Fig. 6

KDM6A promotes ARHGDIB transcription by catalyzing demethylation of H3K27me3. a The indicated protein levels in Puro/KDM6A and NC/shKDM6A T24 cells were detected by Western blot. b and c Western blot (b) and qPCR (c, Puro was set as 1) were performed to detect indicated gene levels in KDM6A catalytic domain wild type or mutant expressed T24 cells. d and e The effect of GSKJ4 on ARHGDIB expression levels in T24 cells was evaluated by Western blot (d) and qPCR (e, DMSO was set as 1). f Schematic diagram showed the location of 12 pairs of primers in ARHGDIB promoter regions (up). ChIP assays were performed using KDM6A and H3K27me3 antibody in T24 cells to detect the binding sites in ARHGDIB promoter regions (down). g ChIP-qPCR assays were performed using KDM6A and H3K27me3 antibody in indicated T24 cells. The normalized expression in Puro/KDM6A cells (input) was set as 1, respectively. h and i The levels of ARHGDIB in EZH2 knockdown T24 cells were detected by Western blot (h) and qPCR (i, NC was set as 1). j The protein levels of total and active Rac1 in T24 cells treated with GSK126 (20 μM) for 48 h were determined by Western blot. k The mRNA levels of ARHGDIB in indicated T24 cells treated with GSK126 (20 μM) for 48 h were measured by qPCR (NC + Control was set as 1). All quantification analyses were based on independent triplicate experiments. Error bars represent SD. l Lung metastasis assays of indicated cells. The number of lung metastasis node were counted (Puro, n = 4, KDM6A, n = 3, NC, n = 3, shKDM6A, n = 3). Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, NS no significant, based on Student’s t test