Fig. 4

Smyca governs a positive feedback regulation of TGF-β signaling. (A) qRT-PCR analysis of Smyca expression in indicated cell lines treated with 5 ng/ml TGF-β for 6 h. Data are normalized with that of untreated group. (B) Upper panel: Smyca gene architecture. The transcription starting site (+ 1), regions corresponding to miR-23a ~ 27a ~ 24–2 clusters (blue), Smyca (orange) and locations of the two SBEs (red) and two sets of PCR primers covering the SBEs are indicated. The corresponding chromosome location of Smyca gene (green) and the DNase I hypersensitive clusters (gray and black) are shown on the top. Bottom panel: ChIRP assay for Smyca occupancy on its own promoter. 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. The locations of two sets of PCR primers covering the two SBE regions of Smyca promoter are shown on the top. (C) ChIP analysis of the recruitment of Smad3 and Smad4 to the indicated SBEs in MDA-MB-231 cells stably expressing control or Smyca shRNAs and treated with 5 ng/ml TGF-β for 2 h. (D, F) qRT-PCR analysis of indicated genes in Hs578T cells stably expressing Smyca shRNAs (D) or M10 cells stably expressing Smyca (F) and treated with or without 5 ng/ml TGF-β for indicated time points. Data are normalized with that of untreated group (0 h). (E, G) Luciferase reporter assay on MDA-MB-231 cells stably expressing Smyca shRNAs (E) or M10 cells stably expressing Smyca (G) and treated with or without 5 ng/ml TGF-β for indicated time points. Data are normalized with that of untreated group (0 h). Data in all panels are mean ± SD, n = 3. P values are determined by unpaired t test (A, B, F, G) or one-way ANOVA with Tukey’s post hoc test (C, D, E), *P < 0.05, **P < 0.01, ***P < 0.001