Leukemogenic fusion proteins induce senescence in human primary fibroblasts. (A) IMR90 cells expressing empty vector, H-RAS V12, or a leukemogenic fusion protein were monitored for proliferation by trypan blue viability assay. Error bars represent SEM. Quantitative reverse transcription–polymerase chain reaction confirmed that the expression level of each oncogene was comparable (supplemental Figure 5). (B) IMR90 cells expressing empty vector, H-RAS V12, or a leukemogenic fusion protein were stained for senescence-associated β-galactosidase. (C) Immunoblot analysis monitoring H3K9 acetylation (H3K9 Ac) in IMR90 cells expressing empty vector, H-RAS V12, or a leukemogenic fusion protein. β-Actin (ACTB) was monitored as a loading control. (D) Immunoblot analysis monitoring p16^INK4a levels in IMR90 cells expressing empty vector, H-RAS V12, or a leukemogenic fusion protein. (E) IMR90 cells expressing empty vector, H-RAS V12, or a leukemogenic fusion protein were stained for γ-H2AX and analyzed by fluorescence microscopy. (Right) Quantification of γ-H2AX staining. Cells with more than 10 γ-H2AX foci were included in the analysis. Error bars represent SEM. (F) IMR90 cells expressing empty vector, H-RAS V12, or a leukemogenic fusion protein were analyzed by comet assay under alkali conditions, and comet tail length was quantified. Error bars represent SEM. (G) Immunoblot analysis monitoring levels of phospho-ATM and, as a control, total ATM in IMR90 cells expressing empty vector, H-RAS V12, or a leukemogenic fusion protein. (H) BrdU incorporation assay in IMR90 cells stably expressing either a nonsilencing (NS) shRNA or one of 2 unrelated shRNAs directed against ATM, and expressing empty vector, H-RAS V12, or a leukemogenic fusion protein. Error bars represent SEM. (I) Immunoblot analysis of phosphorylated p39 (P), total p38 (T), and p16^INK4a levels in IMR90 cells expressing empty vector, H-RAS V12, or a leukemogenic fusion protein treated with dimethyl sulfoxide (DMSO) or the p38 MAPK inhibitor SB203580.