Figure 5. H2A.J is required for normal expression of SASP genes.

(a) GSEA enrichment plot showing the ranking of 38 SASP genes within the set of genes differentially-expressed on knock-down of H2A.J in senescent WI-38hTERT fibroblasts. (b) Corresponding heat maps for the 38 SASP genes. Each row represents an independent biological replicate of cells expressing sh3-H2AFJ or sh-NoTarget. Genes are listed by highest (left) to lowest (right) expression in the sh-NoTarget cells. (c) RT–qPCR verification of the effect of H2A.J knock-down on CXCL1, CXCL2, CXCL3, and IL1B expression in senescent cells (SEN). Values were normalized to proliferating fibroblasts expressing an sh-No Target RNA (PRO-NT). Shown are 3 biological replicates for senescent cells expressing No Target or sh-H2AFJ RNAs. Error bars represent mean±s.d. t-tests with *P<0.05, **P <0.001 or not significant (NS). (d) Time course of CXCL1, CXCL5, CXCL6 derepression during etoposide-induced senescence for cells expressing an sh-NoTarget or the indicated sh-H2AFJ RNA. The maximal expression for each gene as determined by qPCR was normalized to 100% (3 technical replicates). (e) Heat map showing genes significantly down-regulated in senescence after knock-down of H2AFJ by sh2-RNA as determined by RNA-sequencing. (f) Heat map constructed with the pheatmap R package showing the scaled concentrations of 52 human cytokines, chemokines, growth or adhesion factors in the conditioned media of WI-38hTERT fibroblasts expressing sh-NT or sh3-H2AFJ RNAs in proliferation or senescence. See Supplementary Fig. 8 for dot plots showing concentrations (pg ml⁻¹) of secreted factors that are differentially affected by H2A.J knock-down.