Fig. 2. Valproate blocks ER stress-induced activation of SREBP and accumulation of free cholesterol in HepG2 cells. (A) Filipin staining of HepG2 cells pre-cultured for 2 hours in the presence or absence of 0.5 mM valproate and then exposed to 5 µM A23187, 2 µg/ml tunicamycin or 5 mM glucosamine as indicated. As a control, HepG2 cells were exposed to the HMG CoA reductase inhibitor, mevastatin (50 µM) and 5 mM glucosamine as described above. After 24 hours, cells were washed, fixed in paraformaldehyde and stained with filipin. Intracellular filipin-cholesterol complexes were visualized by fluorescence microscopy and images were captured with a digital camera. Representative images are shown. (B) Median fluorescence of filipin-stained cells treated as described in A. *P<0.01 when compared with the fluorescence in the corresponding control. **P<0.05 compared to the relative fluorescence under the same conditions without valproate. (C) Immunoblot analysis of mature SREBP-2 protein levels in HepG2 cells pretreated with 0.5 mM valproate for 2 hours and then exposed to 0, 1 or 5 mM glucosamine for 24 hours as indicated. Total protein lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes and immunostained with antibodies against SREBP-2.