Fig. 3. Knockout of LAMP1 and LAMP2 sensitizes to nutrient depletion-induced cell death. (A) Cytoplasmic vacuolization induced by nutrient depletion in mouse embryonic fibroblasts (MEF) with a normal genotype (WT) or lacking both LAMP1 and LAMP2 (double knockout, DKO). Cytoplasmic vacuoles are visualized as CMFDA-negative holes. (B, C) LC3 accumulation observable in DKO cells. WT or DKO cells were first transfected with LC3-GFP (24 hours) and then nutrient-depleted for 15 hours (B) or for the indicated period (C), followed by quantitation of the frequency of cells with LC3 accumulation in cytoplasmic vacuoles and that of cells with manifest chromatin condensation, as detectable by counterstaining with Hoechst 33342. (D) WT or DKO cells were cultured in complete medium (CM) or nutrient-free (NF) medium for the indicated time, stained with PI and DiOC₆(3) and subjected to cytofluorometric analysis (X±s.d., n=3). (E) Activation of caspase-3 WT and DKO cells, 24 hours after culture in the presence or absence of nutrients. Cells were stained with an antibody recognizing mature, cleaved caspase-3 and the percentage of cells containing active caspase-3 was quantified by immunofluorescence. Bar in A and B, 10 µm.