Journal: Oncology reports

Article Title: The pancreatic cancer secreted REG4 promotes macrophage polarization to M2 through EGFR/AKT/CREB pathway.

doi: 10.3892/or.2015.4357

Figure Lengend Snippet: Figure 3. Involvement of EGFR/AKT/CREB pathway in REG4-mediated macrophage polarization to M2. (A) Representative western blots of phosphorylated EGFR, AKT and CREB in macrophage cultures treated with REG4 (10 nM) for different time intervals indicated. (B) Quantitative analysis of the immunoblots of p-EGFR, p-AKT and p-CREB normalized with EGFR, AKT and CREB, respectively. Data are mean ± SEM from four independent experiments. *p<0.05, **P<0.01 compared to untreated cells. (C) Representative western blotting images showing the phosphorylation of EGFR, AKT and CREB in macrophages cultures treated with or without REG4 (10 nM) for 30 min in the absence or presence of LY294002. (D) Quantitative analysis of the immunoblots of p-EGFR, p-AKT and p-CREB normalized with EGFR, AKT and CREB, respectively, in macrophages cultures treated with or without REG4 (10 nM) for 30 min in the absence or presence of LY294002. Data are mean ± SEM from four independent experiments. *p<0.05 compared to untreated cells; #P<0.05 compared with REG4 treatment alone. (E) Representative western blots of CD163, CD68, and CREB in REG4-treated macrophages which were infected with control shRNA (shCON) or shCREB lentiviral particles. (F) Quantitative analysis of the immunoblots of CD163, CD68 and CREB in REG4-treated macrophages which were infected with control shRNA (shCON) or shCREB lentiviral particles. Data are mean ± SEM from three independent donors. *p<0.05 compared with control.

Article Snippet: The lentiviral CREB shRNA, targeting human CREB (NM_004379) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, uSA).

Techniques: Western Blot, Phospho-proteomics, Infection, Control, shRNA

Journal: Oncology reports

Article Title: The pancreatic cancer secreted REG4 promotes macrophage polarization to M2 through EGFR/AKT/CREB pathway.

doi: 10.3892/or.2015.4357

Figure Lengend Snippet: Figure 4. The conditioned medium of Panc1 cells induces macrophage polarization to M2. (A) Western blots of REG4 in the culture medium or cell lysate of Panc1, AsPC1 and BxPC3 cells. Shown are representatives of three independent experiments with similar results. (B) Representative western blots of CD206 and CD163 in macrophage cultures treated with or without the conditioned medium of Panc1, AsPC1 and BxPC3 cell cultures, respectively. (C) Quantitative analysis of the immunoblots of CD163 and CD206 in macrophage cultures treated with or without the conditioned medium of Panc1 (CMPanc1), AsPC1 (CMAsPC1) and BxPC3 (CMBxPC3) cell cultures, respectively. Data are shown by mean ± SEM from four independent experiments. *p<0.05 compared to untreated cells; #P<0.05 compared with CMPanc1 treatment. (D) Western blots of REG4 in the culture medium and cell lysate of Panc1 cells infected with or without lentiviral REG4 shRNA. Shown are representatives of three independent experiments with similar results. (E) Representative western blots of CD206 and CD163 in macrophage cultures treated with the conditioned medium Panc1 cells infected with lentiviral control shRNA (CMPanc1/shCon) or REG4 shRNA (CMPanc1/shREG4). (F) Quantitative analysis of the immunoblots of CD163 and CD206 in macrophage cultures treated with CMPanc1/shCon or CMPanc1/shREG4. *p<0.05 compared with control.

Article Snippet: The lentiviral CREB shRNA, targeting human CREB (NM_004379) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, uSA).

Techniques: Western Blot, Infection, shRNA, Control

Journal: American journal of physiology. Cell physiology

Article Title: Activation of Nrf2 contributes to the protective effect of Exendin-4 against angiotensin II-induced vascular smooth muscle cell senescence.

doi: 10.1152/ajpcell.00093.2016

Figure Lengend Snippet: Fig. 4. PKA-CREB pathway is required for the Exendin-4 (EX-4)-activated Nrf2/ARE signaling. A: VSMCs were incubated with ANG II for indicated periods of time. Phospho-CREB (p-CREB) and CREB expressions were determined by Western blot. n 3. B: VSMCs were incubated with Exendin-4 for indicated periods of time. Phospho-PKA (p-PKA), PKA, p-CREB, and CREB expression were determined by Western blot. n 3. C: Exendin-4 activated CREB via PKA. VSMCs were preincubated with PKA inhibitor PKI14-22 for 30 min before stimulation with 10 nM Exendin-4 for 30 min. The cell lysates were immunoblotted for p-CREB at Ser 133 and reprobed for total CREB protein. D: effect of PKA inhibition on Exendin-4-mediated increase in Nrf2 activity. Human VSMC cell lines were transfected with the ARE4-luciferase constructs for 24 h before incubation with PKI14-22 for 30 min. The cells were then stimulated with 10 nM Exendin-4 for 12 h, followed by treatment with ANG II for 12 h. Relative ARE4-driven luciferase activity was measured. Values are means SE; n 6. Open bars represent the empty vector group. *P 0.05 vs. untreated. #P 0.05 vs. ANG II Exendin-4. E: representative Western blots of HO-1 and NQO-1 expression. VSMCs were preincubated with PKI14-22 for 30 min before stimulation with 10 nM Exendin-4 for 12 h, followed by treatment with 100 nM ANG II for 12 h. Whole cell lysates were subjected to Western blot analysis with antibodies against HO-1 and NQO-1. n 3. F: representative Western blots of NQO-1 and HO-1 in VSMCs transfected with CREB siRNA or scramble siRNA for 48 h before incubation with 10 nM Exendin-4 for 12 h, followed by treatment with 100 nM ANG II for 12 h.

Article Snippet: Antibodies against Nrf2 (sc-722), HO-1 (sc-7695), NQO-1 (sc-16464), phospho-CREB (sc-7978), CREB (sc-186), p300 (sc584), CBP (sc-25748), and p300/CBP-associated factor (PCAF) (sc8999) were from Santa Cruz.

Techniques: Incubation, Western Blot, Expressing, Inhibition, Activity Assay, Transfection, Luciferase, Construct, Plasmid Preparation

Journal: Oncogene

Article Title: Regulation of cyclin D2 and the cyclin D2 promoter by protein kinase A and CREB in lymphocytes.

doi: 10.1038/sj.onc.1209255

Figure Lengend Snippet: Figure 3 CREB-1, from lymphocyte nuclear extracts, binds to an oligonucleotide from the cyclin D2 promoter. Supershift analysis of the 320 to 270 bp region of the cyclin D2 promoter using CREB-1 and CREB-2 antibodies. Nuclear extracts from (a) IL-2 stimulated Kit225 or (b) EPV-immortalized B cells were untreated (lanes 1 and 6) or incubated with specific antibodies raised to CREB-1 (lanes 2, 3, 7 and 8) or CREB-2 (lanes 4, 5, 9 and 10). The different lanes marked CREB1 and CREB2 represent incubation with two different antibodies. They were then incubated with an oligonucleotide corresponding to the 320 to 270 bp region of the cyclin D2 promoter (lanes 1–5) or a CREB-1 consensus oligonu- cleotide (lanes 6–10). This figure is representative of four experiments. (c) A DNA affinity precipitation was performed using nuclear extracts from EPV-immortalized B cells with oligonucleotides corresponding to consensus STAT, NF-kB or CREB oligonucleotides, as indicated or with an oligonucleotide corresponding to the 320 to 270 bp region of the cyclin D2 promoter. NE designates nuclear extract on which no precipitation has been performed. The precipitated proteins were separated by SDS–PAGE followed by Western blotting. CREB-1 was detected using a specific antibody.

Article Snippet: For supershift assays, the nuclear extracts were preincubated for 30min with 1ml of antibodies for CREB1 or CREB2 from Santa Cruz Biotechnology: CREB-1 (C-21, sc-186); CREB-1 (24H4B, sc-7583); CREB-2 (C20, sc-186) and CREB-2 (C19, sc-7583).

Techniques: Incubation, SDS Page, Western Blot

Journal: Oncogene

Article Title: Regulation of cyclin D2 and the cyclin D2 promoter by protein kinase A and CREB in lymphocytes.

doi: 10.1038/sj.onc.1209255

Figure Lengend Snippet: Figure 4 CREB-1 is phosphorylated and its phosphorylation and binding are important for cyclin D2 promoter activity. (a) Kit225 cells were cotransfected with the human D2 promoter and phRL-SV40 together with various amounts of an expression vector for CREB-1 or S133A CREB-1. The amount of DNA was kept constant by the addition of empty vector (pcDNA3). Cells were stimulated with IL-2 for 18 h. Data were normalized using the Renilla luciferase and is expressed as a fold activation induced by IL-2. Error bars represent the mean and standard error of the mean from three independent experiments. (b) Protein extracts were generated from Kit225 cells that had been pretreated with LY294002 for 30 min, stimulated with IL-2 for 1 h. Protein extracts were analysed by SDS– PAGE, and probed with specific antibodies as indicated. (c) To investigate the phosphorylation status of DNA bound CREB-1, a DNA affinity precipitation was performed on Kit225 cell nuclear extracts, with an oligonucleotide corresponding to the 320 to 270 bp region of the cyclin D2 promoter. The precipitated proteins were resolved by SDS–PAGE followed by Western blotting and detection with specific antibodies. (d) Lymphoblastoid cells were treated with various doses of LY294002 as indicated for 1 h. Protein extracts were generated and analysed as in (b). Representative experiments are shown from at least four experiments.

Article Snippet: For supershift assays, the nuclear extracts were preincubated for 30min with 1ml of antibodies for CREB1 or CREB2 from Santa Cruz Biotechnology: CREB-1 (C-21, sc-186); CREB-1 (24H4B, sc-7583); CREB-2 (C20, sc-186) and CREB-2 (C19, sc-7583).

Techniques: Phospho-proteomics, Binding Assay, Activity Assay, Expressing, Plasmid Preparation, Luciferase, Activation Assay, Generated, SDS Page, Western Blot

Journal: Oncogene

Article Title: Regulation of cyclin D2 and the cyclin D2 promoter by protein kinase A and CREB in lymphocytes.

doi: 10.1038/sj.onc.1209255

Figure Lengend Snippet: Figure 5 Loss of the CREB-1-binding site in the cyclin D2 promoter activity causes a dramatic loss of activity. Two doses of each of the four cyclin D2 promoters, two of which contained a mutation within the CREB-1-binding site (illustrated in schematic (a)), were transfected into Kit225 cells. After 4 h, cells were lysed and luciferase activity was determined (b). IB4 lymphoblastoid cells were transiently transfected with the full-length cyclin D2 promoter and the promoter containing a mutation in the CREB-1 site after 16 h (c).

Article Snippet: For supershift assays, the nuclear extracts were preincubated for 30min with 1ml of antibodies for CREB1 or CREB2 from Santa Cruz Biotechnology: CREB-1 (C-21, sc-186); CREB-1 (24H4B, sc-7583); CREB-2 (C20, sc-186) and CREB-2 (C19, sc-7583).

Techniques: Binding Assay, Activity Assay, Mutagenesis, Transfection, Luciferase

Journal: Oncogene

Article Title: Regulation of cyclin D2 and the cyclin D2 promoter by protein kinase A and CREB in lymphocytes.

doi: 10.1038/sj.onc.1209255

Figure Lengend Snippet: Figure 6 CREB phosphorylation and proliferation of EPV- immortalized cells is inhibited by a PKA inhibitor. (a) EPV- immortalized cells were incubated for 1 h with a variety of protein kinase inhibitors. The inhibitors were Ro-31–8220 (5 mM), Genis- tein (500 mM), KN-93 (10 mM), KT5720 (500 mM), staurosporine (100 nM) and U0126 (500 mM). Protein extracts were generated and CREB phosphorylation was measured with specific antibodies following SDS–PAGE and Western blotting. (b) A range of doses of staurosporine and KT5720 were used to treat cells and CREB phosphorylation was measured as before. (c) EBV-immortalized cells were treated with four doses of KT5720 for 72 h. Cells were counted by flow cytometry. The results are expressed as the percentage of values from untreated cells and are the mean and standard error of the mean of three independent experiments.

Article Snippet: For supershift assays, the nuclear extracts were preincubated for 30min with 1ml of antibodies for CREB1 or CREB2 from Santa Cruz Biotechnology: CREB-1 (C-21, sc-186); CREB-1 (24H4B, sc-7583); CREB-2 (C20, sc-186) and CREB-2 (C19, sc-7583).

Techniques: Phospho-proteomics, Incubation, Generated, SDS Page, Western Blot, Cytometry

Journal: Nature Communications

Article Title: Investigational eIF2B activator DNL343 modulates the integrated stress response in preclinical models of TDP-43 pathology and individuals with ALS in a randomized clinical trial

doi: 10.1038/s41467-025-63031-y

Figure Lengend Snippet: a Schematic depicting the ISR pathway, including kinases mediating the phosphorylation of eIF2α, and activation of eIF2B by DNL343. b Confocal microscopy images showing induced expression of GFP-TDP-43 FL and GFP-TDP-43 (86-414) variant in H4 cells increased immunoreactivity of ATF4 (anti-ATF4, red) compared to GFP control. This ATF4 signal was reduced with 1 µM DNL343 treatment. Scale bar: 20 µm. c Quantification of ATF4 immunoreactivity in ( b ) from n = 3 (GFP Control) or 4 biological replicates (GFP-TDP-43 variants). a.u.: arbitrary units. d ECLIA-based assay result demonstrating elevated ATF4 protein levels with induced expression of GFP-TDP-43 FL and GFP-TDP-43 (86-414) , compared to GFP control cells. Pre-treatment with DNL343 reduced ATF4 protein levels in each cell line ( n = 6 biological replicates). e Volcano plot of differentially regulated genes in H4 cells induced with GFP-TDP-43 FL compared to GFP control. Representative ISR genes with significant expression changes are labeled as red (upregulation) or blue (downregulation) and insignificant genes as gray. Significance cutoff, adjusted p -value < 0.05. f Volcano plot of differentially regulated genes in H4 cells induced with GFP-TDP-43 (86-414) compared to GFP control. Representative ISR genes are labeled as in ( e ). Significance cutoff, adjusted p -value < 0.05. g Gene set enrichment analysis (GSEA) plot for ISR gene comparing truncated GFP-TDP-43 (86-414) from GFP control expressing cells. h Volcano plot of differentially regulated genes 24 hours after DNL343 treatment in H4 cells induced with GFP-TDP-43 (86-414) . The same set of representative ISR genes from ( e ) and ( f ) are labeled. All data are shown as mean ± SEM ( c , d ). Statistical significance was determined with one-way ANOVA, with Tukey’s multiple comparison, ns P > 0.05. Source data are provided in Source Data file.

Article Snippet: A non-competitive sandwich immunoassay employing a biotinylated CREB-2 (ATF4) mAb (Santa Cruz Biotechnology, #sc-390063 LS) as capture and a SULFO TAG labeled (ruthenylated) anti-ATF4 pAb (Proteintech, #10835-1-AP) for detection was used to quantify levels of ATF4 protein.

Techniques: Phospho-proteomics, Activation Assay, Confocal Microscopy, Expressing, Variant Assay, Control, Labeling, Comparison

Journal: Nature Communications

Article Title: Investigational eIF2B activator DNL343 modulates the integrated stress response in preclinical models of TDP-43 pathology and individuals with ALS in a randomized clinical trial

doi: 10.1038/s41467-025-63031-y

Figure Lengend Snippet: a Experimental design of acute DN9058 dosing of rNLS8 transgenic mice. All animals were fed Dox-containing diet until 8 weeks of age, including non-transgenic (nTg) controls, single transgenic (sTg) controls and double transgenic (rNLS8) mice, then Dox was removed from their diet except for group 6 (double transgenic (rNLS8) on Dox for two additional weeks). On 13th and 14th day off Dox, indicated groups were dosed with DN9058 by oral gavage at 50 mg/kg per animal weight. b - c Quantification of p-eIF2α normalized to loading control eIF2α ( b ) and ATF4 level normalized to GAPDH ( c ) from Supplementary Fig. and j ( n = 8 sTg Control or 12 rNLS8 mice). Data are shown as fold-changes relative to vehicle-treated control mice. d Transcriptional fold change of pre-selected ISR genes in rostral cortex of indicated mouse line ( n = 7 (nTg Ctrl), 8 (rNLS8 Dox), 9 (sTg Ctrl) and 12 (rNLS8) mice). Data are shown as mean ± SEM ( b − d ). Statistical significance was determined with Kruskal-Wallis test with Dunn’s multiple comparisons ( b ), ordinary One-way ANOVA with Tukey’s multiple comparison ( c , d ). Source data are provided in Source Data file.

Article Snippet: A non-competitive sandwich immunoassay employing a biotinylated CREB-2 (ATF4) mAb (Santa Cruz Biotechnology, #sc-390063 LS) as capture and a SULFO TAG labeled (ruthenylated) anti-ATF4 pAb (Proteintech, #10835-1-AP) for detection was used to quantify levels of ATF4 protein.

Techniques: Transgenic Assay, Control, Comparison

Journal: Nature Communications

Article Title: Investigational eIF2B activator DNL343 modulates the integrated stress response in preclinical models of TDP-43 pathology and individuals with ALS in a randomized clinical trial

doi: 10.1038/s41467-025-63031-y

Figure Lengend Snippet: a Experimental design of chronic DN9058 dosing for 6 weeks off dox (WOD). DN9058 was formulated at 50 mg/kg per chow for the chronic administration to the indicated conditions. b - c Quantification of p-eIF2α normalized to loading control eIF2α ( b ) and ATF4 level normalized to GAPDH ( c ) from Supplementary Fig. and j, respectively ( n = 8 (sTg Control), 10 (rNLS8 Veh), and 13 (rNLS8 DN9058) mice). Data are shown as fold-changes relative to vehicle-treated control mice. d - e Transcript level changes of pre-selected ISR genes ( d ) and ISR genes indicated in later stage of the disease ( e ) in rostral cortex ( n = 9 (sTg Control), 10 (rNLS8 Dox), 11 (rNLS8 Veh), and 16 (rNLS8 DN9058) mice). f Time (weeks) to show clasping for individual rNLS8 mice that demonstrated the clasping phenotype ( n = 13 (Veh) and 18 (DN9058) mice). g Plasma NfL concentration at baseline (0 WOD), 2, 4, and 6 WOD in rNLS8 mice ( n = 11 (Veh) and 16 (DN9058) mice). The same figure legend for rNLS8 + Veh (yellow) and rNLS8 + DN9058 (orange) is shared with panel ( f ). Source data are provided in Source Data file. Data are shown as mean ± SEM ( b − e ) and individual values overlayed on violin plot ( f , g ). Statistical significance was determined with Kruskal-Wallis test with Dunn’s multiple comparisons ( b , c ), ordinary One-way ANOVA with Tukey’s multiple comparison ( d , e ), Two-tailed Mann-Whitney test ( f ), and Two-way ANOVA with multiple comparisons ( g ).

Article Snippet: A non-competitive sandwich immunoassay employing a biotinylated CREB-2 (ATF4) mAb (Santa Cruz Biotechnology, #sc-390063 LS) as capture and a SULFO TAG labeled (ruthenylated) anti-ATF4 pAb (Proteintech, #10835-1-AP) for detection was used to quantify levels of ATF4 protein.

Techniques: Control, Clinical Proteomics, Concentration Assay, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Investigational eIF2B activator DNL343 modulates the integrated stress response in preclinical models of TDP-43 pathology and individuals with ALS in a randomized clinical trial

doi: 10.1038/s41467-025-63031-y

Figure Lengend Snippet: a − d ATF4 protein ( a ) and CHAC1 gene ( b ) expression in ex vivo stimulated PBMCs from healthy participants. ATF4 protein ( c ) and CHAC1 gene ( d ) expression in ex vivo stimulated PBMCs from ALS participants. PBMCs were freshly isolated from participants in each dose group shown at the times indicated and analyzed by either ECLIA ( a , c ) or multiplex qPCR ( b , d ). Values shown as median (IQR = interval from the first to the third quartile, shown as error bars) percent change from baseline. For MAD healthy participants ATF4 protein, n = 12, 6, 7, 7, 7, 6 participants for placebo, 45 mg, 100 mg, 145 mg, 200 mg and 260 mg groups respectively; CHAC1 gene, n = 11, 6, 7, 5, 7, 6 participants for placebo, 45 mg, 100 mg, 145 mg, 200 mg and 260 mg groups respectively. For ALS participants ATF4 protein, n = 6, 7, 6 participants for placebo, 100 mg, and 200 mg groups respectively; CHAC1 gene, n = 5, 7, 6 participants for placebo, 100 mg, 200 mg groups respectively through Day 28 for each dose group. e Heat map depicting relative change from baseline for a panel of ISR genes. Gene expression measured by multiplex qPCR in freshly isolated ex vivo stimulated PBMCs from ALS patients in each dose group. Values grouped based on median percent change from baseline and genes rank ordered based on percent change from baseline at Day 28 in the highest dose cohort (200 mg). f Percent change from baseline in CSF GDF-15 protein concentration at Day 28 in ALS participants, n = 9, 8, 8 participants for placebo, 100 mg, 200 mg groups respectively per group. Data are shown as box plot with individual percent change values overlayed. The middle line of the boxplot displays the median, the lower and upper limits of the box display the first and third quartiles, and the whiskers extend to the largest and smallest values. Source data are provided in Source Data file.

Article Snippet: A non-competitive sandwich immunoassay employing a biotinylated CREB-2 (ATF4) mAb (Santa Cruz Biotechnology, #sc-390063 LS) as capture and a SULFO TAG labeled (ruthenylated) anti-ATF4 pAb (Proteintech, #10835-1-AP) for detection was used to quantify levels of ATF4 protein.

Techniques: Expressing, Ex Vivo, Isolation, Multiplex Assay, Gene Expression, Protein Concentration

Journal: The Journal of biological chemistry

Article Title: v-src induces prostaglandin synthase 2 gene expression by activation of the c-Jun N-terminal kinase and the c-Jun transcription factor.

doi: 10.1074/jbc.270.46.27622

Figure Lengend Snippet: FIG. 2. c-Jun binds to the CRE site of the pgs2 promoter. A 32P probe from nucleotides 265 to 239 of the pgs2 promoter was used for EMGS assay. E-box competitor (75-fold excess) was used in lanes 2–4 to reduce the contribution of the E-box complexes. The E-box binding complexes and the CREB binding complexes indicated in the figure have been identified previously, both by competition with consensus E-box and CRE sequences, and by EMGS-antibody supershift experi- ments (8). The c-Jun-containing complex is identified on the basis of the c-Jun antibody-dependent supershift.

Article Snippet: The c-Jun and CREB binding protein antibodies were purchased from Santa Cruz Biotech.

Techniques: Binding Assay

Journal: Cell Death & Disease

Article Title: Restoration of WT1/miR-769-5p axis by HDAC1 inhibition promotes MMT reversal in mesenchymal-like mesothelial cells

doi: 10.1038/s41419-022-05398-0

Figure Lengend Snippet: List of PCR primers used in this study.

Article Snippet: Mouse monoclonal antibody (mAb) anti-TGFβ1 1D11.16.8 (BE0057) was from inVivoMab/Bio X Cell (Lebanon, NH); mAbs anti-HDAC (10E2), -SNAIL (L70G2), -ALIX (3A9) were from Cell Signalling (Danvers MA); mAbs anti-PAI-1 (sc-5297), -CD9 (sc-59140), -TUBULIN (sc-32293), -HSP90 (sc-13119), -WT1 (sc-7385), -CALNEXIN (sc-23954) -GAPDH (sc-32233) were from Santa Cruz Biotechnology (Dallas, TX); mAb anti-α−SMA (A52228) was from SIGMA ALDRICH (Saint Louis, MO); -ANNEXIN-VII (610668) was from BD-Transduction Laboratories (Franklin Lakes, NJ); rabbit mAb anti-SYNTHENIN (AB133267) was from ABCAM (Cambridge, UK).

Techniques:

Journal: Cell Death & Disease

Article Title: Restoration of WT1/miR-769-5p axis by HDAC1 inhibition promotes MMT reversal in mesenchymal-like mesothelial cells

doi: 10.1038/s41419-022-05398-0

Figure Lengend Snippet: A left , predicted WT1 binding site on miR-769-5p promoter by PROMO. Right , study of H3K27 acetylation of miR-769-5p promoter from ENCODE. B Left , RT-PCR showing WT1 mRNA expression upon stimulation with TGFβ1, after HDAC1 genetic silencing, and treatment with MS275 (250 nM for 72 h). Bars represent the mean ± SEM of duplicate determinations in three to four independent experiments. Right , WB showing expression of WT1 (top) and HDAC1 (bottom) upon HDAC1 genetic silencing. Tubulin is used as a loading control. Data are representative of three independent experiments. C left , immunoprecipitation of WT1 showing HDAC1-WT1 interactions upon stimulatin with TGFβ1 and treatment with MS275. A WB of input cell lysates showing WT1 expression and HSP90 as a loading control is shown in the right of the figure. Data are representative of three independent experiments. D RT-PCR showing the expression of miR-769-5p ( left ) and of WT1 ( middle ) upon miR-769-5p genetic silencing and treatment with MS-275. Bars represent the mean ± SEM of duplicate determinations in four independent experiments. Right , WB showing the expression of WT1in the same conditions as in D left . Tubulin is used as a loading control. Data are representative of three independent experiments. E qPCR of ChIP assays with anti-WT1 and as control, with normal rabbit IgG on chromatin from MCs from PD patients treated with treated with TGFβ1 for 24 h ( top ) or with MS-275 for 72 h ( bottom ) or left untreated (NT) and when indicated. Data show increased binding of WT1 to specific binding sites (1-3) at miR769-5p promoter. Bars represent the mean ± SEM of duplicate determinations in three independent experiments.

Article Snippet: Mouse monoclonal antibody (mAb) anti-TGFβ1 1D11.16.8 (BE0057) was from inVivoMab/Bio X Cell (Lebanon, NH); mAbs anti-HDAC (10E2), -SNAIL (L70G2), -ALIX (3A9) were from Cell Signalling (Danvers MA); mAbs anti-PAI-1 (sc-5297), -CD9 (sc-59140), -TUBULIN (sc-32293), -HSP90 (sc-13119), -WT1 (sc-7385), -CALNEXIN (sc-23954) -GAPDH (sc-32233) were from Santa Cruz Biotechnology (Dallas, TX); mAb anti-α−SMA (A52228) was from SIGMA ALDRICH (Saint Louis, MO); -ANNEXIN-VII (610668) was from BD-Transduction Laboratories (Franklin Lakes, NJ); rabbit mAb anti-SYNTHENIN (AB133267) was from ABCAM (Cambridge, UK).

Techniques: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Restoration of WT1/miR-769-5p axis by HDAC1 inhibition promotes MMT reversal in mesenchymal-like mesothelial cells

doi: 10.1038/s41419-022-05398-0

Figure Lengend Snippet: In this condition, the antifibrotic miR-769-5p is downregulated. HDAC1 inhibition may promote MMT reversal through WT1-induced miR-769-5p expression.

Article Snippet: Mouse monoclonal antibody (mAb) anti-TGFβ1 1D11.16.8 (BE0057) was from inVivoMab/Bio X Cell (Lebanon, NH); mAbs anti-HDAC (10E2), -SNAIL (L70G2), -ALIX (3A9) were from Cell Signalling (Danvers MA); mAbs anti-PAI-1 (sc-5297), -CD9 (sc-59140), -TUBULIN (sc-32293), -HSP90 (sc-13119), -WT1 (sc-7385), -CALNEXIN (sc-23954) -GAPDH (sc-32233) were from Santa Cruz Biotechnology (Dallas, TX); mAb anti-α−SMA (A52228) was from SIGMA ALDRICH (Saint Louis, MO); -ANNEXIN-VII (610668) was from BD-Transduction Laboratories (Franklin Lakes, NJ); rabbit mAb anti-SYNTHENIN (AB133267) was from ABCAM (Cambridge, UK).

Techniques: Inhibition, Expressing