Fig. 3.

Cytosolic p53 inhibits Parkin-mediated mitophagy in β-cells. (A) The endogenous Parkin–p53 complex in β-cells. Cytosolic lysate of MIN6 β-cells exposed to 30 mM high glucose (HG) and 0.3 mM palmitate (PA) for 1 wk was immunoprecipitated with anti-p53, Parkin, and control IgG antibodies. (B) Representative images of GFP-LC3–overxpressing MIN6 β-cells exposed to HG and PA. Cells were transfected with siRNA targeting p53 and/or Parkin and were treated to underscore mitophagy with 100 nM Baf-A1, a specific inhibitor of H⁺-ATPases of the vacuolar type, for 6 h before the immunostaining of mitochondria with anti-TOM20 (red) (original magnification, ×1,000). (Scale bars, 10 μm.) (Right) Colocalization between LC3 and TOM20. A minimum of 50 GFP-positive cells were scored in three independent experiments. (C and D) MIN6 β-cells were stimulated with 2.8 or 22.2 mM glucose for 30 min. Insulin secretion (C) and ATP contents (D) were measured by ELISA and a luminometer, respectively, in duplicate (n = 4). (E) Glucose-stimulated insulin secretion was measured by ELISA in the context of mitophagy deficiency induced by the transfected with siRNA targeting Atg5 or PINK1. The experiments were performed in duplicate (n = 4). Data are shown as the means ± SD. *P < 0.05; **P < 0.01.