolabeled RNA, 0.2° µg of yeast tRNA, 1 × Structure buffer , and 0.01 U of RNase T1 . Incubation was done at 37 °C for 5 min. Reactions were stopped by the addition of 40 µL of a quenching buffer composed of 10 mM HEPES pH 7.5, 1 mM EDTA, and 3% SDS. Partial alkaline hydrolysis was performed as follows; 10 µL of reaction mix containing 0.5 pmol of RNA, 1 µg of yeast tRNA, and 1× Alkaline Hydrolysis buffer were incubated at 95 °C for 12 min then quenched with 2× denaturing loading buffer and placed on ice. For RNA/protein complexes, 0.5 pmol RNA were previously incubated with a 36 µM protein  at 37 °C for 30 min then treated with RNase T1. After quenching, samples were phenol extracted and ethanol precipitated. After recovering from precipitation, all samples were run on a 15% sequencing polyacrylamide gel in 0.5 × TBE. (Tris Borate EDTA). Results were analysed by phosphorimaging.2, 2 mM β-mercaptoethanol and 5% glycerol) adjusting the final NaCl concentration to 150 mM. Samples were complemented or not with 600 pmol of 3′ end–biotinylated TAR  on ice and proteins were eluted with an SDS loading buffer and analysed on a polyacrylamide 16% (37.5:1) SDS-PAGE. For the Histidine pulldown assay, magnetic streptavidin resin was replaced with HisPur™ Ni-NTA Magnetic Beads  and protein elution was done with a 0.5 M Imidazole containing buffer.The pulldown assays were conducted as described before . Brieflyated TAR  in a fin2, 1 µM ZnSO4, and 0.5 M BSA. For the BLItz platform, we used Streptavidin (SA) Biosensors , and for the Octet RED96e, we used the Streptavidin (SAX) Biosensors  that were hydrated for 10 min in an assay buffer then plunged in a solution containing 1 µM 3′ end–biotinylated TAR RNA in an assay buffer with a 1 × RNAase inhibitor  and 0.1 µM BSA for the RNA loading step. A wash was performed after RNA loading. In the BLItz experiment, the association of the protein to RNA was monitored in real-time for 200 s; the hydrated biosensor tip was placed in a 1.5 mL black assay tube containing 200 µL of the protein solution in an assay buffer as indicated. Afterward, a dissociation step or a second association with Tat was performed for 200 s. Biosensors were discarded after each measurement. All kinetic assays shown in For the Bio-Layer Interferometry (BLI) analysis, we used the BLItz platform (FortéBio). For the high throughput experiments such as shown in E. coli or in mammalian cells), binds TAR RNA  as an RNA binding protein in the very first stages of proviral transcription. In particular, we have shown that full length IN, despite its intrinsic instability and independently of the post-transcriptional modifications , int


ClC-3 chloride channels promote osteogenic differentiation. Transforming growth factor-β1 (TGF-β1) and its receptors are closely related to ClC-3 chloride channels, and canonical TGF-β1 signaling is largely mediated by Smad proteins. The current study aimed to explore the role of the Smad2/3/4 signaling pathway in the mechanism by which ClC-3 chloride channels regulate osteogenic differentiation in osteoblasts.First, real-time PCR and western blotting were used to detect the expression of Smad and mitogen-activated protein kinase (MAPK) proteins in response to ClC-3 chloride channels. Second, immunocytochemistry, coimmunoprecipitation (Co-IP) and immunofluorescence analyses were conducted to assess formation of the Smad2/3/4 complex and its translocation to the nucleus. Finally, markers of osteogenic differentiation were determined by real-time PCR, western blotting, ALP assays and Alizarin Red S staining.ClC-3 chloride channels knockdown led to increased expression of Smad2/3 but no significant change in p38 or Erk1/2. Furthermore, ClC-3 chloride channels knockdown resulted in increases in the formation of the Smad2/3/4 complex and its translocation to the nucleus. In contrast, the inhibition of TGF-β1 receptors decreased the expression of Smad2, Smad3, p38, and Erk1/2 and the formation of the Smad2/3/4 complex. Finally, the expression of osteogenesis-related markers were decreased upon ClC-3 and Smad2/3/4 knockdown, but the degree to which these parameters were altered was decreased upon the knockdown of ClC-3 and Smad2/3/4 together compared to independent knockdown of ClC-3 or Smad2/3/4.The Smad2/3 proteins respond to changes in ClC-3 chloride channels. The Smad2/3/4 signaling pathway inhibits osteogenic differentiation regulation by ClC-3 chloride channels in MC3T3-E1 cells. Bone is a kind of highly mineralized tissue that is always under regulation by dynamic balance between the processes of bone formation and bone resorption. This homeostasis is mainly supported by the coordinated activities of osteoblasts and osteoclasts. The mechanism of bone metabolic regulation is extremely complex, and abnormalities in any step may lead to bone metabolic disorders, such as osteoporosis and osteopetrosis –3.As one of the most abundant anions in organisms, chloride ions regulate a very wide range of physiological activities. The ClC-3 chloride channels, a kind of voltage-gated anion channel, participates in ion homeostasis, the regulation of cell volume, acidification of the internal environment, proliferation, apoptosis and many other processes in biological systems , 5. ReceBased on the above context, TGF-β1 signaling was hypothesized to likely be involved in the functions of ClC-3 chloride channels in bone metabolism. Canonical TGF-β1 signaling can be mediated by Smads. Upon binding to TGF-β1 receptor (TβR) complexes, including type I and type II receptors, Smad2/3 is phosphorylated and activated, and Smad4 is then recruited to the Smad2/3 complex and translocated into the nucleus to direct the osteogenic gene transcriptional response –14. In aNotably, there is crosstalk between the canonical and noncanonical TGF-β1 signaling pathways. The mechanisms of TGF-β1 signaling in bone metabolism are quite complex. Given the importance of TGF-β1 signaling in ClC-3 chloride channels, it is crucial to investigate the specific regulators of TGF-β1 signaling that participate in the regulatory effect of ClC-3 chloride channels on osteoblast differentiation in osteoblasts, as done in the current study.2 at 37 ℃. Before treatment, cells were plated on treated tissue culture plates and grown to 85% confluence. TβR inhibitor LY2109761  was constituted in dimethyl sulfoxide and stored at − 80 °C. The cells were treated with LY2109761 at a concentration of 2 μM for 24 h to inhibit the expression of receptor complexes.MC3T3-E1 cells, a mouse calvaria-derived preosteoblast cell line, were cultured in α-MEM  supplemented with 10% fetal bovine serum (FBS) , 1% penicillin–streptomycin and a 1% L-glutamine solution under 5% COMC3T3-E1 cells were transfected with a specific small interfering RNA (siRNA) and a control siRNA using Lipofectamine 2000  according to the manufacturer’s instructions. The regular medium was replaced at 6 h, and the cells were continuously cultured for 48 h for the next step. The siRNA targeting mouse ClC-3 and Smad2/3/4 were synthesized by GenePharma company . The sense and antisense sequences were as follows, ClC-3: 5’-CGAGAGAAGUGUAAGGACATT-3’, 5’-UGUCCUUACACUUCUCUCGTT-3’; Smad2: 5’-UCUUUGUGCAGAGCCCCAA-3’, 5’-UUGGGGCUCUGCACAAAGA-3’; Smad3: 5’-GGAGAAAUGGUGCGAGAAG-3’, 5’-CUUCUCGCACCAUUUCUCC-3’; Smad4: 5’-GCCUGAUCUACACAAGAAUTT-3’, 5’-AUUCUUGUGUAGAUCAGGCTT-3’; negative control: 5’-UUCUCCGAACGUGUCACGUTT-3’, 5’-ACGUGACACGUUCGGAGAATT-3’.2O in a total volume of 20 μl. Then, the 2-ΔΔCt method was applied to calculate the relative changes in gene expression. The primer sequences used are shown in Table Total RNA was isolated from MC3T3-E1 cells using Trizol reagent , and cDNA was synthesized using PrimeScript™ RT Master Mix  by reverse transcription following the manufacturer’s instructions. Real-time PCR experiments were performed with the SYBR Green PCR kit  utilizing the ABI 7500 sequence detection system. The reaction system consisted of 2 μl of cDNA, 10 μl of SYBR® Premix Ex Taq, 0.8 μl of each PCR primer (forward and reverse), 0.4 μl of ROX and dHAs the protocol described, western blotting was performed with the following steps. Whole-cell lysates were prepared with lysis buffer . The total protein concentration was determined via the BCA method, and proteins were transferred onto PVDF membranes. After blocking with milk (5% w/v) and incubating with p38, Erk1/2, Smad2, Smad3, Alp, Runx2, Osx, Collagen I or β‐actin  antibodies overnight at 4 ℃, the membranes were re‐probed with horseradish peroxidase‐labeled secondary antibodies. The HRP-ECL kit was used to enhance chemiluminescence in the blots, and XAR film exposure was applied to determine the densities of the bands.2O2 in sequence, and blocked with 3% bovine serum albumin at 37 °C. Then, the cells were incubated with primary antibodies  overnight at 4 °C and conjugated secondary antibodies  for 45 min at 37 °C. Finally, the cells were incubated in a SABC wet box for 20 min, and the expression in each group was observed by a microscope.The cells were treated as described above and fixed with ice-cold 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 and 3% HFor coimmunoprecipitation (Co-IP) assays, protein lysates were prepared from MC3T3-E1 cells in precooled RIPA buffer containing protease inhibitors (ab156034). Cells were scraped into PBS and centrifuged for 15 min at 4 °C. The lysates were then incubated with antibody overnight and treated with protein A/G agarose beads to conjugate the antibody. The complex precipitate was washed with lysis buffer 3 ~ 4 times and resuspended in 2 × SDS loading buffer. The sample proteins were eluted from the agarose beads by immersion in boiling water at 95 °C for 5 min. The isolated proteins were measured by immunoblotting after separation by SDS–PAGE.5 cells/ml. Different groups of cells were treated accordingly, and then the cells were fixed with ice-cold 4% paraformaldehyde for 30 min, permeabilized with 0.25% Triton X-100 for 5 min, and blocked with 3% bovine serum albumin for 30 min at 37 °C. The cells were then incubated with primary antibodies  overnight at 4 °C, followed by secondary conjugated antibodies for 45 min at 37 °C after several rinses with PBS. Images were obtained using a laser scanning confocal microscope.MC3T3-E1 cells were plated in culture dishes specific for immunofluorescence at a density of 1 × 104 per well. Then, the cells were cultured for the corresponding number of days in osteogenic induction medium . The induction medium was changed every 3 days.MC3T3-E1 cells were separately transferred into six-well plates at a density of 1 × 10After 7 days of induction, ALP activity was assessed by utilizing an ALP assay kit . The cell lysates were homogenized, and the supernatants were collected. Then, spectrophotometry was used to measure the absorbance at 405 nm to quantify ALP activity.After 21 days of osteogenic induction, 2% Alizarin Red S staining  was applied for cell staining at 37 °C to evaluate mineralization of the cell matrix. An inverted microscope and camera system were used for further imaging. The concentration of Alizarin Red S dye emitted from the cell matrix into the cetylpyridinium chloride solution was determined by measuring absorbance at 540 nm on a spectrometer.All data were analyzed using SPSS statistical software  and are expressed as the mean ± standard deviation (SD). Statistical analysis was conducted using GraphPad Prism (version 6.00) for Windows. Student's t test was used to assess differences between two groups. Comparisons between three or more groups were analyzed by one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test and Student-Newman–Keuls (SNK) q test. Differences with a P value less than 0.05 were considered significantly different. All experiments were repeated more than three times.TGF-β1 signaling is mediated through the Smad-dependent pathway and the non-Smad-dependent pathway. To investigate the specific signal involved in the change in ClC-3 chloride channels, we selected Smad2 and Smad3 as target proteins of the Smad-dependent pathway and p38 and Erk1/2 as targets of the non-Smad-dependent pathway. Then, ClC-3 siRNA and TβR inhibitors were used to knock down the expression of ClC-3 chloride channels and TβR, respectively, and changes in the p38, Erk1/2, Smad2 and Smad3 genes and proteins were observed. The RT-PCR  in the ClC-3 siRNA group, Smads siRNA group and ClC-3 + Smad siRNA group were lower than those in the control group. Runx2 and Osx levels decreased by 80% and 90%, respectively, and Alp levels decreased by 70% when ClC-3 was expressed at low levels. However, the expression of osteogenic genes in the ClC-3 + Smads siRNA group was lower than that in the control group but higher than that in the ClC-3 siRNA group and Smads siRNA group, but there was no significant difference in osteogenic gene expression between the ClC-3 siRNA group and Smads siRNA group (Fig. ALP activity is considered an earlier marker that reflects the mature state of osteoblasts, and mineralized extracellular matrix nodules are a sign of mature osteoblasts. We then detected the ALP activity of MC3T3-E1 cells 7 days after osteogenic induction and found that the activity level in the control group was the highest, followed by that in the ClC-3 + Smads siRNA group, the ClC-3 siRNA group and the Smads siRNA group Fig. a, b. TweBone metabolism is the result of balance between bone resorption and bone formation, and stable bone regulation is the basis for the normal maintenance of biological activities. Ion channel proteins, hormones and many other cytokines participate in the regulation of bone remodeling , 18. ClCIn general, both the Smad and MAPK pathways are activated through TGF-β1 induction during osteoblast differentiation, which is essential for the formation of the skeleton –22. To fSubsequently, we further investigated the nuclear translocation of the Smad complex when ClC-3 chloride channels were expressed at different levels. Phosphorylation of Smad2/3 is a key step and marker in the Smad signaling pathway , 26. OurSince Smad2/3/4 are closely related to the expression of ClC-3 chloride channels, it is particularly important to determine the mechanism by which the Smad2/3/4 signaling pathway regulates ClC-3 chloride channels-mediated regulation of osteogenic differentiation. We knocked down the ClC-3 chloride channels and Smad2/3/4 complex and detected the expression of osteogenic genes and the formation of mineralized nodules. ALP activity is an early marker of osteogenic differentiation, and ALP is highly active in the early stage of bone formation. Runx2 is a key transcriptional regulator that acts as a “switch” to influence the bone formation marker Osx –34. In gIn addition, we continued to detect ALP activity and calcium nodule formation after osteogenic induction in MC3T3-E1 cells , 39, andClC-3 chloride channels affect the regulation of osteogenic differentiation, and Smad signaling can respond to ClC-3 chloride channels. The expression of ClC-3 chloride channels decreased the formation of Smad2/3/4 and the ability of the complex to translocate to the nucleus. Inhibition of Smad2/3/4 abrogated the inhibition of osteogenic markers when ClC-3 chloride channels were knocked down. In conclusion, the Smad2/3/4 signaling pathway may act as a negative regulator of osteogenic differentiation regulation by ClC-3 chloride channels in MC3T3-E1 cells.


In vitro studies demonstrated that Hm3A4-Rap could efficiently kill target cells. In vivo studies demonstrated that Hm3A4-Rap had potent anti-leukemia activity, with dosed mice showing a significant increase in survival time compared to control mice (P < 0.01). In summary, our immunotoxin had excellent biological activity suggesting its potential therapeutic value for treating AML patients. Additional preclinical and clinical studies are needed to develop this immunotoxin as a treatment option for patients with leukemia.CD45RA is a specific marker for leukemia stem cell (LSC) sub-populations in acute myeloid leukemia (AML). Ranpirnase (Rap), an amphibian RNase, has been extensively investigated in preclinical and clinical studies for its antitumor activity. Rap could be administered repeatedly to patients without inducing an immune response. Reversible renal toxicity has been reported to be dose-limiting. In this study, we generated a novel immunotoxin targeting LSCs: Hm3A4-Rap, which was composed of Rap and Hm3A4, a human-mouse chimeric antibody against CD45RA. This immunotoxin was generated recombinantly by fusing Rap to Hm3A4 at the Fc terminus and then produced by stably transfecting CHO cells. The immunotoxin was purified using Ni-NTA and then evaluated using RT-PCR, SDS-PAGE, antibody titer assays, competitive inhibition assays, and internalization assays. In addition, the purity, molecular integrity, and affinity to the CD45RA antigen were determined.   In addition, we constructed a human-mouse chimeric antibody Hm3A4 by linking the murine ScFv3A4 to the human IgG1 Fc region. We found that the Hm3A4 antibody could eliminate leukemia cells including LSCs in vitro and in vivo [Chemotherapy-resistant leukemic stem cells (LSCs) are thought to be responsible for relapses after therapy in acute myeloid leukemia (AML). CD45RA is expressed on leukemic cells in most AML patients. CD45RA has been used as a marker to specifically identify LSC and HSC and improve LSC quantification. Compared to other markers , CD45RA is the most reliable marker . In our Immunotoxins (ITs) are hybrid proteins comprising a targeting moiety of mostly single-chain Fv (ScFv) or Fab antibody fragments fused or chemically conjugated to a cytotoxic moiety ,7. The aMr, 11,817; 104 amino acids), is an amphibian ribonuclease (RNase) that belongs to the RNase A superfamily. Native Rap, isolated from Rana pipiens eggs [in vitro and in vivo studies [Ranpirnase (Rap), a monomeric protein  tagged with a humanized, internalizing anti-Trop-2 antibody recognizes a cell surface glycoprotein that is overexpressed in a variety of epithelial cancers. It has been shown to induce potent cytotoxicity against several types of epithelial cancer cells . Recentlll lines . Rap hasll lines . Althougll lines .Based on the above findings, our hypothesis was that the recombinant Hm3A4-Rap immunotoxin can effectively kill the 3A4 positive AML cells, including leukemia stem cells (LSC). The aims of this study were to test the targeting activity of Hm3A4-Rap on AML cells and their LSCs both in vitro and in vivo. The goals of our work were to evaluate the biological activity of Hm3A4-Rap and provide a potential agent for AML therapy.2.2.1KG1a cells  were cultured in IMDM medium with 20% (v/v) heat-inactivated fetal calf serum (FCS) . Nalm-6 C Patrick Reynolds at the children’s hospital of Los Angeles) were cultured in PRMI-1640 medium with 10% (v/v) heat-inactivated fetal calf serum (FCS) . All cel2.2The pHMCH3/Hm3A4 plasmid was constructed in our laboratory as previously described . The Rap2.33PO4. The supernatants were then passed through the column that had been previously equilibrated with a wash buffer 1 . The column was washed with wash buffer 1 and then wash buffer 2 . Next, proteins on the column were eluted using elution buffer . The eluted fraction was collected and dialyzed against phosphate-buffered saline (PBS). Crude and purified culture supernatants were run on a 10% SDS–PAGE and analyzed by western blot using the horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc antibody . The concentration of Hm3A4-Rap protein was determined using the BCA kit  [The pHMCH3/Hm3A4-Rap plasmid was transfected into CHO cells using Lipofectamine™ 2000 . Transfected cells were selected in serum-free medium (Invitrogen) containing 600 µg/ml of G418 (Sangon) to establish stable cell lines. After 2–3 weeks of selection, the supernatants of pHMCH3/Hm3A4-Rap plasmid transfected CHO cells were harvested and centrifuged at 6000 g for 10 min to remove cells and cell debris. The supernatants were then concentrated by ultrafiltration and purification using an immobilized nickel-iminodiacetic acid affinity chromatography column  . First, , China) .2.4CHO cells expressing Hm3A4-Rap were grown on glass slides for approximately 24 h and then fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked in PBS containing 10% calf serum. The cells were then stained with TRITC-conjugated anti-mouse Fab specific IgG antibody  and FITC-conjugated goat anti-human IgG Fc antibody (GAHFc FITC) (Invitrogen) for 1 h in the dark. This was followed by incubation with DAPI (Invitrogen) nucleic acid (nuclear) staining for 1 min in the dark. The cells were visualized and imaged using confocal immunofluorescence microscopy . Non-transfected CHO cells were used as the negative control. The expression of Hm3A4-Rap in CHO cells was detected by immunofluorescence microscopy. The ScFv3A4 fragment was detected using GAM-Fab-TRITC and the Fc fragment was detected using GAH-Fc-FITC .2.57/mL. 100 μL (106 cells) of cell suspension was then incubated with various amounts  of purified Hm3A4-Rap for 30 min at 25°C in the dark. The cells were then incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-human Fc  for an additional 15 min at 25°C. Afterward, the cells were washed twice with PBS before flow cytometry analysis (FCM) . We also performed competitive inhibition assays to identify the relative binding affinity of Hm3A4-Rap in comparison with the parental antibody, 3A4 mAb. Briefly, KG1a cells were incubated with 3A4-FITC at a fixed sub-saturated concentration of 0.5 µg, either alone or in the presence of varying amounts of Hm3A4-Rap  for 30 min at 25°C. The same amounts of mouse IgG1 isotype antibody  was used as the control. After three washes with PBS, stained cells were analyzed by FCM. Experiments were performed in triplicates.To identify the binding capacity of Hm3A4-Rap, we performed titration assays. KG1a cells were harvested at their logarithmic growth phase and adjusted to a concentration of 102.65 of KG1a cells were incubated with 1 μg of Hm3A4-Rap or Hm3A4 at 37° for 0.5 h, 1 h, 2 h, or 4 h. Then, 0.1 mol/L glycine (pH = 2.5) was added to the experiment tube to remove any unbound antibody on the surface of KG1a cells and then washed twice with PBS containing 1% BSA. Cells in the control group were washed with PBS containing 1% BSA to remove unbound antibodies. Cells were then resuspended and fixed with 4% paraformaldehyde for 10 min, then permeabilized with 0.2% Triton X-100 for 5 min and incubated with 2 μL of GAH-Fc-FITC antibody for 30 min. Cells were then washed twice with PBS, resuspended in PBS, and analyzed by FCM. Experiments were performed in triplicates.Internalization of the Hm3A4-Rap antibody in KG1a cells was determined and compared to that of the Hm3A4 antibody. 0.1 mol/L glycine (pH = 2.5) was used to remove unbound antibodies on the surface of KG1a cells . Cells w2.76 cells/mL).ADCC function was evaluated using the CytoTox 96 ® Non-Isotopic Cell Killing Test Kit . Briefly, peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy volunteer by Ficoll-Hypaque separation  to be used as effector cells. Cells were washed twice with Hanks Balanced Salt Solution (Invitrogen) to remove platelets and then resuspended in RPMI-1640 media supplemented with 10% heat-inactivated fetal bovine serum (HIBS) (5 × 10The following assay conditions were set up: 1) Effector spontaneous wells; 2) Target spontaneous wells; 3) Target Maximum wells  Experimental wells (effector cells with target cells and Hm3A4-Rap or Hm3A4). Three replicate wells were set for each condition. Step1: Hm3A4-Rap and Hm3A4 antibodies were diluted to a concentration of 0.1 mg/mL using phenol red-free RPMI-1640 media. Step2: Target cells were added and incubated at 4°C for 30 min. Cells were then washed twice with PBS and resuspended in 300 μL RPMI-l640 media. Step3: Cell suspensions with antibodies were added to a 96-well plate at 100 μL/well. Step4: 100 μL/well of effector cells were added at a ratio of 25:1 or 50:1 KG1a cells. Cells were then placed in a 5% CO2 incubator for 12 h to 24 h. Step5: Cells were centrifuged and then 50 μL of the supernatant was transferred from each well to a new 96-well plate. The absorbance at 490 nm of each well was measured using the Epoch 2 Microplate Spectrophotometer . Cytotoxicity was calculated based on the following formula:6 cells/mL. 50 μL of the cell suspension was added to each well of a 96-well plate, and then 25 μL of different concentrations of Hm3A4-Rap and Hm3A4 were added to each well and incubated for 1 h. 25 μL of human serum as a complement source from healthy volunteers was added per well and incubated at 37°C for 2 h. Wells with no antibodies containing equal volumes of culture medium and complement was used as the negative controls. Three replicate wells were performed for each condition. 10 μL of CCK-8 (Beyotime Institute of Biotechnology) was added to each well and incubated for an additional 2 h at 37°C. The absorbance at 450 nm of the formazan dye produced by metabolically active cells from each well was measured using the Epoch 2 Microplate Spectrophotometer. Cytotoxicity was calculated based on the following formula..Complement-dependent cytotoxicity (CDC) assay was performed using the Cell Counting Kit-8 (CCK-8) method, which is a rapid, reliable, and sensitive measurement for cell viability, details of which have been described previously . BrieflyWhere E was the absorbance of the medium group, C was the absorbance of the complete lysis group, and T was the absorbance of the experimental group.6 cells/mL. Add 5 μL of Annexin V-FITC and 5 μL of PI to 100 μL of the cell suspensions. Mix gently and keep tubes on ice and incubate for 15 minutes in the dark. Then, add 400 μL of ice-cold binding buffer and mix gently. Analyze cell preparations within 30 minutes by FCM. The Annexin+/PI- and Annexin+/PI+ cells were identified as apoptotic and necrotic cells. 2) The cytotoxic effect of Hm3A4-Rap or 3A4 on KG1a or Nalm-6 cells was measured using the traditional cell counting method. The concentration of Hm3A4-Rap and 3A4 were both set at 10 μg/mL and 20 μg/mL in 2 mL volume/well of a 24-well plate. Control wells were treated with equal volumes of PBS. Three replicate wells were performed for each condition. Cells were incubated for 24, 48, 72, and 96 h at 37°C, 5% CO2. Cell numbers were measured using Countstar . Cytotoxicity was calculated based on the following formula:Direct killing effect of Hm3A4-Rap on KG1a: 1) We compared the cytotoxic effects of Hm3A4-Rap with Hm3A4 using Annexin-V/PI Kit(Beyotime Institute of Biotechnology). The concentration of Hm3A4 and Hm3A4 were both set at 2.5 μg/mL, 5 μg/mL and 10 μg/mL in 4 mL volume/well of a 6-well plate. Three replicate wells were performed for each condition. Cells were incubated for 48 h at 37°C, 5% CO2. Then, cells were washed twice with ice-cold PBS and centrifuge for 5 minutes at 300 g at 4°C. Discard supernatant, and resuspend the cells in ice-cold binding buffer to 1 × 102.8in vivo antitumor activity of Hm3A4-Rap, we established a xenograft model by inoculating KG1a cells into non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice . The model was established as follows: Twenty specific pathogen-free 4-week-old female NOD/SCID mice  were used for model generation and were divided into four groups with 5 mice for each group (n = 5): (1) the control group, (2) PBS treatment, (3) Hm3A4-Rap treatment, and (4) Hm3A4 treatment group. Mice were administered cyclophosphamide (CTX) by intraperitoneal injection 1 day before xenografting and were randomly assigned to the control or the xenograft group. The xenograft group was further subdivided into the PBS, Hm3A4-Rap, and Hm3A4 treatment groups.To determine the 6 exponentially growing KG1a cells in 100 μL or an equal volume of PBS were tail vein injected into the xenograft or the control group, respectively. Mice in the xenograft group were treated intravenously with PBS, Hm3A4-Rap, or Hm3A4 on d1, d4, and D7, respectively. Disease symptoms  were monitored daily. Mice with disease symptoms were euthanized by cervical dislocation.On day 0 of the study, 1 × 102.9.In vitro data were analyzed using a t-test. In vivo data between the groups was analyzed using the Kaplan–Meier method. P < 0.05 was considered statistically significant.3.We generated the Hm3A4-Rap immunotoxin using recombinant DNA techniques. The potential therapeutic efficacy of the immunotoxin Hm3A4-Rap on target cells was evaluated for its binding capacity, internalization function, and most importantly the ability to kill target cells.3.1All PCR products were verified to be correct in size and sequence (Supplementary file). Sangon sequencing demonstrated no mutations in the Hm3A4-Rap gene and was of the correct size, i.e., 7181bp. We are still in the patent application stage, so we cannot publish all sequences, please understand.3.2Immunofluorescence microscopy for red and green fluorescence was detected in the cytoplasm of CHO/Hm3A4-Rap cells. Images were superimposed, in which, yellow images indicated colocalization indicating that CHO/Hm3A4-Rap cells successfully expressed the recombinant protein. No FITC and TRITC fluorescence was observed in transfected CHO cells .Figure 3.36 KG1a cells . The antigen-antibody binding decreased by 50% when Hm3A4-Rap was 2.682 μg  was Kd = 5.0 × 1010M−1.300 mL of CHO/Hm3A4-Rap cell culture supernatant was harvested and then concentrated by ultrafiltration to obtain 3 mL (100:1) of concentrated supernatant. 15 mL of the purified antibody were collected and concentrated to 5:1 using ultrafiltration to a final concentration of 1 mg/mL. The amount of Hm3A4-Rap secreted in the CHO/Hm3A4-Rap cell culture supernatant was estimated to be 10 μg/mL. Titration results indicated that 0.25 μg of Hm3A4-Rap resulted in 97% positivity in 101a cells . Competi2.682 μg . The proInternalization studies demonstrated that the degree of internalization of Hm3A4-Rap at different time points were 2.1% (0.5 h), 13.3% (1 h), 39.6% (2 h), and 68.4% (4 h), while they were 2.1% (0.5 h), 8.41% (1 h), 27.1% (2 h), and 60.8% (4 h) for Hm3A4. These results indicated that Hm3A4-Rap could be efficiently internalized into cells with prolonged incubation times. The internalization degree was similar to Hm3A4 .Figure 3.4Both Hm3A4-Rap and Hm3A4 were able to effectively kill CD45RA-positive KG1a cells as determined by ADCC and were enhanced with increased ratios of PBMC: KG1a. Hm3A4-Rap  and Hm3A4 . The ADCC of Hm3A4-Rap was comparable to that of the Hm3A4 antibody (p > 0.05) . The CDC3.5We observed a significant reduction in overall survival in the model (PBS treatment) group compared to the Hm3A4-Rap treatment group. Mice in the model group died on the following days: day55, day108, day112, day134, and day136. For mice in the Hm3A4-Rap treated group, one mouse died at day165, and the remaining four survived with no disease symptoms. For mice in the Hm3A4 treated group, one mouse died at day112, and another died at day182, while the remaining three mice survived with no disease symptoms. Both groups were statistically significant compared to the control group (p < 0.05). We extended the observation time to 200 days, during which, all remaining mice survived .Figure 4.in vitro and in vivo studies.LSCs are the root cause of leukemia relapse and drug resistance. The development of antibodies that can recognize LSCs is of great significance for AML treatment. At present, studies performed on LSCs are limited. KG1a cells are myeloid leukemia cell lines, a subset of cells with tumor stem cell-like biological characteristics. They express CD34 (98%) and CD123 (90%), and some do not express CD38, which is consistent with the currently recognized immunophenotypic characteristics of LSCs. Consistent with this, KG1a cells could be used as LSCs for research studies. In our study, we use KG1a cells for 10M−1, indicating that it had good binding capacity and affinity. ADCC of Hm3A4-Rap was comparable to that of the Hm3A4 antibody, and significantly higher compared to IgG. This indicated that the fusion of Rap with the Fc fragment did not affect the binding of the Fc fragment to effector cells, which is an important mechanism for antibody-based targeting therapy [In vivo studies showed that the survival time in mice treated by Hm3A4-Rap was significantly longer than those treated by either Hm3A4 and PBS treated mice. The 150-day and 200-day survival rates of the Hm3A4-Rap group were 100% and 80%, respectively, and those of the Hm3A4 group were 80% and 60%, respectively. The death time of the first dead mouse in the Hm3A4-Rap group was 53 days later than that in the Hm3A4 group. Although there is no statistical difference between Hm3A4-Rap group and Hm3A4 group due to the small number of mice, a clear trend can be found. And finally, the immunotoxin generated by the fusion of Rap and the Hm3A4 antibody increased the molecular weight of Hm3A4 and prolonged its in vivo half-life [Titration and competitive binding studies demonstrated that Hm3A4-Rap could effectively bind to the target antigen and inhibit the binding of the 3A4 antibody to antigen. Ka was found to be 5.0 × 10 therapy . CDC stualf-life . TherefoRegarding MAb-targeting drugs, intracellular transport, or internalization of the conjugated MAb is believed to be one of the major mechanisms responsible for tumor cell killing. Our results demonstrated that Hm3A4-Rap could be internalized into cells rapidly after binding to the target antigen in a time-dependent manner. The result was based on a single cell line KG1a and but if we can find a chance to test more cell lines in the future, the results will be more meaningful.The direct killing effect of Hm3A4-Rap was not as high as expected. In our previous study, we generated Norcantharidin-conjugated 3A4 (3A4-NCTD) using the active ester method. The inhibition percentage of KG1a cells by 3A4-NCTD was 61.10% after 96 h of co-culture  They devRegarding the structure of the immunotoxin, we believe that the 6× His-Tag epitope may interfere with the immune response. For future studies, we may consider making changes to the immunotoxin to that of N`-Rap-ScFv-Fc-C` . Purific5.In vitro and in vivo studies have showed that Hm3A4-Rap has a good antitumor effect. We believe that the immunotoxin developed by us is an effective therapeutic candidate for clinical development.We have successfully generated the fusion protein Hm3A4-Rap using recombinant DNA techniques to target leukemia cells. The fusion protein retains the affinity and the antigen recognition specificity as its parental antibody and has the toxicity of Rap. Furthermore, the immunotoxin can kill target cells through ADCC.


Pseudomonas aeruginosa has two functional DksA paralogs: DksA1 is constitutively expressed and has a zinc-finger motif, while DksA2 is expressed only under zinc starvation conditions and does not contain zinc. DksA1 stimulates the production of virulence factors in vitro and is required for full pathogenicity in vivo. DksA2 can replace these DksA1 functions. Here, the role of dksA paralogs in P. aeruginosa tolerance to H2O2-induced oxidative stress has been investigated. The P. aeruginosa dksA1 dksA2 mutant showed impaired H2O2 tolerance in planktonic and biofilm-growing cultures and increased susceptibility to macrophages-mediated killing compared to the wild type. Complementation with either dksA1 or dksA2 genes restored the wild type phenotypes. The DksA-dependent tolerance to oxidative stress involves, at least in part, the positive transcriptional control of both katA and katE catalase-encoding genes. These data support the hypothesis that DksA1 and DksA2 are eco-paralogs with indistinguishable function but optimal activity under different environmental conditions, and highlight their mutual contribution to P. aeruginosa virulence.In Gram-negative pathogens, the stringent response regulator DksA controls the expression of hundreds of genes, including virulence-related genes. Interestingly,  These molecules are ubiquitous in the environment, and some of them are also generated by aerobically growing bacterial cells. During infection, pathogenic bacteria must cope with immune response cells, which produce particularly high levels of ROS that are either released in the extracellular milieu or used to kill engulfed bacteria1. After phagocytosis of bacteria, professional phagocytic cells, including macrophages, activate the NOX2 NADPH oxidase, a multicomplex enzyme assembled in the phagosomal membrane, which catalyses the reduction of molecular oxygen to superoxide. The latter can directly kill the bacteria within phagosomes and is converted to H2O2 by superoxide dismutases (SODs)3. Interestingly, bacterial SODs contribute to the production of microbicidal H2O2 within macrophages, as recently observed in Pseudomonas aeruginosa4. H2O2 can be further converted to hydroxyl free radical via Fenton reaction or halogenated ROS. This quick ROS production in the phagosome, a process known as oxidative burst, plays a key role in the host innate immune response6. Bacteria must also cope with extracellular ROS. As an example, the oxidases DUOX1 and DUOX2 generate H2O2 on the extracellular side of the apical membrane of the airway epithelium7.Bacteria constantly contend with toxic reactive oxygen species (ROS) such as hydrogen peroxide  for which novel treatment approaches are urgently needed9.P. aeruginosa expresses an array of ROS-scavenging enzymes to counteract endogenous and exogenous ROS, including the four catalases KatA, KatB, KatE and KatN12. The KatA and KatB catalases are crucial for P. aeruginosa adaptation to the high levels of H2O2 encountered in the host14. Expression of katA is constitutive and increases in the presence of H2O2. Conversely, the expression of katB is induced only upon exposure to exogenous H2O215. Up to now, it is unclear if also the KatE  and KatN  catalases are involved in H2O2 detoxification in P. aeruginosa.In addition to a wide arsenal of virulence factors, Salmonella enterica and Haemophilus ducreyi19. DksA also plays a key role in the control of virulence-related genes. In some pathogenic bacteria  dksA mutants are less virulent than the wild type both in vitro and in vivo25.The bacterial stringent response regulator DksA contributes to ROS tolerance in bacterial pathogens such as P. aeruginosa is the only bacterium known to express two functional DksA paralogs, encoded by the dksA1 and dksA2 genes. The DksA1 and DksA2 proteins share a similar overall structure containing: a RNAP-interacting coiled-coil domain, a globular domain and a C-terminal α-helix. However, the globular domain of DksA1 contains a canonical zinc-finger structure, with a four-cysteine motif (4-Cys) for zinc binding, while the DksA2 globular domain has only two cysteine residues (2-Cys), and does not coordinate zinc27. The constitutively expressed DksA1 protein broadly affects the P. aeruginosa transcriptome, and positively controls virulence genes expression24. Conversely, DksA2 is exclusively expressed under conditions of zinc starvation28.Up to now, 24. Only a small number of functionally unrelated genes seems to be exclusively controlled by each one of the two DksA paralogues under our artificial conditions24. Since the zinc-containing 4-Cys motif is required for the proper folding and functionality of P. aeruginosa DksA1, our study supports the previous hypothesis that the structural stability and functionality of DksA1 could be impaired in zinc-poor environments, where the zinc-free DksA2 protein could replace or adjuvate DksA1 function28.By artificially inducing DksA2 expression in Luria–Bertani Broth (LB), our group has recently shown that the DksA2 protein can replace DksA1 in the control of about sixteen hundred genes, including those involved in virulence and biofilm formationP. aeruginosa DksA1 and DksA2 proteins has also been proposed by Crawford et al.19, who showed that a S. enterica ∆dksA mutant complemented with the P. aeruginosa dksA2 gene was less resistant to H2O2 and more susceptible to macrophage-mediated killing than the same strain complemented with the P. aeruginosa dksA1 gene. Although intriguing, the differential role of DksA1 and DksA2 in oxidative stress tolerance has not yet been tested in P. aeruginosa.A difference in the functionality of P. aeruginosa tolerance to H2O2 and macrophages-mediated killing, and to verify to which extent DksA2 can replace DksA1 function. Evidence is here provided that DksA1 is required for H2O2 tolerance in both planktonic and biofilm growing cells, and that this protein protects against macrophages-mediated killing. One of the mechanisms involved in DksA-dependent resistance to oxidative stress implies the positive control of katA and katE genes expression. Finally, in the homologous P. aeruginosa system, DksA2 fully replaced the protective function of DksA1 against oxidative stress, in contrast with previous observations in the heterologous Salmonella system19.The objective of this study has been to investigate the role of DksA1 in P. aeruginosa, the dksA1 gene is constitutively expressed while the dksA2 gene is strongly repressed by Zur in the presence of available zinc28. To achieve comparable expression levels of the two dksA paralogs in a rich laboratory medium, such as LB, we previously generated a set of four P. aeruginosa PAO1 recombinant strains: the wild type strain (PAO1) and the dksA1 dksA2 double mutant (∆dksA1-2), both carrying the pME6032 empty vector, and the ∆dksA1-2 mutant carrying either pDksA1 or pDksA2. The latter are pME6032-derivative plasmids for isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible expression of dksA1 and dksA2, respectively  than in the ∆dksA1-2(pME6032) mutant  and ΔdksA1-2(pDksA2) restored H2O2 tolerance, with no significant difference between the two dksA paralogs.The minimum inhibitory concentration (MIC) of HdksA1 and dksA2 in P. aeruginosa biofilm tolerance to H2O2 was also investigated. To this purpose, the MIC of H2O2 was determined in M9-glu-CAA, a chemically-defined medium suited for P. aeruginosa biofilms studies24. In M9-glu-CAA, the MIC of H2O2 was 0.1 mM for all the tested strains, thus tenfold lower than that determined in LB. Hence, pre-formed P. aeruginosa biofilms were challenged with supra-MIC concentrations of H2O2, ranging from 50 to 200X MIC, and compared to the respective untreated controls.The role of 2O2 treatment, higher levels of biofilm biomass were detected by crystal violet (CV) staining in the ∆dksA1-2(pME6032) mutant compared to PAO1(pME6032) and the ΔdksA1-2(pDksA1) and ΔdksA1-2(pDksA2) complemented strains  assay showed that the metabolic activity of PAO1(pME6032) and ∆dksA1-2(pME6032) biofilms were comparable in the absence of H2O2  mutant there could be an increased production of extracellular biofilm matrix components compared to PAO1(pME6032). After challenging with H2O2, a dose–response biofilm disrupting effect was detected for all the tested strains by using both CV and FDA detection methods, though ∆dksA1-2(pME6032) biofilms were significantly more reduced by the treatment compared to the wild type and complemented strains . Flow cytofluorimetry analysis showed that the percentage of fluorescent macrophages was similar in both infections, revealing that the ∆dksA1-2 double mutation has no impact on P. aeruginosa internalization by macrophages  than for the ∆dksA1-2(pME6032) mutant  than in ∆dksA1-2(pME6032), both grown in LB  disclosed a 44% reduction of catalase activity compared to PAO1(pME6032)  and ΔdksA1-2(pDksA2) fully restored wild type catalase activity and ROS levels, providing evidence that also in this case DksA2 can replace DksA1.The results presented above show that both  LB Fig. a. Hence,dksA gene products on the expression of different catalase-encoding genes, relative mRNA levels of katA, katB, katE and katN were investigated by means of Real Time PCR analyses in P. aeruginosa cultures grown in LB containing 0.1 mM IPTG, treated or not with 1 mM H2O2 (1X MIC) for 20 min, to measure the expression of the H2O2-inducible genes. Preliminary experiments showed that the expression of dksA1 and dksA2 was not affected in PAO1 in response to 1 mM H2O2  relative to PAO1(pME6032) in the presence of H2O2, respectively  relative to PAO1(pME6032), irrespective of H2O2 presence  grown in LB, the above results show that at least DksA1 is required for the HdksA1-2(pDksA1) and ΔdksA1-2(pDksA2) strains restored wild type mRNA levels for all the tested genes ppGpp and DksA are required for full stringent response, even if the two factors can also work independently35. While previous studies showed that (p)ppGpp positively affects P. aeruginosa tolerance to H2O237, the contribution of DksA1 and DksA2 to this process remained unknown so far.The stringent response controls bacterial adaption to nutrient starvation and other stressful conditions. In Gram-negative bacteria, (p)ppGpp binds the ß’ subunit of RNAP at the interface with the ω subunit and in a second site, sandwiched between the ß′ subunit and the DksA protein, which is strictly required for this interaction24, dksA2 expression is strongly repressed by zinc in the P. aeruginosa PAO1 wild type strain. Hence, the comparison of the wild type and dksA1 dksA2 double mutant phenotypes only reveals the effect of DksA1 depletion. However, complementation of the double mutant with either DksA1 or DksA2 expressed by the corresponding gene under the control of an IPTG-inducible promoter allows comparing the activity of the two DksA paralogs. Overall, the dksA1 and dksA2 expression levels obtained by this approach are adequate to restore wild type levels of all the tested phenotypes.In the culture media used in this and our previous studydksA1 dksA2 double deletion does not affect P. aeruginosa long-term exposure to H2O2 in both LB and M9-glu-CAA , DksA1 increases P. aeruginosa tolerance to transient H2O2 exposure, both in planktonic and in biofilm growing cultures. In line with these results, the deletion of both dksA1 and dksA2 undermines P. aeruginosa survival within macrophages. The dksA1 dksA2 double deletion leads to an increase in P. aeruginosa biofilm biomass, as already shown in our previous study by using different biofilm models 24. In the same study, it was shown that c-di-GMP levels were lower in the ΔdksA1-2 mutant compared to the wild type, indicating that DksA1 negatively controls biofilm formation through a c-di-GMP-independent pathway. Furthermore, the RNA-Seq analysis showed that some of the known P. aeruginosa biofilm-related genes were up-regulated and others were down-regulated in the ΔdksA1-2 mutant compared to the wild type strain24. This study showed that the biofilm cells of the ΔdksA1-2 mutant are less viable after H2O2 exposure relative to wild type biofilm cells, despite the increased biofilm biomass. This suggests a defect in the ΔdksA1-2 biofilm matrix composition and development. In the future, it could be interesting to further investigate this issue.While the 2O2, the ΔdksA1-2 mutant showed an increase in the endogenous ROS content and a decrease in the overall catalase activity, compared to wild type PAO1. This could be due to the reduced expression of katE in the ΔdksA1-2 strain, since the expression of the other P. aeruginosa catalase genes was not affected by dksA1 dksA2 double deletion in the absence of oxidative stress. However, since DksA1 controls more than one thousand genes, including primary metabolism pathways24, the ROS increase measured in the ΔdksA1-2 strain could also be due to a generalized metabolic effect. Moreover, in the presence of exogenous H2O2, katA and katE genes were downregulated while katB was up-regulated in the double mutant compared to the wild type PAO1.In the absence of exogenous HkatA and katE genes, combined with an altered metabolism leading to increased levels of endogenous ROS, could overcome the effects of the increased katB expression, ultimately reducing the tolerance of the ΔdksA1-2 mutant to exogenous H2O2 and macrophages-mediated killing, relative to wild type PAO1. In this context, it is to note that the role of the KatE catalase in P. aeruginosa H2O2 tolerance has not been clarified yet. Indeed, KatE seems not to be involved in H2O2 detoxification in the PA14 strain11, while a protective role of KatE against oxidative stress only at elevated temperatures was described in the PAO6049 strain38.Overall, it is plausible that the defective expression of P. aeruginosa mutant lacking the relA and spoT genes, required for (p)ppGpp synthesis, showed several phenotypes similar to those caused by the dksA1 dksA2 double deletion, i.e., increased susceptibility to H2O2, impaired overall catalase activity and high levels of endogenous ROS with respect to the parental strain39. However, different from what we observed for DksA1, (p)ppGpp positively controls the expression of both katA and katB genes. Interestingly, this (p)ppGpp-dependent regulation is independent of exogenous H2O2 for katA and H2O2-dependent for katB37.Interestingly, a P. aeruginosa tolerance to endogenous and exogenous oxidative stress, even if they differently regulate the expression of H2O2-scavenger enzymes. Further studies should be carried out to unravel the regulatory mechanisms underlying the different effects of DksA1 and (p)ppGpp on katA, katB and katE genes expression, as well as the impact of KatE-mediated H2O2 detoxification in P. aeruginosa PAO1.By combining our data with previous findings, it can be argued that both (p)ppGpp and DksA1 play a major role in S. enterica and H. ducreyi. Each one of these pathogens expresses only one DksA protein, containing a zinc finger domain, as it is for DksA118. Hence the possibility that DksA2 could replace DksA1 deserves special attention, also considering that a previous study showed that a S. enterica ∆dksA mutant expressing the P. aeruginosa dksA2 gene had an impaired tolerance to H2O2 in vitro, reduced survival in macrophages, and reduced virulence in mice relative to the S. enterica ∆dksA mutant expressing the P. aeruginosa dksA1 gene. The different behaviour of DksA1 and DksA2 in the heterologous host Salmonella was supported by experiments carried out with the purified proteins, showing that DksA1 was more resistant to oxidation than DksA2, likely due to the presence of zinc in the globular domain19. Different from what observed in S. enterica, we showed by multiple experimental strategies that DksA2 replaces all DksA1 functions related to P. aeruginosa H2O2 tolerance when expressed in its isogenic background, at least under our experimental conditions.To our knowledge, the importance of DksA in oxidative stress tolerance has so far been documented only for dksA2 overexpression may mask a higher sensitivity of DksA2 to ROS than DksA1 cannot be rule out, and this issue should be further studied under conditions of zinc starvation, mimicking the environmental conditions where dksA2 is naturally expressed. In the wild type genetic background, under zinc-limiting conditions, both dksA1 and dksA2 genes are expressed from their natural promoters28 and they may have a different impact on P. aeruginosa oxidative stress response. However, while dksA2 expression levels are related to the extent of zinc limitation, DksA1 activity might be impaired under conditions of extreme zinc deficiency, due to incorrect folding of the Zn-finger domain28.Certainly, the possibility that dksA2 is expressed in the sputum of cystic fibrosis patients colonized by P. aeruginosa41, supporting a role for DksA2 at least in this kind of infection. While further studies should be carried out to better define the relative importance of DksA1 and DksA2 in the protection from H2O2 exposure under zinc starvation conditions, this study provides the evidence that DksA2 has the intrinsic ability to replace the dksA1 gene product under oxidative stress conditions.Zinc depletion is recognised as an innate immunity mechanism faced by pathogens during the infection. Interestingly, 24 strengthening the hypothesis that DksA1 and DksA2 are eco-paralogs as defined by Sanchez-Perez et al.42, i.e., paralogs with the same overall function, having optimal activity under different environmental conditions.Overall, the results of this study are in line with our previous observation that DksA2 can replace DksA1 function in controlling the expression of almost all the DksA1-regulated genes and DksA1-dependent virulence phenotypesP. aeruginosa strains were routinely grown at 37 °C in shaking conditions in LB43, or LB supplemented with 1.5% (w/v) agar. When required, the media were supplemented with 0.1 mM IPTG or 100 µg/mL tetracycline (Tc). M9 medium 44 supplemented with 0.39% (w/v) glucose and 0.5% (w/v) casamino acids (CAA) as carbon sources (M9-glu-CAA)24 was used for specific assays. H2O2  was added to the media at the concentrations indicated in the text.Bacterial strains used in this study are listed in Supplementary Table 2O2 was evaluated as previously detailed45 with the standard microdilution method, according to the Clinical and Laboratory Standards Institute guidelines46. Cultures of P. aeruginosa were incubated at 37 °C with shaking in LB containing 100 μg/mL Tc or in M9-glu-CAA containing 50 μg/mL Tc, both supplemented with 0.1 mM IPTG. After overnight growth, cultures were diluted to an optical density at 600 nm wavelength (OD600) of ≈ 0.0005 (ca. 5 × 105 CFU/mL) in 100 μL of LB or M9-glu-CAA, both supplemented with 0.1 mM IPTG, in the presence of increasing concentrations of H2O2. The MIC values were evaluated after 24 h of static incubation at 37 °C.The MIC of H2O2 killing assays were performed as previously described36, with minor modifications. Late exponential phase cultures (OD600 ≈ 2.5) were normalized to an OD600 of 0.5 in LB supplemented with 0.1 mM IPTG and 50 mM H2O2 before incubation at 37 °C in shaking conditions. At the same time, untreated cultures were incubated at 37 °C as a control. After 30 min, 0.2% (w/v) sodium thiosulfate was added to neutralize residual H2O2. Colony forming units (CFU) were determined by the standard microdilution technique on LB agar plates. Survival was reported as percentage of CFU counted in the treated sample relative to the CFU counted in the untreated sample.H49, with slight changes. Bacterial cells were grown in M9-glu-CAA with 50 µg/mL Tc and 0.1 mM IPTG for 8 h in shaking conditions and subsequently diluted to OD600 ≈ 0.015 in fresh M9-glu-CAA medium supplemented with 0.1 mM IPTG. Aliquots of 100 µL were transferred to sterile 96-well polystyrene microtiter plates and incubated at 30 °C for 15 h in static conditions. After the removal of the planktonic phase, biofilms were refreshed with 100 μL of fresh medium supplemented with 5, 10 or 20 mM H2O2 (treated biofilms) or without the addition of H2O2 (untreated biofilms). Microtiter plates were incubated at 30 °C for 6 h. After removing the liquid phase, biofilm mass was quantified by CV binding assay, while biofilm viability was determined by FDA assay. Briefly, CV binding assay was performed staining the attached cells with 1% (w/v) CV for 15 min. Hence, all wells were washed four times with distilled water and air dried. Finally, the biofilm-bound dye was solubilized with 200 µL of ethanol for 10 min and measured as A595 in an automated luminometer-spectrometer plate reader (Tecan Spark 10 M). To perform the FDA assay, the FDA stock solution  was diluted 1:100 in 100 mM 3-(N-morpholino)-propanesulfonic acid  and 200 μL of FDA working solution were dispensed in all wells. The microtiter was incubated in the dark at 37 °C in static condition. Fluorescence was measured at 485 nm excitation and 535 nm emission wavelengths after 1 h through an automated luminometer-spectrometer plate reader (Tecan Spark 10 M).The crystal violet (CV) binding assay and the fluorescein diacetate (FDA) assay were performed in microtiter plates as already detailed in previous studies2. For the infection assay, the day before infection 5 × 104 cells/well were seeded in 48-well plates and incubated over-night in antibiotic-free medium. Each well was infected with different PAO1 strains at a multiplicity of infection (MOI) of 10. After 30 min of incubation at 37 °C, extracellular bacteria were killed by gentamycin (600 µg/mL) treatment during 15 min of incubation. For counting intracellular bacteria, infected RAW264.7 cells were washed with phosphate-buffered saline (PBS), lysed with 1% (v/v) Triton X-100, and dilutions plated on Pseudomonas isolation agar (PIA).Murine macrophages RAW264.7 (ATCC TIB71) were grown in Dulbecco’s modified Eagle’s medium  high glucose supplemented with 10% (v/v) fetal bovine serum and 1% (w/v) glutamine  at 37 °C in 5% COdksA1-2 with the pUCP30T-GFPmut3 plasmid . Phagocytosis was evaluated by the fraction of GFP positive cells in the bulk populations. Data were analysed using the CellQuest software and images processed using FlowJo.For the phagocytosis assay, macrophages were infected as described above, using GFP expressing bacteria obtained by transformation of PAO1 and ∆51, with minor modifications. P. aeruginosa strains were grown in LB with 0.1 mM IPTG to the late exponential phase (OD600 of ≈ 2.5). Then, cells were collected by centrifugation, washed with PBS, and resuspended in PBS at an OD600 of about 1.25. Bacteria were incubated with 10 µM 2′,7′-dichlorodihydrofluorescein diacetate  for 20 min at 37 °C in the dark. Cells treated with an equal volume of DMSO were used as a negative control. Aliquots (200 µL) of each bacterial suspension were dispensed in a 96-well black microtiter plate. Fluorescence was measured at 485 nm excitation and 535 nm emission wavelengths with an automated luminometer-spectrometer plate reader (Tecan Spark 10 M) and normalized to the OD600 of each sample.The intracellular ROS levels were measured as previously described52, with minor modifications. Bacteria were grown in LB with 0.1 mM IPTG to the late exponential phase (OD600 of ≈ 2.5). Hence, cells were collected by centrifugation, resuspended in 50 mM potassium-phosphate buffer , lysed by sonication and subsequently centrifugated to collect the supernatants. Total proteins from the soluble fraction were quantified by the Bradford assay53, with bovine serum albumin as the standard. Aliquots containing 10 µg of proteins were added to 50 mM PPB, and catalase activity was monitored by following the decomposition of 20 mM H2O2 in 50 mM PPB at 240 nm (OD240) for 1 min (readings every 10 s) by using UV-transparent disposable cuvettes. One unit of catalase activity corresponded to 1 μmol of H2O2 hydrolysed per min by 1 mg of total proteins at 25 °C.Catalase activity was measured as described previouslyP. aeruginosa strains were grown at 37 °C with shaking at 200 rpm in 10 mL of LB supplemented with 0.1 mM IPTG until reaching an OD600 of ≈ 2.5. Exponential phase cells were incubated with or without 1 mM H2O2 for 20 min in shaking conditions and 1 mL of each culture was mixed with 2 mL of RNA Protect Bacteria Reagent (Qiagen) for RNA extraction. Total RNA was extracted as previously described54. Briefly, RNA isolation was performed using RNeasy Mini Kit (Qiagen), including the on-column DNase I digestion step, followed by 1 h treatment at 37 °C with TURBO DNase  and SUPERase-In , and subsequent purification with the RNeasy Column Purification Kit (Qiagen). After confirmation of the lack of contaminating chromosomal DNA by PCR with the primer pair FWPpqsL and RVPpqsL  were calculated from two independent experiments each performed on three technical replicates.p value of < 0.05 was considered statistically significant.Statistical analysis was performed with the software GraphPad Prism 6.01, using one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison test. Statistical analysis of macrophages infections used two-way ANOVA. A Supplementary Information.


Cholangiocarcinoma (CCA) is difficult to cure due to its ineffective treatment and advanced stage diagnosis. Thoroughly mechanistic understandings of CCA pathogenesis crucially help improving the treatment success rates. Using three-dimensional (3D) cell culture platform offers several advantages over a traditional two-dimensional (2D) culture as it resembles more closely to Here, we aimed to establish the 3D CCA spheroids with lowly (KKU-100) and highly (KKU-213A) metastatic potentials to investigate the CCA migratory process and its EMT-associated galectin-3 in the 3D setting. Firstly, the growth of lowly metastatic KKU-100 cells was slower than highly metastatic KKU-213A cells in both 2D and 3D systems. Hollow formation was observed exclusively inside the KKU-213A spheroids, not in KKU-100. Additionally, the migration activity of KKU-213A cells was higher than that of KKU-100 cells in both 2D and 3D systems. Besides, altered expression of galectin-3 were observed across all CCA culture conditions with substantial relocalization from inside the 2D cells to the border of spheroids in the 3D system. Notably, the CCA migration was inversely proportional to the galectin-3 expression in the 3D culture, but not in the 2D setting. This suggests the contribution of culture platforms to the alternation of the CCA cell migration process. in vivo.Thus, our data revealed that 3D culture of CCA cells was phenotypically distinct from 2D culture and pointed to the superiority of using the 3D culture model for examining the CCA cellular mechanisms, providing knowledges that are better correlated with CCA phenotypes  The global incidence of CCA is on the rising trend with the highest rate in Southeast Asia, especially in the northeastern part of Thailand  , 6. Asymin vitro studies using CCA cell lines have been implemented as tools to discern the cellular and molecular alteration of the disease without the involvement of patient participants or animal studies  culture has therefore been developed to overcome these problems and bridges the gap between the 2D culture and the animal model. The 3D culture system has been established to be more resembling to in vivo tumor properties, including histomorphology, functions, and microenvironments in terms of cellular heterogeneity, nutrients and oxygen gradients, cell-cell interaction, matrix deposition, and gene expression  basement membrane matrix and cell recovery solution were acquired from Corning . Ribonuclease A and phenylmethylsulphonyl fluoride (PMSF) protease inhibitor were purchased from Sigma Chemicals . Bradford solution was purchased from Bio-Rad . Hoechst 33342, TRITC-conjugated phalloidin, primary antibodies against galectin-3, ZEB-1, N-cadherin, E-cadherin, β-catenin, vimentin, horseradish peroxidase (HRP)-conjugated secondary antibodies against anti-rabbit and anti-mouse antibodies, Alexa Fluor® 647-conjugated secondary antibody against anti-rabbit antibodies and SignalFire™ ECL Reagent were purchased from Cell Signaling Technologies . The primary antibody against β-actin was purchased from Sigma-Aldrich. Triton X-100 was obtained from Bio-Rad. Recombinant human galectin-3  was purchased from Merck .Ham’s F12 nutrient mix, Roswell Park Memorial Institute (RPMI) 1640, fetal bovine serum (FBS), penicillin/streptomycin, and Lab-Tek II 8-well chamber slide were purchased from Thermo Fisher Scientific . Corning2.22 incubator.Human poorly differentiated KKU-100 and mixed papillary and non-papillary KKU-213A CCA cell lines, derived from Thai CCA patients, respectively, were employed to represent lowly and highly invasive CCA models in this study . Both ce2.33 CCA cells were seeded into Matrigel-coated wells and were further incubated for 15 min to allow cell attachment. Ham’s F12 nutrient mix supplemented with 10% FBS, 2% of 5 mg/ml Matrigel, 100 U/ml penicillin and 100 μg/ml streptomycin were added at a total volume of 120 µl each well. The 3D spheroids were formed and incubated 37°C in a 5% CO2 incubator and the culture medium were changed every two days.To produce tumor spheroids of CCA cell lines, 35 µl of GFR Matrigel at a concentration of 5,000 µg/ml was filled into a 96-well plate and incubated at 37°C for 2 h. Then, 5 x 102.43 cells were seeded in a 96-well plate and incubated for 24 h at 37°C in 5% CO2 incubator. The cells were collected by trypsinization. The number of cells was counted by a dye exclusion method using 0.4% trypan blue. For 3D culture proliferation assay, the diameters of tumor spheroids were observed under a phase-contrast inverted microscope  with 40x magnification for 5 fields per well and were measured using an ImageJ program . The cellular morphology was observed under a phase-contrast microscope using 100x magnification.For monolayer culture, ~5 x 102.55 CCA cells were seeded in a 24-well plate and incubated for 24 h. Then, culture medium was discarded, and the cells were washed with phosphate buffer saline (PBS). Scratches were created, and scattered cells were removed. Cells were then supplemented with Ham’s F12 nutrient mix containing 0.1% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. Wound images were captured under a phase-contrast microscope at 0 and 12 h to reduce the contribution of cell proliferation to fill the gap. The migration areas were determined using ImageJ analysis software version 1.8.0. The relative migration was calculated as the ratio between the difference in migration wound area at time 12 h relative to 0 h and migration wound area at time 0 h.Wound healing assay was performed by creating a scratch on the monolayer cells as our previous study . Migrati2.64) CCA cells were then seeded on Matrigel-pre-coated wells and grew for 4 days. They were then collected and pipetted into Matrigel-coated wells. After that, the CCA spheroids were supplemented with 180 µl of Ham’s F12 nutrient mix, 0.1% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Spheroid images were taken at 0 and 12 h to prevent the effect of cell proliferation under a phase-contrast microscope. Next, cell migration areas were determined using ImageJ analysis software. The relative cell migration was subsequently calculated as the difference in the migrating area between time at 12h and 0 h.The 3D migration assay for CCA spheroids was modified from a previously published protocol . Briefly2.7-ΔΔCq method. The gene primer sequences of galectin-3 F: 5’ GCCAACGAGCGGAAAATGG 3´, R: 5´ CAGGCCATCCTTGAGGGTTT 3´ and actin F: 5´ GCACAGACCTCGCCTT 3´, R: 5´ CTTGCACATGCCGAG 3´ were used.Total RNA was extracted from CCA cells in 2D and 3D culture systems using TRIzol reagent . Then, 1 µg of the extracted RNA was used as a template to synthesize first strand cDNA using iScript™ cDNA synthesis kit . Then, the relative gene expression of galectin-3 was evaluated using quantitative real-time polymerase chain reaction using specific galectin-3 primers and iTaq Universal SYBR Green Supermix . All reactions were performed in triplicate using the CFX Connect Real-Time system  with the following thermocycling conditions: initial denaturation at 95°C for 5 min, extension at 95°C for 30 s, 60°C for 30 s, 70°C for 60 s (40 cycles), and final extension at 70°C for 1 min. Actin gene expression was used as an internal housekeeping gene control to normalize gene expression between samples. The relative fold change was calculated using the 22.8g for 10 min. The supernatant as protein lysate was collected for further analysis. For tumor spheroids, spheroids were collected using cell recovery solution and transferred into a 1.5 ml-microcentrifuge tube. The tumor spheroids were washed three times with cell recovery solution to eliminate ECM. The cell pellet was then filled with 50 µl of protein lysis cocktail and sonicated for 10 min on ice. The protein concentrations were determined using the Bradford assay. Then, 20 µg of protein sample was subjected to 10% SDS-PAGE at 100 V. The separated proteins were transferred to a nitrocellulose membrane using semi-dry electroblotting . The membrane was washed 3 times with tris buffer saline containing 0.1% tween and then incubated with 5% skim milk for 1 h to block non-specific binding. Primary antibodies for ZEB1 , E-cadherin , N-cadherin , β-catenin (1:500), vimentin (1:500), galectin-3 (1:500) and β-actin  were probed on the membranes at 4°C overnight. The membranes were then washed 3 times and incubated with HRP-conjugated secondary antibodies (1:500) for 1.5 h. The immunoreactivity of protein bands was determined using an enhanced chemiluminescent (ECL) substrate. Protein intensities were evaluated using ImageJ analysis software.Total proteins were retrieved using RIPA lysis buffer and a PMSF protein inhibitor. One hundred microliters of lysis cocktail were pipetted onto monolayer cells, which were harvested using a cell scraper. The supernatant was transferred into a microcentrifuge tube and was centrifuged at 4°C 15,000 x 2.9g for 5 min. A tissue clearing protocol was adopted to improve image quality  and incubated for 48 h. Then, cells were fixed using 4% paraformaldehyde for 15 min and washed with PBS for 5 min three times. The slide was blocked with 5% BSA for 30 min and then washed with PBS. Galectin-3 antibody (1:500) was added to the cells with incubation at 4°C overnight. The supernatant was discarded, and the cells were washed PBS for 5 min three times. Then, Alexa 647-conjugated secondary antibody was added to the cells with 1 h incubation in the dark. Hoechst 33342 (5 µg/µl) was added to the cells for 10 min in the dark. Cells were then washed with PBS for 5 min thrice and observed using a confocal laser scanning microscope . For 3D culture, tumor spheroids were formed using a 3D Matrigel overlay culture for 6 days. Tumor spheroids were then retrieved with ice-cold PBS and centrifugation at 3,500 x  quality . Briefly2.10The galectin-3 shRNA plasmid (sc-155994-SH) containing a pool of three to five lentiviral vector plasmids each encoding galectin-3-specific 19-25 nucleotides (plus hairpin) shRNAs, and scramble shRNA plasmid-A (sc-108060) were purchased from Santa Cruz Biotechnology . The knockdown experiment was slightly modified from our previous study . Briefly2.11To investigate the effect of galectin-3 in CCA cell mobility in 2D culture, galectin-3-knockdowned CCA cells were pre-plated in 24-well plate and wound scratches were generated, followed by an incubation with 1.0 or 5.0 µg/mL rGal-3 containing medium (Ham’s F12 nutrient mix containing 0.1% FBS and 100U/ml penicillin/100 µg/ml streptomycin) for 6 or 12 h prior to experiment. For the 3D culture experiment, galectin-3-knockdowned CCA spheroids were pre-grown on Matrigel-coated wells for 4 days as previously described. Then, the galectin-3-knockdowned CCA spheroids were transferred to newly Matrigel-coated wells and incubated with 1.0 or 5.0 µg/mL rGal-3 containing medium (Ham’s F12 nutrient mix or RPMI 1640 containing 0.1% FBS and 100U/ml penicillin/100 µg/ml streptomycin) for 6 or 12 h prior to experiment. The culture medium containing 1% PBS was used as a control for the rescue experiments.2.12To evaluate the correlation between galectin-3 expression and survival in CCA patients, publicly available web-based tools for analyzing RNA sequencing data (GEPIA), the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases were used . The RNA2.13p < 0.05.All experiments were performed at least in three biological replicates. All data were evaluated using two-tailed unpaired student’s T-test and presented as the mean ± standard error of the mean. The GraphPad Prism version 6.0 software was used for statistical analysis. Significant value cutoffs were set at 33.1To determine the growth rate of both CCA cell lines in 2D and 3D culture systems, the numbers of cells and the diameters of CCA tumor spheroids were determined, respectively. For 2D monolayers, the numbers of both KKU-100 and KKU-213A cells gradually increased from 0 to 48 h. Then, a drastic growth of KKU-213A cells was observed from 72 h onwards, whereas the growth pattern of KKU-100 cells was much slower. The doubling time of KKU-100 and KKU-213A cells was 168 h and 54.6 h, respectively, confirming that highly metastatic KKU-213A exhibited a higher growth rate than lowly metastatic KKU-100 , and mesenchymal markers  were investigated at a protein level in both CCA monolayers and spheroids B. For 23.4p < 0.01)  rescue assays were employed to investigate the correlation between galectin-3 and migration ability in CCA cells. KKU-213A was chosen as a CCA model due to the most distinguishable galectin-3 expression and migration ability. Gal-3-shRNA plasmids were transfected into the KKU-213A cells and showed a diminution in galectin-3 in both transcriptional and translational levels (3.5in vivo condition, provided knowledge on the probable negative association between galectin-3 and cell migration, leading to clinically lower survival outcome.As previously demonstrated, the expression and localization of galectin-3 in 3D CCA spheroids were explicitly deviated from that in 2D CCA monolayers and it was negatively correlated with the CCA migration ability. According to the high metastatic phenotype, poor prognosis and low overall survival were witnessed in a variety of cancers , 32. We 4in vitro system in CCA research could greatly fulfil the fundamental knowledge on cellular mechanisms of CCA (in vivo (in vitro 3D culture in which the cells are surrounded by ECM proteins, could potentially recapitulate in vivo-like environment and render in vivo characteristics, bridging and overcoming the experimental gap (CCA is a cancer arising from the epithelial cells of biliary trees. The incidence of CCA in Thailand is extremely high, especially in the Northeast part of Thailand , 3. Treas of CCA , 41, 42.(in vivo . Recentlntal gap . Althougin vitro, which are more physiologically relevant to tumor tissues than the traditional 2D culture. While the cells in 2D culture are uniformly enriched with both oxygen and nutrient, tumor spheroids receive differential amounts of oxygen and nutrients, of which cells inside the core are exposed to less amount than the cells at the border, resulting in the necrotic core (3D culture produces cell aggregates or tumor spheroids tic core –45. At atic core . The grotic core . The strtic core , 49. Thetic core , 50, 51.tic core .Our data showed that the KKU-213A compared to KKU-100 cells exhibited higher migration activity in both 2D and 3D settings. Our scratch wound healing results in 2D culture were comparable to the previous data using the Transwell migration assay . AdditioThe migration of tumor cells is regulated by various pathways. The central pathway of migration is EMT, which is the process that allows non-motile epithelial cells to become motile mesenchymal cells. EMT is contributed by many proteins and transcription factors. To migrate, E-cadherin expression, which is an epithelial marker, has been reported to be reduced to suppress epithelial phenotypes . Then, βvia siRNA in 2D highly metastatic KKU-213B cells, formally recognized as KKU-M214, led to the promotion of cell migration (in vivo conditions.The verification of the association between galectin-3 and CCA cell migration was performed in KKU-213A cells, due to its most distinguishable galectin-3 expression and migratory activity. The galectin-3 gene knockdown showed contradictory observations between 2D and 3D cell migration which was already discussed in the abovementioned paragraph together with galectin-3 localization and intensity. In addition, other cancer studies also reported contrast cell mobility between 2D and 3D cultures , 60, 61.igration . These cigration . Furtherigration . Also a igration . Hence, 5in vivo conditions in investigating the CCA pathogenesis and carcinogenesis. It could potentially be developed as a CCA drug screening platform that offers a stronger correlation and more accurate interpretation to in vivo conditions.In summary, we established and characterized the 3D lowly- and highly-metastatic CCA spheroids. The 3D CCA spheroids demonstrated distinct patterns of growth and migration, compared to the 2D CCA monolayers. We further revealed differential expression and localization of galectin-3 and other EMT proteins in different culture settings. Galectin-3 uniquely accumulated at the outer surface of the 3D CCA spheroids, potentially facilitating the cell attachment and cellular binding characteristics. The expression of galectin-3 produced by the 3D CCA spheroids displayed a negative correlation with cell migration, which coincided well with previous observations in the clinical data. Altogether, the disparity in the culture systems described here illustrates the advantage of employing a 3D culture, which more resembles to The original contributions presented in the study are included in the article/SS and TJ designed and conceived the study. Material preparation, data collection and analyses were performed by SS, PK, SK. Analyses of the data were performed by SS, PK and TJ. The first draft of the manuscript was written by SS and PK and all authors commented on previous versions of the manuscript. All authors contributed to the article and approved the submitted version.


Myocardial ischemia-reperfusion injury in diabetic patients leads to an increased incidence of complications and mortality. Secreted frizzled-related protein 4 (SFRP4) plays a critical role in diabetic myocardial ischemia-reperfusion. This paper aims to uncover the underlying mechanisms of SFRP4 in hypoxia/reoxygenation (H/R) injury of diabetic myocardial cells. An in vitro ischemia/reperfusion (I/R) injury model was established using high glucose-induced H9c2 cardiomyocytes. Expression of SFRP4 was detected by real-time reverse transcriptase-polymerase chain reaction and Western blotting. After transfection of SFRP4, the binding of SFRP4 to protein tyrosine phosphatase nonreceptor type 12 (PTPN12) was predicted by database and verified by co-immunoprecipitation assay. P13 K/AKT protein levels were examined by Western blotting. PTPN12 levels were tested by RT-qPCR and Western blotting, cell viability by Cell Counting Kit-8, lactose dehydrogenase kit, terminal dUTP nick-end labeling assay, and cell inflammation and oxidative stress by Western blotting and enzyme linked immunosorbent assay. After overexpression of PTPN12, the experiments for cell viability, inflammation and oxidative stress were repeated once more. SFRP4 expression was upregulated in a high-glucose-stimulated H/R cardiomyocyte model. The interference of SFRP4 promoted cell viability, inhibited the inflammatory and oxidative stress response of H/R cardiomyocytes induced by high glucose. SFRP4 interacted with PTPN12 and inhibited the PI3K/AKT signaling pathway. PTPN12 overexpression reversed the inhibitory effect of sh-SFRP4 on H/R cardiomyocyte damage induced by high glucose. Downregulation of SFRP4 inhibited H/R cell damage in diabetic cardiomyocytes by binding to PTPN12.  The incidence of diabetes is increasing year by year. Cardiovascular diseases, mainly ischemic heart disease, are the leading cause of morbidity and mortality in diabetics . Diabetihttps://mint.bio.uniroma2.it/) and the BioGRID database (https://thebiogrid.org/) revealed its ability to bind to protein tyrosine phosphatase nonreceptor type 12 (PTPN12).Secreted frizzled-related protein 4 (SFRP4) is a protein coding gene belonging to the SFRP family. It binds extracellularly to Wnt ligands or Frizzled receptors, thereby regulating the Wnt cascade . There ihttp://gepia.cancer-pku.cn/), the expressions of SFRP4 and PTPN12 were found to be positively correlated in the heart.PTPN12 is a newly identified molecule involved in tumorigenesis and development . ExtensiTherefore, in this study, we aimed to identify the role and mechanism of SFRP4 in diabetic cardiomyopathy. We made the conjecture that downregulation of SFRP4 inhibited diabetic cardiomyocyte H/R injury through activation of the PI3K/AKT signaling pathway by PTPN12.2.The rat embryonic ventricular cardiomyocytes H9c2 cell line was obtained from Sigma-Aldrich  and kept in Dulbecco’s modified Eagle’s medium (DMEM)  containing 10% fetal bovine serum (FBS) , 100 U/mL penicillin and 100 lg/mL streptomycin at 37°C in 5% CO2, 5% CO2 and 1% O2 at 37°C for 12 h to simulate anoxia. Finally, cells were exposed in a normoxic chamber for 12 h reoxygenation [H9c2 cells were placed in six-well plates up to 50%-60% confluence. Then, these cells were cultured in a serum-free medium overnight before treatment and stimulated with high glucose at a concentration of 30 mmol/L for 24 h. Afterward, cells were subjected to an incubator with a mixture of 95% Ngenation .5/well and grown to 70–80% confluence. The above shRNAs were transfected into H9c2 cells using 2.5 μl/ml Lipofectamine 2000 reagent  according to the manufacturer’s instructions.Short hairpin RNAs (shRNAs) against SFRP4 namely sh-SFRP4-1 (5′-GGAAGTTAGTGATATATAA-3′) and sh-SFRP4-2 (5′-GCAGAAGAATGATACATAA-3′), sh-NC (5′-GGTACGCAATAGGAGTGTGTG-3′), as well as PTPN12 overexpression vector and overexpression negative control were all purchased from Gene Pharma . Then, H9c2 cells were seeded into 6-well plates at a density of 4 × 10−ΔΔCT method [The extracted total RNA of H9c2 was carried out following the standard procedures of vendor’s instructions by TRIzol® reagent . A NanoDrop 3000 spectrophotometer  was used to confirm the sufficient quality of total RNA according to the manufacturer’s protocol. PrimeScript RT Master Mix  was used to reverse transcribe 2 μg of RNA into cDNA. Quantitative real-time PCR analysis was implemented to determine the mRNA expression using SYBR Premix Ex Taq kit  in line with the instructions of manufacturer. The amplification conditions for this reaction were as follows: 95°C for 10 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 60 seconds. The primer sequences for PCR are presented as below: SFRP4, 5′-CACCTATCCCTCGAACGCAA-3′ (forward) and 5′-TCCTGTAAGGGTGGCTTCAAC-3′ (reverse); PTPN12, 5′-CTGCCATTTGATCACAGCCG-3′ (forward) and 5′-TGCTCTCGGCCCATATACAC-3′ (reverse); 5′-CACCTATCCCTCGAACGCAA-3′ (forward) and 5′-TCCTGTAAGGGTGGCTTCAAC −3′ (reverse); GAPDH, 5′-GTCGTGGAGTCTACTGGCGTCTTCA-3′ (forward) and 5′-TCGTGGTTCACACCCATCACAAACA-3′ (reverse). The relative expression of genes was normalized to the expression of GAPDH  be means of 2T method . The expThe proteins extraction was prepared from H9c2 cells . Then, pThe 96-well plates were used to load H9c2 cells with a density of 1 × 104 cells/well. The plates were placed in an incubator at 37°C for 2 h after the addition of 10 μl CCK-8 solution . The measurement of the absorbance was carried out at 450 nm with a microplate reader  .LDH levels in the supernatant of H9c2 cells were examined with an LDH assay kit  with reference to the manufacturer’s instructions . SubsequH9c2 cells were first fixed with 1% formaldehyde for 1 h at 37°C in the dark and subsequently rinsed with PBS for three times. These cells were then mixed with 0.2% Triton X-100. The assay of cell apoptosis was performed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) . The cells were observed with the aid of a fluorescence microscope  .The content of MDA, SOD and GSH in H9c2 cells were tested by means of corresponding commercial kits  in accordance with the directions of manufacturer. Briefly, RIPA lysis buffer was applied to lyse these H9c2 cells and then the supernatant was taken. The levels of MDA, SOD and GSH were determined using a microplate reader  by measuring absorbance at 532, 550 and 412 nm, respectively.H9c2 cells were collected in lysis buffer and placed on ice for 30 min. Then, these treated cells were centrifuged at 15, 000 rpm for 15 min at 4°C. The supernatant was gathered. Immunoprecipitation experiments were performed using 5 μg antibody and 500 μg protein. The collection, washing and elution of precipitated proteins were carried out. Finally, Western blotting was adopted to treat these protein blots .The experimental data are analyzed by SPSS Vision 19.0  and mainly presented in the means mean ± SD. Differences among multiple groups were analyzed using one-way ANOVA. When P was less than 0.05, it was regarded as statistically significant difference.In this study, we explored the biological functions of SFRP4 and the potential mechanism in high-glucose-stimulated H/R cardiomyocyte model. The data showed that the silencing of SFRP4 promoted the viability and suppressed high glucose-induced inflammation and oxidative stress under H/R condition. In addition, SFRP4 was found to be bound with PTPN12 and inhibit PI3K/AKT signaling pathway. Moreover, PTPN12 overexpression reversed the effects of SFRP4 silencing on high-glucose-stimulated H/R cardiomyocyte injury.H/R model in H9c2 cells was established under HG conditions. The experiment was divided into three groups: normal, HR, and HR+HG. Subsequently, SFRP4 expression in the cells was assayed by RT-qPCR and Western blotting. Cell viability at pre-transfection and post-transfection in H9c2 cells was determined by RT-qPCR, Western blotting, CCK-8, LDH kit and TUNEL. As shown in To verify the effect of SFRP4 interference on cell damage, the protein levels of inflammatory factors and oxidative stress-related factors were detected by both Western blotting and ELISA kit. To confirm the correlation between SFPR4 and PTPN12 and their influence on PI3K/ Akt signaling pathway, we demonstrated the relationship between SFPR4 and PTPN12 with the help of two databases MINT and the BioGRID as well as immunoprecipitation assay, and confirmed their effect on P13 K and AKT through the assays of RT-qPCR and Western blotting. As seen from To demonstrate whether PTPN12 overexpression can affect high-glucose-stimulated H/R cardiomyocyte injury, we examined cell viability and expressions of inflammatory factors, oxidative stress factors. Diabetic cardiomyopathy is one of the leading causes of death in diabetic patients. As mentioned previously, SFRP4 may pose potential implications on diabetic myocardial ischemia-reperfusion. The database search revealed that SFRP4 can bind to PTPN12 and was highly positively correlated. And PTPN12 can activate the P13 K/APK signaling pathway. Therefore, we made a speculation that downregulation of SFRP4 inhibits H/R injury in diabetic cardiomyocytes through activation of PI3K/AKT signaling pathway by PTPN12. To simulate the diabetic environment, cells were stimulated with high glucose at a concentration of 30 mmol/L for 24 h. In addition, to establish an H/R cell model, these cells were cultured in a serum-free medium overnight and were subjected to an incubator with a mixture of 95% N2, 5% CO2 and 1% O2 at 37°C for 12 h to simulate anoxia. Finally, cells were exposed in a normoxic chamber for 12 h reoxygenation. In the present study, the experimental data showed that SFRP4 expression was elevated in the H/R model. SFRP4 interference promoted the cell viability of high glucose-stimulated H/R cardiomyocytes and inhibited inflammatory and oxidative stress responses. In addition, SFRP4 could interacte with PTPN12 to inhibit the P13/APK signaling pathway. However, PTPN12 overexpression reversed the inhibitory effect of sh-SFRP4 on high-glucose-stimulated H/R cardiomyocyte injury.SFRP4 is the largest member of the SFRP family  and is aNumerous articles related to PTPN12 have focused on its intrinsic association with tumors. PTPN12 is a tumor suppressor gene that is involves in tumorigenesis and development . For insOverall, our study elucidated that downregulation of SFRP4 attenuated cardiomyocyte injury in H/R models and regulated the activation of the P13 K/AKT signaling pathway through PTPN12, which possibly provides a novel therapeutic target for patients with diabetic cardiomyopathy.Click here for additional data file.