Fig. 2

TGFβ represses RSK3 expression through SMAD3. A RSK3 expression decreased after TGFβ treatment. Transcriptome analyses (n = 3) were performed 48 h after cell treatment with TGFβ and RSK3 mRNA levels are shown. Statistical significance was determined with unpaired t-test. B Western blot validation of RSK3 protein decrease. C, D Cells were infected with pLPCX (Ctrl), or pLPCX/SMAD3 (SMAD3). C SMAD3 constitutive expression (left) and RSK3 gene expression (right). RT-QPCR was performed 72 h after infection (n = 3 independent experiments). D Western blot to assess RSK3 decrease induced by SMAD3 constitutive expression 3 days after infection (representative of 2 experiments). E ChIP-seq data analysis of SMAD3 binding in HMEC treated by TGFβ (TGFβ) or not (Ctrl) were visualized in UCSC genome browser after BigWig files uploading (GSM2807302 for non-treated cells and GSM2807304 for TGFβ-treated cells). F-G HMECT were infected with pWZL or pWZL/RSK3 and selected for 1 week with neomycin and next were infected with pLPCX or pLPCX/SMAD3. F Crystal violet assay for cell density 10 days after overexpression of SMAD3 (representative of 2 experiments). G Levels of mRNA encoding SASP factors. RT-QPCR was performed 3 days after infection with SMAD3 (n = 3 independent experiments). Ratio-paired t-test was used to determine p-value