Figure 6

Ceramide induces ER stress-mediated cell death. (A) ACC-M and ACC-2 cells were treated with indicated concentration (10–100 μM) of ceramide for 6 and 12 h, total RNA was subjected to Real-time PCR. Relative mRNA expression levels for CHOP were calculated by normalizing to GAPDH. *P < 0.05 (one-way ANOVA) versus control cells. (B) Cells were treated as in A. After 12 h treatment with ceramide, whole cell lysates were collected and subjected to western blot. The level of phosphorylated JNK, total JNK, cleaved caspase-3 and total caspase-3 was analyzed with primary antibody. Actin were used as loading controls. The experiment was repeated several times and the representative result is shown. (C) ACC-M and ACC-2 cells were seeded into 60 mm culture dishes and the next day cells were treated with indicated concentration (10–100 μM) of ceramide for 12 h and further grown for 3 weeks. Colonies were stained and counted. The experiment was repeated 3 times and the representative result is shown. (D) Cells were treated as in C. Cells were pretreated with 5 mM 4-PBA or 1 mg/ml TUDCA. After 3 h incubation, 100 μM ceramide was added for further 12 h incubation in full medium. As showed in images, ER stress inhibitor TUDCA rescued cells from ceramide-induced cell death. The experiment was repeated 3 times and the representative result is shown.