Figure 4.

PJA1 inhibits SMAD3 transcriptional activity. (A) Genes co-regulated by PJA1 and TGF-β1. RNAseq analyses of genes regulated by PJA1 knockdown or exposure to TGF-β1 in HepG2 cells. (B) Negative association between PJA1 mRNA expression and the TGF-β target gene, FOS and SERPIN1 in HCC patients. Wurmbach Liver and Mas Liver mRNA microarray datasets from Oncomine were downloaded and analyzed. Significance was determined by Pearson correlation. (C) The expression of TGF-β-regulated genes in PJA1 knockdown cells. Transcripts were quantified by quantitative RT-PCR from HepG2-shCtrl or HepG2-shPJA1 cells (Supplementary Materials and Methods). *: P < 0.05, two-tailed Student’s t- tests. (D) Effect of PJA1 on SMAD3-dependent reporter gene activity. HepG2 cells were cotransfected with the luciferase reporter constructs controlled by 4 copies of the SBE (SBEx4) (left panel) or 3TP (right panel) along with Flag-tagged SMAD3 and HA-tagged PJA1 as indicated. Where indicated, cells were exposed to 200 pM TGF-β1 for 1 hour. (E) Effect of PJA1 knockdown on binding of SMAD3 and β2SP at the TERT promoter. HepG2-shCtrl or HapG2-shPJA1cells were treated with 200 pM TGF-β1 for 1 hour. Chromatin immunoprecipitation (ChIP) analysis was performed with antibodies against the indicated proteins and the interaction with the TERT promoter was assessed. IgG served as a negative control (Supplementary Materials and Methods). For D and E*: P < 0.05, **: P < 0.01, ***: P < 0.001, 1-way ANOVA with post-hoc Bonferroni’s test. For C, D and E, data are shown as mean ± standard deviation of three independent experiments. (F) A model for the regulation of SMAD3 and β2SP by PJA1.