Figure 3.

The essential role of the AR and H3K27me3 complex in the regulation of TNIK expression

(A) LNCaP cells were cultured in steroid-depleted medium for 24 h and subsequently treated with DHT (10 nM) for the indicated time. Protein levels were analyzed by western blot.

(B) The levels of TNIK protein were measured by western blot in LNCaP and C4-2 cells upon AR knockdown.

(C) qPCR analysis mRNA expression of TNIK after LNCaP cells were treated with MDV3100 for 24 h. Data are shown as mean ± SD. ∗∗∗p < 0.005.

(D) The levels of TNIK protein were measured in LNCaP cells treated for 24 h with the AR antagonist MDV3100 (enzalutamide, 100 nM).

(E) Coimmunofluorescence (co-IF) analysis of TNIK and AR proteins in LNCaP treated with MDV3100. Scale bars: 50 μm.

(F) Immunoprecipitation of AR or H3K27me3 in LNCaP and C4-2 cells followed by immunoblot analysis of H3K27me3 or AR. IgG represents a control antibody used for IPs.

(G) ChIP–PCR analysis of H3K27me3 and AR binding to the TNIK gene promoter in LNCaP cells treated with MDV3100. ChIP–PCR primer in Table S3. Data are shown as mean ± SD. ∗∗∗p < 0.005.

(H and I) LNCaP and C4-2 cells were treated with DZNep (0, 5, 10 μm) and GSKJ1 (0, 5, 10 μm) for 8 h, WB analysis of TNIK protein expression.

(J) Western blot showed that treatment of the indicated LNCaP or C4-2 cells with GSK-J1 could reverse the effects of AR knockdown on TNIK.