Figure 1.

H-ras^V12-expressing cells are dependent on autophagy to survive starvation. (A) Autophagy-competent (atg5^+/+ and atg7^+/+) or authophagy-deficient (atg5^−/− and atg7^−/−) cells expressing H-ras^V12 or vector were transiently transfected with the fluorescent autophagosome marker p-tFL-LC3 and subjected to starvation. Representative images depict RFP-LC3 localization. Numbers indicate percentage of cells with LC3 translocation to autophagosomes (punctate localization). (B) Evaluation for processing of endogenous LC3-I to LC3-II indicative of autophagy induction and activation of caspase-3 (apoptosis). (C) Ras-expressing cells were treated with HBSS for 12–20 h, and cell viability was examined by a trypan blue exclusion-based cell viability analyzer and normalized to untreated cells at the time of initiation of starvation. (D) Cells treated as in C were allowed to recover in normal medium for 2 d and assessed for clonogenic survival.