Figure 6
Inhibition of metabolic pathways providing substrates for the TCA cycle induces apoptotic cell death in PDAC.
(A) 8988Tcells were plated in the complete medium, which was replaced by glutamine-free medium the following day and then incubated for another 24 h with or without EIPA (25 μM). Glutamine levels were monitored by using LC-MS/MS. Error bars represent the s.d. of triplicate wells from a representative experiment. (B and C) PDAC cells were plated in the complete medium which was replaced by glutamine-free medium the following day and then incubated for another 24 h with or without EIPA (25 μM) for 24 h. Cell death was assessed by using the annexin V/PI assay (B) and cleaved-3, -9 and PARP expression was determined by immunoblotting (C). Error bars represent the s.d. of three separate experiments. (D) 8988 T cells expressing a control shRNA (shGFP) or TFEB shRNAs (shTFEBs) were plated in the complete medium, which was replaced by glucose or glutamine-free medium the following day and then incubated for another 24 h. Cell lysates were immunoblotted for cleaved caspase-3 and PARP. TFEB knockdown was confirmed by western blot. (E) TCA metabolite pools were analyzed by using LC-MS/MS in 8988 T cells expressing a control (shGFP) or ATG7 shRNA. Error bars represent the s.d. of triplicate wells from a representative experiment. (F) 8988 T cells were plated in the complete medium, which was replaced by glutamine-free medium supplemented with NAC (2 mM), GSH (2 mM), or MAKG (2 mM) in the absence or presence of CQ (10 μM) the following day and then incubated for another 24 h. NAC, N-acetylcysteine; GSH, glutathione; MAKG, dimethyl α-ketoglutarate. Error bars represent the s.d. of triplicate wells from a representative experiment. *p < 0.05; **p < 0.01.
