Fig. 5

Smyca promotes c-Myc/Max complex recruitment to its target promoters and c-Myc/TRRAP binding. (A) GSEA plots for the match of Smyca signature with Myc signature from indicated sources. (B, C) qRT-PCR analysis of indicated c-Myc targets in MDA-MB-231 cells stably expressing Smyca shRNAs (B) or MCF7 cells stably expressing Smyca (C). Data are normalized with that derived from control cells. (D, F, G) RNA pull-down analysis by incubating MDA-MB-231 nuclear extracts (D, G) or indicated amounts of recombinant c-Myc protein (F) with biotinylated sense or antisense Smyca (D, F) or its deletion mutants (G). (E) RIP assay for the enrichment of Smyca in c-Myc immunoprecipitates derived from MDA-MB-231 cells. Data are normalized with that derived from Neat1. The presence of c-Myc in the immunoprecipitates is shown on the right panel. (H) ChIRP assay for detecting Smyca occupancy on indicated c-Myc target loci. Tilling biotinylated oligonucleotides complementary to LacZ or Smyca were used to pull down the RNA-associated chromatins from MDA-MB-231 cells, followed by qRT-PCR analysis. Data are normalized with that of inputs. (I) ChIP analysis of the recruitment of c-Myc or Max to indicated promoters in MDA-MB-231 cells stably expressing Smyca shRNAs. The enrichment folds are normalized with that from control cells. Data in (B), (C), (E), (H) and (I) are mean ± SD, n = 3. P values are determined by one-way ANOVA with Tukey’s post hoc test (B, I) or unpaired t test (C, E, H), *P < 0.05, **P < 0.01, ***P < 0.001. (J) Immunoprecipitation analysis of c-Myc binding to TRRAP in MDA-MB-231 cells stably expressing Smyca shRNA or Smyca