HMGB2 expression is altered during senescence. (A) Using publically available microarray databases, the most significantly altered chromatin-related genes during senescence were identified. Only genes that were significantly (P < 0.05) altered more than twofold in all four datasets were considered “hits.” Study #1: GEO accession no. GSE28464; study #2: GEO accession no. GSE40349; and study #3: GEO accession no. GSE60652. FC, fold change; FDR, false discovery rate; OIS, oncogene-induced senescence; RS, replicative senescence. (B) IMR90 cells were infected with retrovirus encoding oncogenic RAS to induce senescence or control. Cells were selected for 3 d with 1 µg/ml puromycin. 4 d later, HMGB2 mRNA expression was determined. (C) Same as B, but HMGB2 protein expression was determined in total protein lysates by immunoblot. β-Actin was used as a loading control. (D) HMGB2 mRNA expression was determined in young (PD24) and old senescent (PD60) IMR90 fibroblasts. (E) HMGB2 mRNA expression was determined in senescent G4 mTerc^−/− or wild-type control ear fibroblasts. (B, D, and E) B2M expression was used as an internal control. (F) Same as B, but total cell lysates (TCL), cytoplasmic fraction (Cyto), nuclear soluble fraction (Nuc), and chromatin-bound fraction (Chromatin) were isolated, and HMGB2 and HMGB1 protein expression was determined. Long and short film exposures are shown for HMGB1. Histone H3 and β-actin were used as loading controls. For all panels, graphs shown are the mean and SEM of triplicates from a representative experiment that was independently repeated at least three times. *, P < 0.05 versus control.