Fig. 6

Simultaneous inhibition of AR and PDEF expression further suppresses tumour cell proliferation compared with the inhibition of AR alone. a AR, PDEF, MAD1 and MYC protein levels in only AR-downregulated (AR KD), simultaneous AR- and PDEF-downregulated (AR KD/PDEF KD) and control MDA-MB-453 cells (NS) were determined by performing western blotting (KD: knockdown; NS: non-specific). b Flow cytometry analysis was performed to detect the proliferation of the above three MDA-MB-453 cell clones; **P < 0.05. c Colony-forming assay was conducted to determine the clone-initiating ability of the above three MDA-MB-453 cell clones; **P < 0.05. d and e Wound-healing (left) and Transwell (right) assays were performed to detect the invasion and migration potential of the above three MDA-MB-453 cell clones (wound-healing assay: original magnification, × 100; Transwell assay: original magnification, × 200); **P < 0.05. f CCK-8 assay was performed to detect the proliferation of the above three MDA-MB-453 cell clones; **P < 0.05. g Images of tumours removed from the nude mice subcutaneously injected with control (NS), stable AR-shRNA-expressing (AR KD) and simultaneous AR-shRNA- and PDEF-shRNA-expressing stable MDA-MB-453 cell clones (AR KD/PDEF KD). h Representative tumour growth curves for the three groups. Data are presented as mean ± SD; **P < 0.05. i H&E staining of tumours removed from the mice in the three groups (magnification, × 400). Results of the IHC staining for detecting Ki67 and MYC expression in tumour samples obtained from the mice in the three groups (Ki67 and MYC; magnification, × 400). j and k Statistics of the percentage of Ki67- and MYC-positive cells in the tumour samples removed from the mice in the three groups; **P < 0.05. l A model showing the role of the AR–PDEF and MAD1–MYC pathways in ER-negative BC cell proliferation