Fig. 2

Smyca enhances TGF-β signaling. (A) qRT-PCR analysis of the ratios of nuclear and cytoplasmic Smyca from indicated cells. NEAT1 and GAPDH were used as controls. (B) Representative image for Smyca subcellular distribution analyzed by in situ hybridization on MDA-MB-231 cells. Bar, 10 μm. (C) Comparison of RNA-seq data derived from MDA-MB-231 cells expressing control shRNA and Smyca shRNA #1. DEGs are marked by blue dots. (D) GSEA Hallmark Pathway analysis of DEGs shown in (C). The top enriched and depleted hallmarks are shown by the order of FDR (bottom to top). (E) Representative GSEA plots for the match of Smyca signature with the indicated signatures. Enrichment score (ES) and normalized enrichment score (NES) are indicated. The full set of GSEA data is shown in Additional file 1: Fig. S3A. (F, H) qRT-PCR analysis of indicted genes in MDA-MB-231 cells stably expressing Smyca shRNAs (F) or M10 cells stably expressing Smyca (H) and treated with or without 5 ng/ml TGF-β for 24 h. Data are normalized with that of untreated group in each cell. (G, I) Luciferase reporter assay on MDA-MB-231 cells stably expressing Smyca shRNAs (G) or M10 cells stably expressing Smyca (I), transfected with indicated reporters and treated with or without 5 ng/ml TGF-β for 24 h. Data in (F), (G), (H), and (I) are normalized with that of untreated control and expressed as mean ± SD from three independent experiments. P values are determined by one-way ANOVA with Tukey’s post hoc test (F, G) or unpaired t test (H, I), **P < 0.01, ***P < 0.001. (J) Representative correlation plots of Smyca expression with the expression of indicated TGF-β target genes by analyzing HCC or breast cancer data sets from TCGA (n = 369 for HCC and 1099 for breast cancer). Pearson’s coefficients and P values are indicated. Additional correlative data are shown in Additional file 1: Fig. S3F