Figure 7. ATF4 is activated through the ISR. (A) Western blot analysis showing the increased phosphorylation of eIF2α (Ser51) upon 6 h of treatment with the different mitochondrial stressors. Bottom, ratio between P-eIF2α and eIF2α total levels. (B and C) mRNA expression analysis of ATF4 and its target genes, CHOP (DDIT3), ASNS, CHAC1, PCK2, and the ER stress marker BIP, upon 6 h of treatment with the different mitochondrial stressors and the ER stressor tunicamycin (Tn at 2.5 µg/ml) in HeLa cells, together with the inhibitor of the integrated stress response (ISRIB at 500 nM). (D) Boxplots showing an increase in basal and ATP-dependent respiration of HeLa cells treated with 500 nM of ISRIB for 24 h. OCR: oxygen consumption rate. (E) mRNA expression analysis of eIF2α kinases upon knock down with specific shRNAs. Data are presented as mean ± SEM of two independent shRNAs for each gene. Statistical differences were calculated compared with pLKO1. (F) mRNA expression analysis of ATF4 and some of its target genes upon knock down of the eIF2α kinases and 6 h of treatment with FCCP. Data are presented as mean ± SEM of two independent shRNAs for each gene. No statistical differences were found between the FCCP treated conditions. All experiments were independently performed at least two times, using triplicates for each condition; data are presented as mean ± SEM of a representative experiment; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Acti, actinonin; Dox, doxycycline; MB, MitoBloCK-6.
Figure 7.

ATF4 is activated through the ISR. (A) Western blot analysis showing the increased phosphorylation of eIF2α (Ser51) upon 6 h of treatment with the different mitochondrial stressors. Bottom, ratio between P-eIF2α and eIF2α total levels. (B and C) mRNA expression analysis of ATF4 and its target genes, CHOP (DDIT3), ASNS, CHAC1, PCK2, and the ER stress marker BIP, upon 6 h of treatment with the different mitochondrial stressors and the ER stressor tunicamycin (Tn at 2.5 µg/ml) in HeLa cells, together with the inhibitor of the integrated stress response (ISRIB at 500 nM). (D) Boxplots showing an increase in basal and ATP-dependent respiration of HeLa cells treated with 500 nM of ISRIB for 24 h. OCR: oxygen consumption rate. (E) mRNA expression analysis of eIF2α kinases upon knock down with specific shRNAs. Data are presented as mean ± SEM of two independent shRNAs for each gene. Statistical differences were calculated compared with pLKO1. (F) mRNA expression analysis of ATF4 and some of its target genes upon knock down of the eIF2α kinases and 6 h of treatment with FCCP. Data are presented as mean ± SEM of two independent shRNAs for each gene. No statistical differences were found between the FCCP treated conditions. All experiments were independently performed at least two times, using triplicates for each condition; data are presented as mean ± SEM of a representative experiment; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Acti, actinonin; Dox, doxycycline; MB, MitoBloCK-6.

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