Figure 5.

VCP inhibitors disrupt intracellular amino acid homeostasis. (a) VCP inhibitors trigger EIF2AK4 phosphorylation. Representative immunoblots with antibodies against phosphorylated (Thr889) and total EIF2AK4 (GCN2) on whole-cell extracts from A549 cells treated with DBeQ, NMS-873, bortezomib (Btz), or tunicamycin (Tm). (b) Cell viability of A549 and OPM-2 cells growing in complete media (+AA) or media deficient in L-glutamine, L-methionine, and L-cystine (–AA). Cells were treated with DBeQ (15 μM for A549 cells and 10 μM for OPM2 cells) or NMS-873 (10 μM for A549 cells and 5 μM for OPM-2 cells) for 16 h. (c) Representative images of A549 cells treated as in (b). Images shown were taken with an ECOS core digital microscope at × 100 magnification (size bar: 25 μm). (d) mRNA levels of the indicated genes relative to untreated controls determined by real-time quantitative PCR after treatment of A549 cells as described in (b). Data shown in (b) and (d) are the mean±S.E.M. from three independent experiments. *P<0.05, **P<0.01, ***P<0.001