Figure 2.

CQ sensitisation to cell death is independent of autophagy. (a) 4T1 cells were treated under full nutrient conditions with CQ (25 μM) for up to 72 h. Cell lysates were analysed for LC3 and Sequestosome 1/p62 protein levels by immunoblotting. Quantification is expressed as fold change in LC3-II/actin or p62/actin. (b) 4T1 cells were treated as above with CQ for 24 h and stained for LC3- and p62-associated membranes. (c–e) 4T1 cells expressing ATG7 shRNA were treated to amino-acid starvation (EBSS incubation) for 2 h. Cell lysates were analysed for ATG7 levels and lipidation of LC3 (c) or autophagosome number by staining for LC3 (d; scale bars: 10 μm). Autophagosomes in wild-type or shATG7-4T1 cells were quantified (expressed as mean±S.D. from >80 cells; two experiments). ***P<0.001 by paired t-test as compared with wild-type starved cells. (f) Wild-type or shATG7-expressing 4T1 cells were incubated in full-nutrient (Ctrl) or serum-free DMEM for 24 h before exchange back into fresh full-nutrient media to assess clonogenic viability. Genetic targeting of autophagy did not sensitise cells to serum starvation. All viability shown as Clonogenic Index relative to control (n=3 experiments ±S.E.M.). (g) Wild-type (left) or shATG7-(right) expressing 4T1 cells were incubated in full-nutrient (Ctrl) or serum-free DMEM ±CQ (25 μM) for 24 h before exchange back into fresh full-nutrient media to assess clonogenic viability. ***P<0.001 by one-way ANOVA as compared with paired no CQ condition. CQ shows efficacy in autophagy-deficient 4T1 cells. (h) Wild-type or ULK1/2 DKO MEFs were analysed for LC3 autophagosome number following 1.5 h starvation in EBSS as in (d and e). Mean±S.D. from >90 cells; three experiments. ***P<0.001 by paired t-test as compared with wild-type starved cells. (i) Wild-type or ULK1/2 DKO MEFs were analysed as in (g). Genetic targeting of autophagy did not sensitise MEF, and ULK1/2-deficient cells are still targeted by CQ