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

Figure 3

Impairment of antioxidant enzymes and elevation of ROS in a condition of inhibited autophagy is mediated by the decrease of FOXO1/3 transcription factors, and scavenging ROS by NAC not only fails to rescue the loss of FOXOs or antioxidant enzymes but even exacerbates their loss. (a) HEK293T cells were treated with autophagy inhibitors Bafi A1 (100 nM), CQ (50 μM), or 3-MA (10 mM) for 24 h or with 50 μM CQ for 0, 12, 24, or 48 hours before harvest, then protein levels of FOXO transcription factors and antioxidant enzymes were determined by W.B. (b and c) HEK293T cells were treated with 50 μM CQ for 24 hours before transfection with FOXO1 or FOXO3 plasmids, then followed by treatment with 50 μM CQ for another 48 hours before harvest, and then protein and mRNA levels of FOXOs and antioxidant enzymes were relatively determined by W.B. and q-RT-PCR () (b), and intracellular ROS levels were measured with a DCFDA probe (c) (). (d) Protein levels of FOXO1 and FOXO3 in Atg7-/- MEF cells compared with WT MEF cells, in siAtg5- or siBeclin1-transfected HEK293T cells compared with negative control siRNA transfected ones were determined by W.B. (e) Atg7-/- MEF cells were treated with 1 mM NAC for 48 hours before harvest, HEK293T cells were treated with 1 mM NAC for 48 hours and/or CQ for 24 hours before harvest, then protein levels of FOXOs and antioxidant enzymes were determined by W.B. in both cells, and mRNA levels of antioxidant enzymes were determined by q-RT-PCR () in MEF cells. Values are represented as . ^,#; ^,##; ^,###; vs. control group or WT MEF cells, ^# vs. treatment with CQ alone or Atg7-/- MEF cells.

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