Fig. 6: Sirt5 is a transcriptional target of ATF4 necessary for survival. | Cell Death Discovery

Fig. 6: Sirt5 is a transcriptional target of ATF4 necessary for survival.

From: Elucidation of an mTORC2-PKC-NRF2 pathway that sustains the ATF4 stress response and identification of Sirt5 as a key ATF4 effector

Fig. 6

A Western blot analysis of WCL from control or ATF4 KD MDA-MB-231 cells treated with CB-839 (1 μM) for 8 h or without glutamine for 24 h. Blots are representative of three independent experiments. B Western blot analysis of WCL from control or ATF4 KD BT549 cells treated with CB-839 (1 μM) or without glutamine for 24 h. Blots are representative of two independent experiments. C RT-qPCR quantification of Sirt5 mRNA in control and ATF4 KD MDA-MB-231 and BT549 cells treated with CB-839 (1 μM) for 8 h (MDA-MB-231) or 24 h (BT549). Data shown are representative of two independent experiments and expressed as means ± SD for triplicate measurements. D RT-qPCR quantification of Sirt5 and ATF4 mRNA in control and ATF4 KD BT549 cells treated with CB-839 (1 μM) ± AI-1 (10 mM) for 24 h. Data shown are representative of two independent experiments and expressed as means ± SD for triplicate measurements. Differences were analyzed with two-way AVOVA. E ChIP analysis of ATF4 binding to the Sirt5 promoter in MDA-MB-231 cells treated CB-839 (1 μM) or without glutamine (2% dialyzed FBS) for 8 h. Data shown are representative of two independent experiments and expressed as means ± SD for triplicate measurements. F The percentage of TUNEL positive cells (n = 4). Data shown are representative of two independent experiments. G CCK8 assay showing the percent inhibition of cell viability for control, ATF4 knockdown and ATF4 knockdown cells with the ectopic expression of Sirt5. MDA-MB-231 cells were cultured in glutamine free medium for 48 h. Data shown are representative of two independent experiments and expressed as means ± SD for triplicate measurements.

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