Figure 5: NXTAR over-expression causes an increase in repressive methylation on AR promoter.

A, Location of primers with respect to AR gene shown in a graphical format. B, LNCaP cells were infected with either pBabe control vector or NXTAR expressing constructs and grown in androgen-free medium for 48 hrs. ChIP was performed using H3K27me3 antibody or IgG (Supplementary Fig. S9G-K and L-O), followed by qPCR with using primers spanning regions upstream and downstream of AR TSS (−0.7 kb to +3.4 kb) as shown in (A). C, LNCaP cells infected with pBabe or NXTAR expressing constructs and grown in androgen free medium for 48 hrs and ChIP was performed using EZH2 and IgG antibodies (Supplementary Fig. S9P-R), followed by qPCR. D, Biotinylated oligos complementary to NXTAR (or lacZ as control) were used to pull down NXTAR from cell lysates prepared from VCaP cells retrovirally-infected to over-express NXTAR, followed by immunoblotting for EZH2. E, VCaP cells were infected with NXTAR expressing construct and treated with EZH2 inhibitor, EPZ6438 (1μM) overnight, followed by qRT-PCR. F, NXTAR (or lacZ as control) immobilized onto streptavidin beads were incubated with purified EZH2. Pull-down were washed, followed by immunoblotting with EZH2 antibodies. Data (B, C and E) are represented as mean ± SEM. *p ≤0.05; ** p<0.01; ***p<0.001, two-tailed Student’s t-test.