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. 2017 May 10;8:14995. doi: 10.1038/ncomms14995

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Copyright © 2017, The Author(s)

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(a) Experimental schema of samples for microarray, RNA-seq, and RT-qPCR analyses. (b,c) Volcano plots summarizing the differentially expressed (DE) genes for WI-38hTERT fibroblasts induced into senescence with etoposide for 3 weeks versus proliferating fibroblasts. (b) Red dots: FDR<0.01. Blue dots: genes that are transcriptionally up-regulated in senescence and down-regulated by sh-H2AFJ (see below). (c) DE genes for senescent WI-38hTERT fibroblasts expressing sh-H2AFJ RNAs versus a control sh-NoTarget. Only genes that were affected by both the sh2-H2AFJ and sh3-H2AFJ RNAs are shown. Red dots: FDR<0.05. Blue dots: genes that are up-regulated in senescence in sh-NT cells. (d) Heat maps of log2 fold change for 71 genes that are up-regulated at least 2 fold in senescent versus proliferating fibroblasts (Sen/Pro) and down-regulated in senescence by knock-down of H2A.J (sh-H2AFJ Sen). These 71 genes are represented as blue dots in c. Also shown are the effects of ectopic expression of an sh2-resistant H2AFJ cDNA in senescent cells in which endogenous H2AFJ RNA is knocked-down with sh2 RNA (Rescue Sen), and the effect of ectopic expression of the sh-resistant H2AFJ cDNA in proliferating fibroblasts (Over-exp Pro). The asterisks indicate genes encoding proteins that are bound to the cell surface or secreted from cells, involved in immune response, or implicated in cellular senescence. (e) Volcano plot of DE genes in proliferating cells over-expressing H2AFJ. The blue dots show 110/346 up-regulated ‘immune system process' genes belonging to GO:0002376 (Supplementary Data 2). (f) GSEA enrichment plot (left panel) showing the ranking of interferon gamma response genes in the set of differentially expressed genes on over expression of H2AFJ in proliferating fibroblasts. Right panel: the corresponding heat map of differential expression for the top 50 IF-gamma-response genes is shown for proliferating cells over-expressing H2AFJ (H2AFJ OE) versus proliferating cells expressing sh-NoTarget or sh-H2AFJ RNAs (sh2 or sh3). Each row shows an independent biological replicate. NES, normalized enrichment score. FWER, family-wise error rate.