Fig. 3

Smyca enhances Smad3/Smad4 complex formation and promoter recruitment. (A, G) RNA pull-down assay using MDA-MB-231 nuclear extracts and biotinylated sense or antisense Smyca or Smyca deletion fragments. Antibodies that recognize Smad2/3 and Smad3 only were used in (A) and (G), respectively. (B) RIP analysis for the enrichment of indicated lncRNAs in Smad3 or Smad4 immunoprecipitates derived from MDA-MB-231 cells. Data are normalized with that from the control antibody. The presence of Smad3 or Smad4 in the immunoprecipitates is shown on the right. (C) Bacterially purified GST-Smad3 or GST-Smad4 bound on beads was incubated with sense or antisense Smyca. The bound Smyca was analyzed by qRT-PCR and normalized with that from the GST only group. The input GST fusion proteins are shown on the bottom and marked by arrows. (D) Smad4-associated complex was immunoprecipitated from MDA-MB-231 cells transfected with Flag-Smad4 and treated with 5 ng/ml TGF-β for 2 h. The immunocomplex was eluted and further precipitated with anti-Smad3 antibody to isolate the Smad3/Smad4 complex. Smyca enrichment in this complex was analyzed by qRT-PCR. (E, F) Immunoprecipitation analysis of Smad3 and Smad4 interaction in MDA-MB-231 cells stably expressing Smyca shRNAs (E) or M10 cells stably expressing Smyca (F) and treated with or without 5 ng/ml TGF-β for 1 h. (H) ChIRP assay for Smyca occupancy on the indicated Smad target loci. GAPDH was used as a control. Tilling biotinylated oligonucleotides complementary to LacZ or Smyca were used to pull down the RNA-associated chromatins from MDA-MB-231 cells treated with 5 ng/ml TGF-β for 2 h, followed by qRT-PCR analysis. Data are normalized with the inputs. (I, J) ChIP analysis of the recruitment of Smad3 or Smad4 to the indicated promoters in MDA-MB-231 cells stably expressing Smyca shRNAs (I) or Smyca (J) and treated with or without 5 ng/ml TGF-β for 2 h. The enrichment folds are normalized with that from untreated group. Data in (B), (C), (D), (H), (I), and (J) are mean ± SD, n = 3. P values are determined by one-way ANOVA with Tukey’s post hoc test (B, C, I) or unpaired t test (D, H, J), *P < 0.05, **P < 0.01, ***P < 0.001