Ischemic preconditioning (IPC) upregulated GRP78 and induced autophagy in PC12 cells. (A) PC12 cells were exposed to oxygen glucose deprivation (OGD) for 30 min to induce IPC. Twelve hours after IPC, the cells were subjected to OGD for 10 h. The cell viability was examined with cell counting kit-8 (CCK8) and an optical microscope. Scale bar = 100 μm. (B)-(D) The cells were harvested 0, 6, 12 and 24 h after IPC. (B) GRP78 was upregulated after IPC. (C) LC3II/LC3I was upregulated after IPC. (D) Beclin1 was upregulated after IPC. (E) Autophagic flux was examined by comparing accumulation of LC3-II with and without NH4Cl. NH4Cl 20 mM treatment was given during the IPC episode. Cells were harvested at 12 h after IPC. Bar represents mean ± SD, n = 3. * P < 0.05, ** P < 0.01, ***P < 0.001 compared with the control group; $$ P < 0.01 compared with the OGD group. (F) Representative electron microscopic images show increased number of double-membrane vacuolar structures in PC12 cells after IPC. PC12 cells were harvested at 12 h after IPC. The cells were fixed in 0.25% glutaradehyde and examined with a transmission electron microscope. N: nucleus. Arrows indicate typical autophagosomes. Insets in the pictures were enlarged autophagosomes taken from the areas indicated by arrows. Scale bar = 100 nm.