Fig. 7

FOXA1 binds to the KDM6A promoter and active KDM6A transcription. a Schematic diagram of KDM6A promoter fragments cloned into pGL3-basic vector (left). Transcriptional activities of KDM6A promoter fragments were examined by dual-luciferase reporter assays in T24 cells (right). b The relative luciferase activities of P5 reporter in T24 cells transiently transfected with indicated plasmids were examined by dual-luciferase reporter assays. c The relative luciferase activities of wild type (P5) and FOXA1-binding site mutant (mP5) KDM6A promoter fragments containing reporters in T24 cells transiently transfected with pc3.1 of pc3.1-FOXA1 were examined by dual-luciferase reporter assays. d Schematic diagram showed the locations of target primer (B primer) and two pairs of negative control primers (A and C primers) on KDM6A promoter region (up). ChIP assays was performed using FOXA1 antibody in wild type T24 cells (down). e and f KDM6A and ARHGDIB expression in indicated T24 cells were detected by Western blot (e) and qPCR (f, siNC was set as 1). g-i Migration and invasion capacities of indicated cells were examined by wound-healing assays (g), Transwell migration assays (h) and Transwell invasion assays (i). The normalized wound area of control cells at 0 h was set as 1. j Lung metastasis assays of indicated T24 cells. Representative gross lung images of indicated group were shown (left). The number of tumours per lung were counted (right, siNC vs siFOXA1–1, n = 4). Error bars represent SEM. k The FOXA1 and KDM6A protein levels in indicated T24 cells were detected by Western blot. l and m The migration and invasion capacities of indicated cells were examined by wound healing assays (l) and Transwell invasion assays (m). n The FOXA1 and ARHGDIB protein levels in indicated T24 cells were detected by Western blot. o The migration capacities of indicated cells were examined by wound healing assays. p and q The corelation between FOXA1 and KDM6A (p), FOXA1 and ARHGDIB (q) mRNA levels in 12 fresh human BCa tissues was assessed by Spearman’s correlation analysis. All quantification analyses were based on independent triplicate experiments. Error bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001, NS no significant, based on Student’s t test