Figure 5. Linc-RAM facilitates formation of the MyoD–Baf60c–Brg1 complex by interacting with MyoD.

(a) Interaction between Linc-RAM and MyoD, Baf60c and Brg1 determined by RNA immunoprecipitation (RIP). C2C12 cell lysates were immunoprecipitated using anti-MyoD, anti-Baf60c or anti-Brg1 antibodies, and Linc-RAM in immunoprecipitates was detected by RT–qPCR. IgG antibodies served as a control. (b) Expression of Baf60c and Brg1 in C2C12 cells with overexpression or knockdown (KD) of Linc-RAM analysed by RNA-Seq. (c) Expression of Baf60c and Brg1 in C2C12 cells with overexpression or KD of Linc-RAM determined by RT–qPCR. (d) Co-immunoprecipitation of Linc-RAM and the components in the MyoD/Baf60c/Brg1 complex determined by RIP analysis. Linc-RAM-overexpressing (OE) and Linc-RAM KD C2C12 cell lysates were immunoprecipitated using MyoD antibodies; Baf60c, Brg1 and MyoD in immunoprecipitates were detected by western blotting, and Linc-RAM was detected by RT–PCR. GAPDH served as a negative control. (e) Quantification of the immunoprecipated products in d. (f) ChIP assays were performed using chromatin from stable Linc-RAM-OE and Linc-RAM KD C2C12 cell lines and negative control (NC) cells cultured in growth medium (GM) or differentiation medium (DM). Chromatin was immunoprecipitated using antibodies against MyoD, H3K4me3, and RNA Pol II. The immunoprecipitated DNA was amplified using primers specific for MyoG and miR-206 gene promoters. (g) Gapdh gene promoter were amplified using the same samples presented in f. (h) The resulted immunoprecipitates in f were applied for detection of MyoD by western blotting. The data are presented as mean±s.e.m. from three independent experiments. The statistical significance was calculated with the t-test, *P<0.05, **P<0.01.