Figure 4.

CDDO-Me induces autophagy in Ec109 and KYSE70 cells determined by flow cytometry and confocal microscopy.

Notes: (A and B) Percentages of specific cell populations showed in dot plots and autophagic cells showed in bar graphs for Ec109 and KYSE70 cells treated with CDDO-Me at 0.25–1.0 μM for 24 hours, respectively; and percentages of specific cell populations showed in dot plots and autophagic cells showed in bar graphs for Ec109 and KYSE70 cells treated with CDDO-Me at 0.5 μM for 4–48 hours, respectively. (C) CDDO-Me-induced autophagic death in Ec109 and KYSE70 cells determined by confocal microscopy. The level of autophagy was evaluated using a lysosome-specific fluorescence dye. Cells were treated with CDDO-Me at 0.25–1.0 μM for 24 hours or at 0.5 μM for different incubation times from 4 hours to 48 hours. The confocal microscopic images of autophagic Ec109 and KYSE70 cells (stained in green) are also shown. (D) Effects of CDDO-Me treatment on the expression levels of p-PI3K at Tyr458, PI3K, p-AMPK at Thr172, AMPK, p-p38 at Thr180, p38, p-Akt at Ser473, Akt, p-mTOR at Ser2448, mTOR, PTEN, beclin 1, LC3-I, and LC3-II in Ec109 and KYSE70 cells. Cellular lysates were analyzed by immunoblotting with respective first antibody followed by the second antibody. (E) Bar graphs showing the levels of the proteins in Ec109 and KYSE70 cells. The ratios of p-PI3K, AMPK, p38, Akt, and mTOR over the corresponding total protein and ratios of LC3-II over LC3-I in Ec109 and KYSE70 cells are also shown. β-actin was used as the internal control. Data represent the mean ± SD of three independent experiments. *P<0.05, **P<0.01, and ***P<0.001 by one-way ANOVA and Tukey’s post hoc test to compare the levels of the protein measured with the control cells treated with the vehicle only.

Abbreviations: CDDO-Me, methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; SD, standard deviation; ANOVA, analysis of variance; mTOR, mammalian target of rapamycin; hr, hour.