Autophagy Deficiency Leads to Impaired Antioxidant Defense via p62-FOXO1/3 Axis

Figure 2

Mitochondrial dysfunction, increased ROS, decreased capacity to survive oxidative stress, and impairment of antioxidant enzymes also occur when Atg7 is deleted or other autophagy components are knocked down by siRNA. (a) Mitochondrial membrane potential (MMP) in WT and Atg7-/- MEF cells () was detected with the JC-1 probe. (b) Relative mRNA levels of subunits of mitochondrial respiratory complexes were determined by quantitative RT-PCR (), relative protein levels of subunits of mitochondrial respiratory complexes were determined by W.B., and relative activities of mitochondrial respiratory complexes I and II were measured in isolated mitochondria from WT and Atg7-/- MEF cells (n=8). (c) HEK293T cells were transfected with negative control siRNA, siAtg5, or siBeclin1, then OCR was determined using the Seahorse XF24 Analyzer (). (d) ROS level changes and mitochondrial superoxide level changes in WT and Atg7-/- MEF cells were detected by flow cytometry using 5 μM DHE or 5 μM MitoSOX Red Mitochondrial Superoxide Indicator. (e) WT and Atg7-/- MEFs were treated with 1 mM tBHP for the indicated time period, and their cell viability was determined by MTT (). (f) mRNA levels of Cu-ZnSOD, MnSOD, and catalase in WT and Atg7-/- MEF cells were determined by q-RT-PCR (); protein levels of antioxidant enzymes in Atg7-/- MEF cells in comparison to WT or siBeclin1- or siAtg5-transfected HEK293T cells in comparison to negative control siRNA were determined by W.B.; relative enzymatic activities of antioxidant enzymes were measured in WT and Atg7-/- MEF cells (). Values are represented as . ; ; ^,###; vs. control group or WT MEF cells, ^# vs. WT MEF cells with the same treatment time period.

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