Supplementary Figure 1: Plasma membrane proteomics (PMP) defines the cell surface signature of senescence including NOTCH1.
From: NOTCH1 mediates a switch between two distinct secretomes during senescence
(a) ER:HRASG12V IMR90 cells with RAS-induced senescence (RIS), induced by 6 days of 4OHT or DNA-damage induced senescence (DDIS) induced by Etoposide, were analysed for SA-β-GAL staining (upper panels and lower left), presence of senescence-associated heterochromatic foci (SAHF) and DNA synthesis by BrdU incorporation (lower right). Manual counting of >200 cells; bars are means; unpaired t-test; n = 3 biologically independent experiments; ∗∗∗P ≤ 0.001. Scale bar 200 μm. (b) Correlation of log2 fold expression changes of 521 high-confidence PMP ‘hits’ at cell surface protein level by PMP (x axis) with transcriptomic changes by mRNA-Seq (y axis). Hits are colour-coded by significance level: green dots indicate a proteomic significance of P < 0.05; yellow indicate a transcript q < 0.01; red indicates significant in both PMP and transcript datasets. Correlation was calculated for all 521 (all), those with significance at transcript or protein level (anySig) or both transcript and protein (Commsig). FDR, false discovery rate. (c) Network enrichment analysis, with networks defined by the presence of a PMP hit and populated by transcript level data revealed the highest enriched network contained NOTCH1 as a major network hub. Other network members are shown by subcellular localisation; red indicates upregulation and green downregulation. Among four NOTCH receptors, only NOTCH1 was significantly upregulated in both PM proteomic and transcriptomic data from RIS cells. (d) ESF and WI38 human diploid fibroblast cell lines stably expressing oncogenic HRASG12V were analysed for cell surface NOTCH1 expression by flow cytometry. (e) ΔMEK1:ER IMR90 cells with and without induction by 4OHT were analysed for cell surface NOTCH1 expression by flow cytometry. Statistics source data for A are provided in Supplementary Table 2.
