Fig. 2

CHTM1 deficiency-associated metabolic stress-induced cell death is caspase-independent. CHTM1 knockdown and scrambled A549 lung cancer cells were growing in regular media or glucose/glutamine-depleted media (for 4 h). Western blot analyses (a) showing increase in PARP cleavage but no effect on procaspase levels in glucose/glutamine-starved CHTM1 knockdown cells. (b) MTT assay showing decreased cell survival of CHTM1 knockdown cells compared to scramble cells under glucose/glutamine-deprived conditions in the presence or absence of 20 μM Z-VAD-FMK (pan-caspase inhibitor). (c) Representative fluorescent photomicrographs showing increase in DCF-DA (red) stained reactive oxygen species in CHTM1 knockdown A549 cells. Scale bar, 50 μM (d) Relative levels of ROS and RNS in glucose/glutamine starved (for 4 h) CHTM1 knockdown A549 cells. (e) Relative levels of ROS and RNS in 50 mM metformin treated (12-h) CHTM1 knockdown A549 cells. DCF-DA for ROS and DAF-FM for RNS were used and analyses done by spectrophotometry. (f) Western blot analyses showing increased phosphorylation H2AX in CHTM1 knockdown cells under glucose/glutamine-deprived condition