Fig. 3

RSK3 reverts senescence by modulating NF-κB activity. A, B Transcriptome analyses (n = 3) were performed 48 h after cell treatment with TGFβ. A Strategy to identify transcriptional program altered by TGFβ and RSK3 expression. Identification by Venn diagram and TFacts software of NF-κB target genes found in common between up-regulated genes in TGFβ-treated HMECT-pWZL (Ctrl TGFβ) versus non-treated HMECT-pWZL (Ctrl NT) conditions and down-regulated genes in TGFβ-treated HMECT-pWZL/RSK3 (RSK3 TGFβ) versus TGFβ-treated HMECT-pWZL (Ctrl TGFβ) conditions. B GSEA enrichment plots related to NF-κB signaling. C, D HMECT were infected with pBABE (Ctrl) or pBABE/IκBα super repressor (IκBα) and selected for 3 days with puromycin, then plated for indicated assays, followed by TGFβ treatment on the next day when indicated. C Western blot validation of IκBα overexpression. D Cell counts (n = 4 independent experiments). E RT-QPCR of SASP factor expression (n = 4 independent experiments). (F–H) HMECT were infected with pWZL/RSK3 (RSK3) or control pWZL (Ctrl) vectors, selected for 1 week with neomycin and plated for indicated assays, followed by TNFα treatment (TNFα) or not (NT) on the next day. F EdU pulse and cell count. EdU was added 2 days after treatment with TNFα (n = 3 independent experiments). Cell counts were performed 7 days after TNFα treatment (n = 4 independent experiments). G SA-β-Gal assay was performed 7 days after treatment with TNFα (n = 3 independent experiments). H RT-QPCR was performed on the indicated SASP encoding mRNAs (n = 3 independent experiments). Ratio-paired t-test was used to determine statistical significance