Figure 4.

HRAS^G12V-induced senescence and C/EBPβ activation in MEFs is dependent on AMPKα2. (a) RAS-induced growth arrest and senescence is independent of AMPKα1 but requires AMPKα2. WT and Ampka1^−/− MEFs were infected with control or HRAS^G12V-expressing retroviruses, and cell proliferation (left panel) and senescence (SA-β-Gal staining, right panel) were analyzed. n=1 experiment. b) RAS-induced growth arrest and senescence requires AMPKα2. WT and Ampka2^−/− MEFs were infected with control or HRAS^G12V-expressing retroviruses, and cell proliferation (left panel) and senescence (SA-β-Gal staining, right panel) were analyzed. n=3 experiments for growth curves (assayed in triplicate), n=2 for SA-β-Gal assays; error bars represent S.E.M. Statistical differences were determined by Student’s t test; *p<0.05. (c) C/EBPβ DNA binding is not affected by loss of AMPKα1. AMPKα1 was depleted in WT MEFs by shRNA knockdown, without or with expression of HRAS^G12V. Nuclear extracts were analyzed by EMSA using a canonical C/EBP site probe. (d) RAS-induced activation of C/EBPβ DNA binding is disrupted in Ampka2^−/− MEFs. Nuclear extracts were analyzed by EMSA using a canonical C/EBP site probe. Levels of C/EBPβ, p-ERK and total ERK are also shown. (e) Analysis of SASP gene expression in WT and Ampka2^−/− MEFs by qRT-PCR. n=2 biological replicates, each sample assayed in triplicate. Values are averages and error bars represent S.D. Statistical significance was calculated using Student’s two-tailed t test; *p<0.05.