Fig. 2

Enzalutamide elicits GATA2-mediated CDC6 suppression in responsive cells. A RNA sequencing analysis in control and enzalutamide-treated LNCaP cells identified GATA2 as one of the most significantly upregulated factors (P < 0.01). See also Table S1. GATA elements have been identified in the CDC6 promoter, adjacent to putative Androgen Response Element (ARE) sites [16]. B Western blotting in LNCaP cells transfected with the indicated siRNAs and/or treated with DMSO or enzalutamide. siCTRL indicates non-targeting siRNA. Cell lysates were probed with the indicated antibodies. CDC6 is upregulated upon GATA2 depletion regardless of treatment. See also Fig. S2A. C qPCR for GATA2 mRNA levels validating successful GATA2 KD in B. Endogenous GATA2 levels are upregulated in response to enzalutamide. D Immunofluorescence for dual GL13/p21.^WAF1/Cip1 staining (top) and quantification (bottom) confirms senescence induction only in enzalutamide-treated cells in B. E Ki67 immunocytochemical staining (top) and quantification (bottom) of cells in B indicate proliferation levels proportional to CDC6 expression. Magnification: 100x (Objective 10x), scale bars: 60 μm. Inset magnification: 400x (Objective 40x). F qPCR for EMT markers CDH1, ZEB1 and SNAI1 in the indicated conditions of cells from B. GATA2 depletion is accompanied with an EMT marker expression profile regardless of treatment. Data are normalized to GAPDH expression. Enzalutamide was used at a 10 μΜ concentration. **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)