Figure 2. MEP50 is not required for PRMT5-mediated activation of AR transcription in CRPC cells.
A-B, Representative western blot images (A) and quantification (B) of protein expression in cell lysates of 22Rv1 cells with doxycycline-inducible MEP50 knockdown (22Rv1-shMEP50) incubated in the presence (Dox (+)) or absence (Dox (–)) of doxycycline for 6 days. C, qPCR analysis of gene expression in cells from A. D-E, Representative western blot images (D) and quantification (E) of protein expression in cell lysates of LNCaP cells with doxycycline-inducible MEP50 knockdown (LNCaP-shMEP50) incubated in the presence (Dox (+)) or absence (Dox (–)) of doxycycline for 6 days. F, qPCR analysis of gene expression in cells from D. G-H, ChIP-qPCR assay of MEP50 binding to the proximal AR promoter or control gene IVL promoter was performed with non-specific IgG binding as a control in 22Rv1 (G) and LNCaP (H) cells. I, Trypan blue cell viability analysis in 22Rv1-shMEP50 cells after 6 days of MEP50 knockdown. J, Flow cytometry analysis of cells following PI staining at Day 6 of MEP50 knockdown in 22Rv1-shMEP50 (sub-G1 cells were gated out). For western blotting, cell cycle, cell viability, and qPCR analysis, statistical significance of group difference was determined for ‘Dox (–) vs Dox (+)’. For ChIP-qPCR, values were normalized to the corresponding IgG control, and indicated statistical significance of group difference was determined for ‘specific IP vs IgG IP’. Results are mean ± SD from 3 independent experiments. For western blotting of AR, the AR N-20 antibody (sc-816, Santa Cruz) was used. Student’s t-test with Welch’s correction was performed to determine statistical significance. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001.