Inhibition of ER stress-induced autophagy by pharmacological inhibitors triggered caspase–3/7 activation and apoptotic cell death in LLC-PK1 cells.

<p><b>A, B, and C.</b> LLC-PK1 cells were treated with the ER stress inducer tunicamycin (TN) for 12 h before treatment with the pharmacological autophagy inhibitors 10 mM 3-MA (<b>A</b>), 200 nM wortmannin (Wort) <b>(B),</b> or 50 μg/ml chloroquine (CHL) (<b>C</b>) for varying lengths of time as specified. Cell lysates (25 μg protein) were used for spectrofluorimetric caspase–3/7 activity assays. Control is untreated cells. Results are presented as mean±SEM of three independent experiments. *<i>P<</i> 0.05 compared with control or tunicamycin-treated cells. **<i>P</i> <0.01 compared with tunicamycin-treated cells. ***<i>P</i> <0.001 compared with tunicamycin-treated cells. <b>D.</b> LLC-PK1 cells were treated with the ER stress inducer tunicamycin (TN) without and with the indicated autophagy inhibitors. Cell lysates (50 μg protein) were processed for western blots to detect active caspase–3 using a cleaved caspase-3-specific antibody. β-actin served as a loading control. <b>E and F.</b> LLC-PK1 cells were treated with the ER stress inducer tunicamycin (1 μg/ml) for 12 h before treatment with 10 mM 3-MA (<b>E</b>) or 50 μg/ml chloroquine (<b>F</b>) for varying lengths of time as specified. The percentage of apoptotic cells under these treatments were determined by FACS analysis using the propidium iodide staining method. Results are represented as mean±SEM of three independent experiments. *<i>P<</i> 0.05 compared with control or tunicamycin-treated cells. **<i>P</i> <0.01 compared with tunicamycin-treated cells. ***<i>P</i> <0.001 compared with tunicamycin-treated cells.