OGD-Rep induces global mitochondrial loss in neurons. (A and B) Primary cultured neurons were treated with PBS as a vehicle control or 50 µM chloroquine for 4 h and then subjected to either 1 h of OGD or 20 min of OGD plus 40 min of reperfusion (O-R) in DIV8. (A) The COX IV, Tim23, p62, LC3B, and GAPDH levels in cultured neurons were determined by Western blot. (B) Semiquantitative analysis of COX IV, Tim23, p62, and LC3B-II bands is shown. (C and D) Primary cultured neurons were subjected to 20 min of OGD in DIV8. The mitochondrial marker Hsp60 and neuronal marker β-tubulin III were stained by immunocytochemistry at 40 min after reperfusion. (C) Images show the representative straightened cultured neurons and the line-scan analysis of axonal mitochondria (lower panel). (D) Columns represent the somatic mitochondrial area (left panel) and axonal mitochondrial area per 100-µm axon (right panel). n = 30–60 cells in each group from three independent experiments were included. The data are expressed as means ± SEM. Statistical comparisons were performed with one-way ANOVA with Sidak’s multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001 versus the indicated group. Scale bar, 10 µm. A.U., arbitrary units; Ctrl, control; Mito, mitochondrial.