Ceramide markedly increases Grp78 mRNA expression and eIF2α phosphorylation. (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 Grp78 were calculated by normalizing to GAPDH. *P < 0.05 (one-way ANOVA) versus control cells. (B) Cells were seeded into 60 mm culture dishes and the next day cells were treated with ceramide. After 3 h incubation, whole cell lysates were collected and subjected to western blot. The level of phosphorylated eIF2α was analyzed with anti-phosphorylated eIF2α antibody. Unphosphorylated eIF2α were measured and actin were used as loading controls. The experiment was repeated several times and the representative result is shown.