Fig. 6: ATF4 leads to TM cell death in a CHOP and GADD34-dependent manner.

GTM3 cells were transduced with Ad5.Empty, ATF4, or CHOP for 36 h. a Cellular lysates were subjected to Western blot analysis for apoptotic markers (n = 3 independent experiments). b Fixed GTM3 cells transduced with Ad5.Empty, ATF4, or CHOP were analyzed by TUNEL assay. Increased number of TUNEL-positive cells (green) were observed in Ad5.ATF4-transduced TM cells (n = 3 independent experiments). Scale bar is 50 µm. c Increased number of TUNEL-positive cells (green) were observed in the TM region of 2 weeks Ad5.ATF4-injected mice compared to Ad5.Empty or Ad5.CHOP (n = 3 in each groups). Bright field images (bottom panel) merged with DAPI shows TM orientation and the TM area is represented in a white box (scale bar = 50 µm). d and e GTM3 cells were transduced with Ad5.Empty, ATF4, CHOP, or ATF4 + CHOP KO (CRISPR-Cas9 vector targeting CHOP) for 36 h. Western blot d and densitometric analyses e demonstrated that depletion of CHOP significantly reduced ATF4-induced cleaved PARP suggesting that CHOP is required for ATF4-mediated cell death. n = 3 independent experiments, data are presented as mean ± SEM, *p < 0.05, one-way-ANOVA. f and g Western blot f and densitometric analyses g of cleaved PARP in GTM3 cells transduced with ATF4 and treated with mild dose of CHX (2.5 μg/ml) demonstrated that reduction of protein synthesis prevents ATF4-induced TM cell death. n = 3 independent experiments, data are presented as mean ± SEM, one-way-ANOVA. h GTM3 cells expressing ATF4 were transfected with plasmid expressing CRISPR-Cas9 targeting GADD34. Puromycin (10 µg/ml) was added to cells for 30 min before harvesting cell lysates. Total cellular lysates were subjected to Western blot analysis using anti-puromycin and anti-GAPDH antibodies (shown in Supplementary Fig. 17). Densitometric analysis demonstrated that GADD34 knock down significantly reduces ATF4-induced protein synthesis. n = 4 independent experiments, data are presented as mean ± SEM, two-tailed unpaired t-test. i and j Western blot i and densitometric analyses j of cleaved PARP and GADD34 of GTM3 cells expressing ATF4 and plasmid expressing CRISPR-Cas9 targeting GADD34 demonstrated that knockdown of GADD34 significantly reduced ATF4-induced cleaved PARP and GADD34 protein levels. n = 3 independent experiments, data are presented as mean ± SEM, **p < 0.01, two-way-ANOVA.