Figure 3.

Bnip3 induces autophagy independent of Ca²⁺, ROS and mPTP opening. (A) Starvation-induced autophagy is inhibited in the presence of 0.2 µM BAPTA-AM, 1 µM CsA or 1 mM NAC in adult myocytes (n = 3, *p < 0.05 compared with non-starved cells). (B) Bnip3-mediated autophagy is not inhibited in the presence of 0.2 µM BAPTA-AM, 1 mM NAC or 1 µM CsA. (C) Quantification of Bnip3-mediated autophagy in the presence of DMSO or inhibitors (*, **, *** and ****p ≤ 0.05 compared with control, n = 3). (D) The presence of CsA failed to inhibit Bnip3-mediated mitochondrial autophagy. Representative image of an adult myocyte infected with Bnip3 for 24 h in the presence of 1 µm CsA. Maximum intensity projection of a cardiac myocyte observed along the XY (center), XZ (top) and YZ (right) planes. Colocalization scatter plot of mitochondria (quadrant 1) and LC3-GFP (quadrant 2). Quadrant 3 represents colocalization of mitochondria and LC3GFP. (E) Mouse embryonic fibroblasts isolated from WT and CypD^-/- mice were infected with adenoviruses encoding µ-gal or Bnip3 plus GFP-LC3 for 24 hours and then examined by fluorescence microscopy. Shown are representative images. (F) Quantitation of autophagy (*p ≤ 0.05 compared with control, n = 3).