Fig. 1

RSK3 inhibits TGFβ-induced senescence. A Schematic diagram of the genetic screen used. hTERT-immortalized human mammary epithelial cells (HMECT) were seeded in 96-well plates and infected with one kinase per well (n = 2 per kinase). After 24 h, cells were treated with TGFβ, and 72 h later cells were stained with Hoechst 33342 nucleic acid stain. Plates were read under a Cytell system and ranked according to the nuclei count. B-F HMECT were infected with pWZL/RSK3 (RSK3) or control vector pWZL (Ctrl), selected for 1 week with neomycin and plated for indicated assays, followed by TGFβ treatment (TGFβ) or not (NT). B Western blot validation of RSK3 overexpression. C Crystal violet assay for cell density 10 days after treatment with TGFβ (representative of 5 experiments). D GSEA enrichment plots related to cell proliferation. GSEA has been done on transcriptome data comparing global gene expression between TGFβ-treated HMECT-pWZL and non-treated HMECT-pWZL conditions (Ctrl TGFβ vs NT) or TGFβ-treated HMECT-pWZL/RSK3 and TGFβ-treated HMECT-pWZL conditions (RSK3 TGFβ vs Ctrl TGFβ). For each condition three independent experiments have been analyzed by transcriptomic (n = 3). False Discovery Rate (FDR) pValue and Normalized Enrichment Score (NES) are indicated. E RT-QPCR was performed 5 days after TGFβ treatment (n = 4 independent experiments). F SA-β-Gal assay, 7 days after treatment with TGFβ (n = 5 independent experiments). Scale bar = 50 μM. G Domains of RSK3 and mutated sites to generate kinase-deficient mutants are represented. Two kinase domain mutants were generated. H HMECT were infected with pWZL/RSK3 (RSK3), pWZL/RSK3 K100R (RSK3 K100R), pWZL/RSK3 K464R (RSK3 K464R) and pWZL control (Ctrl). Crystal violet assay for cell density 10 days after treatment with TGFβ (representative of 3 experiments). Ratio-paired t-test was used to determine statistical significance