Figure 4
Glutamine deprivation and autophagy inhibition exacerbate Ras mutant cell survival.
(A and B) 8988 T cells were plated in the complete medium, which was replaced with glucose or glutamine-free medium the following day and then incubated for another 24 h with or without CQ (10 μM). Cell death was assessed by using the annexin V/PI assay (A) and cleaved caspase-3 and -9 expression was assessed by immunoblotting (B). Error bars represent the s.d. of three separate experiments. (C) 8988 T cells were treated with CQ (10 μM) and BPTES (10 μM) alone, or in combination for 24 h and cell death was determined by using the annexin V/PI assay. Error bars represent the s.d. of three separate experiments. (D and E) 8988 T cells expressing a control (shGFP) or ATG7 shRNAs (shATGs) were plated in the complete medium, which was replaced with glucose or glutamine-free medium the following day and then incubated for another 24 h. Cell death was assessed by using the annexin V/PI assay (D) and cleaved caspase-3 and -9 expression was assessed by immunoblotting. ATG7 knockdown was confirmed by western blot (E). Error bars represent the s.d. of three separate experiments. (F and G) WT MEFs and atg5−/− MEFs expressing KRas G12V were plated in the complete medium, which was replaced with glutamine-free medium the following day and then incubated for another 24 h. Cell death was assessed by using the annexin V/PI assay (F) and immunoblotted for PARP and cleaved caspase-3 expression was assessed by immunoblotting (G). Error bars represent the s.d. of three separate experiments. (H) Subcutaneous MIAPaCa2-driven tumors were established in 6-week old male mice. CQ (20 mg kg−1 per day), BPTES (4 mg kg−1 per day) alone, or in combination, were administered daily via intraperitoneal injection. Tumor growth was assessed once the tumor volume reached 150 mm3. Data are shown as the mean of five mice in each group ± SEM. NS, not significant. **p < 0.01.
