Ceramide activates XBP1 mRNA splicing in ACCs. (A) ACC-M or ACC-2 cells were subjected to the indicated concentration (10–100 μM) of C2-ceramide, and the total RNA was isolated 6 and 12 h after ceramide treatment. Reverse transcription to cDNA and RT-PCR was performed to detect spliced (XBP1S) and unspliced (XBP1U) forms of XBP-1. GAPDH was used as a loading control. RT-PCR products were digested by PstI restriction enzyme in 37°C for 1 h, and then separated on 2% agarose gel and visualized in an UV image system. (B) Cells were treated as in A, and the total RNA was subjected to Real-time PCR. Relative mRNA expression levels for XBP1S and XBP1U were calculated by normalizing to the signal for GAPDH mRNA in each sample and comparison with cells cultured in a control medium. The figure presents mean fold over control change in experimental groups ± S.D. *P < 0.05 (one-way ANOVA). (C) Cell were treated with 1–10 μM Thapsigargin or 0.03-3 μg/ml Tunicamycin (positive control for XBP1S expression), RT-PCR for XBP1 mRNA splicing detection was performed as in A.