Extended Data Fig. 3: Evolution of transcriptomic changes during progression of cellular senescence.

RNA-seq was performed on early proliferating LF1 cells and cultures at 8 weeks (SEN-E) and 16 weeks (SEN-L) in senescence (points C and D, respectively, in Extended Data Fig. 1a). Data were analysed using a three-way comparison: EP versus SEN-E, EP versus SEN-L and SEN-E versus SEN-L (see Methods for details). a, Area-proportional generalized Venn diagrams depicting the intersections of the three comparisons for the following datasets. i–ii, Significantly upregulated and downregulated genes (row ‘2×’ in b). iii–iv, Significant KEGG pathways identified by gene set enrichment analysis (GSEA). Note the considerable evolution of the transcriptome in late senescence, exemplified by large changes (especially upregulated) in differentially expressed genes as well as pathways. v–vi, Significantly changing genes in the IFN-I and SASP gene sets (see Supplementary Table 4 for annotation of gene sets). Note that most changes in SASP genes occur early, whereas a large component of IFN-I changes is specific for late senescence. b, Summary of significantly changing genes using a fixed false discovery rate (FDR) (<0.05) and variable fold-change cut offs (2×, 1.75× and 1.5×). c, GSEA analysis of KEGG pathways. Heat map representation shows significantly upregulated pathways in red (also see e) and downregulated pathways in blue. Non-significant comparisons are shown in black; vertical annotations refer to Venn diagrams in a, iii–iv. Note that the SASP gene set is upregulated early, whereas the IFN-I gene set is upregulated late. d, Heat maps of significantly changing genes in the IFN-I and SASP gene sets. Vertical annotations refer to Venn diagrams in a, v–vi. e, List of significantly upregulated KEGG pathways identified using GSEA (see Supplementary Table 5 for a list of all pathways). NES, normalized enrichment scores. IFN-I and SASP gene sets are highlighted in yellow. Note the significant upregulation of IFN-I between early and late senescence. Red font identifies KEGG pathways indicative of cytosolic DNA sensing and a type I interferon response at late times. f, g, GSEA profiles of the IFN-I and SASP gene sets for all comparisons; FDR is highlighted in yellow. Note that the upregulation of IFN-I is significant for EP_SEN-L and SEN-E_SEN-L but not for EP_SEN-E, and that the upregulation of SASP is significant for EP_SEN-E and EP_SEN-L but not SEN-E_SEN-L. n = 3 independent biological samples. Differential expression data were analysed for significance using the GSEA GenePattern interface and the outputs were corrected for multiple comparisons by adjusting the nominal P values using the Benjamini–Hochberg method (see Methods for details).