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. 2012 Jul 30;109(33):13325–13330. doi: 10.1073/pnas.1120193109

Fig. 1.

Fig. 1.

N-terminal ASPP2 mediates oncogenic RAS-induced senescence and inhibits autophagy. (A) (Upper) Schematic representation of truncated ASPP2 mutants. HRAS V12-expressing ASPP2(Δ3/Δ3) MEFs, infected with retroviruses expressing full-length or mutant ASPP2 as indicated, were subjected to SA-β-gal staining to identify senescent cells. Images were taken with a ×20, air objective lens. (Right) Percentages of positive cells are shown in the graph. Error bars indicate SD. (B and C) HRAS V12-expressing ASPP2(+/+) or ASPP2(Δ3/Δ3) MEFs infected with retroviruses expressing either full-length or truncated ASPP2 mutants were injected s.c. into the flanks of nude mice. (B) Tumor volume was monitored twice weekly over 17 d. Each point represents the mean volume ± SD of four tumors. (C) After 17 d, tumors were removed and weighed. Results are shown as the mean ± SD of tumor weights. (D) ASPP2(+/+) or ASPP2(Δ3/Δ3) MEFs with or without HRAS V12 expression were treated with 50 μg/mL rapamycin for 24 h to induce autophagy. Long-lived protein degradation was scored as the percentage of trichloroacetic acid-soluble counts out of total radioactivity incorporated in a standard protein degradation assay. Error bars represent SD of three independent experiments. (E) Induction of LC3II in ASPP2(Δ3/Δ3) MEFs. Western blot shows LC3 expression in lysates from ASPP2(+/+) or ASPP2(Δ3/Δ3) MEFs, with or without HRAS V12 expression, treated with the indicated compounds. β-Tubulin was used as a loading control. A.A.S., amino acid starvation; FM, full medium containing 1% FBS. (Lower) Ratio of LC3II against LC3I was calculated by densitometry. Error bars indicate SD. (F) Representative confocal images of EGFP-LC3–infected MEFs, treated with or without 50 μg/mL rapamycin for 24 h. Images were taken with a ×63, oil-immersion objective lens. (G) Western blot of LC3 expression in lysates from HRAS V12-expressing ASPP2(+/+) or ASPP2(Δ3/Δ3) MEFs infected with retroviruses expressing full-length ASPP2 or truncated mutants as indicated. Positive controls of LC3 were obtained from ATG5(−/−) and WT (wt) MEFs.