Fig. 3

Loss of CDC6 rescues the oncogenic potential of ezalutamide-resistant cells and establishes senescence. A Western blotting in C4-2B cells receiving or not additional treatment with enzalutamide. Cell extracts were blotted with the indicated antibodies. CDC6 is stabilized upon treatment. See also Fig. S2B. B GL13 immunocytochemical staining of C4-2B cells to assess senescence induction upon enzalutamide treatment. Magnification: 200x (Objective 20x), scale bars: 30 μm. See also Fig. S1C and D. C Quantification of cells in B displaying non-significant differences between the indicated conditions. D Western blotting in C4-2B and PC-3 cells transfected with the indicated siRNAs and/or treated with DMSO or enzalutamide. Cell lysates were probed with the indicated antibodies. CDC6 is stabilized upon enzalutamide treatment at the expense of p21^WAF1/Cip1. See also Fig. S2C and D. E Immunofluorescence for dual GL13/p21^WAF1/Cip1 staining (top) and quantification (bottom) confirms senescence induction only in CDC6-depleted cells in D. Magnification: 100x (Objective 10x), scale bars: 60 μm. See also Fig. S1E-F and S3. F MTS proliferation assay in C4-2B cells from D displaying a proliferation decrease following CDC6 depletion. See also Fig. S4A-D. G qPCR for EMT markers CDH1, ZEB1 and SNAI1 in the indicated conditions of cells from D. CDC6 depletion rescues EMT marker expression, while enzalutamide-mediated CDC6 stabilization has the opposite effect. Data are normalized to GAPDH expression. Enzalutamide was used at a 10 μΜ concentration. *P < 0.05, **P < 0.01 and ***P < 0.001, of Student’s t-test; n.s., non-significant. Error bars indicate s.e.m. Data shown are representative of at least 3 biological experiments (n ≥ 3)