CHX caused the CE increase even in autophagy-deficient cells.

<p>(A) 3Y1 cells were treated without or with either 10 µg/ml CHX or 0.4 mM OA for 18 hr. CHX caused a significant increase in phospho-S6K. (B) The autophagic flux was examined by incubating 3Y1 cells with 20 µM chloroquine for 1 hr immediately before sample preparation. Chloroquine caused a significant increase in LC3-II in the control, but not in cells pretreated with 10 µg/ml CHX for 18 hr. (C) Wild-type and Atg5-null MEF were treated without or with 10 µg/ml CHX for 18 hr. The increase in phospho-S6K was observed in a comparable degree in both cell lines. GAPDH was probed as a loading control. (D) Wild-type and Atg5-null MEF were treated without or with 10 µg/ml CHX for 18 hr. CE increased significantly in response to CHX treatment in both cell lines. Mean ± SD is shown. The difference from the respective control was examined by Student’s <i>t</i> test (*<i>p</i><0.01).