Fig 2: Both TGFß1 and HDAC1 limit WT1 activity in mesenchymal-like MCs.In this condition, the antifibrotic miR-769-5p is downregulated. HDAC1 inhibition may promote MMT reversal through WT1-induced miR-769-5p expression.

Fig 3: WT1-mediated induction of miR769-5p is repressed in fibrotic MCs by HDAC1 activity.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 TGFß1, 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 TGFß1 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 TGFß1 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.

Fig 4: Expression of a stable form of ß-catenin prevents epithelial nephron progenitors from further differentiating. (A) Nascent (left) and mature (right) S-shaped bodies are shown. In the control kidney, Jag1 and Wt1 mark the medial and proximal segments of the S-shaped body, respectively. In the ß-catenin gain-of-function mutant kidney by Wnt4GFPcre, the Jag1 expression domain expands into the proximal segment of the S-shaped body. Pax2 marks the collecting duct, the cap mesenchyme, and nascent developing nephrons. (B) In the control kidney, Pax2 expression in epithelial nephron progenitors is downregulated after S-shaped body stage. As a result, most of the Hnf4a+ cells are negative for Pax2. In the ß-catenin gain-of-function mutant kidney, Pax2 is persistently expressed in the nephron lineage and all Hnf4a+ cells are also positive for Pax2, suggesting that ß-catenin gain-of-function mutant cells fail to exit from their progenitor status. Images are representative of three independent experiments. Stage E18.5. Scale bar: 100 µm.

Fig 5: WT1 knockdown increases the rate of mitotic abnormalities(a) WiT49 cells were transfected with control, WT1, or MAD2 siRNA and 48 hours later immunofluorescence analysis was performed with anti-WT1 antibodies to study the anaphase stage of mitosis. DNA was stained with Hoechst. The scale bar is 10 microns. (b) The percentage of WiT49 and M15 cells exhibiting mitotic defects (chromosome lagging/bridges) was scored over total number of cells that were in anaphase for >100 optical fields and plotted graphically. Error bars are standard deviation from the mean for three independent experiments and statistical significance was determined by Student’s t test (P < 0.05). (c) WiT49 cells were transfected with control, WT1 or MAD2 siRNAs for 48 hours followed by treatment with either DMSO or nocodazole (60 ng/ml) for another 24 hours. Whole cell extracts were prepared and immunoblotted with anti-WT1, MAD2, SECURIN and TUBULIN antibodies (d) Mitotic indices were calculated for WiT49 cells transfected with control, WT1 or MAD2 siRNA followed by DMSO or nocodazole (60 ng/ml) treatment. The results were plotted as graphs after analyzing >100 optical fields for at least three independent experiments. Error bars are standard deviation from the mean. (e) M15, WiT49 and MCF7 cells were transfected with control, MAD2 or WT1 siRNAs for 48 hours followed by immunoblotting with anti-WT1, anti-MAD2, anti-TUBULIN, anti-phosphorylated Histone H3 serine10 (H3S10p) and anti-Histone H3 antibodies. (f) Quantitative RT-PCR analysis was performed to analyze the expression of MAD2, WT1, AREG, EREG and CYCLIN E genes in WiT49 cells transfected with control, MAD2 siRNA or WT1 siRNA for 48 hours. Error bars denote standard deviation from the mean of three independent experiments and statistical significance was analyzed by Student’s t test (P < 0.05).