Fig. 4

Glucose stimulates PCSK9 expression via ROS and SREBP-1 but does not alter LDLR. a Measurement of ROS level in HepG2 cells treated with LG and HG medium for 12 h. b Immunofluorecence staining of SREBP-1 in HepG2 cells cultured in LG and HG for 12 h (Scale bar = 10 μm). c HepG2 cells were pre-treated with 1500 U/ml of catalase (CATA) for 3 h; subsequently, HG treatment was given for 6 h. Whole cell lysates were resolved by SDS-PAGE and SREPB-1; PCSK9 and β-actin protein levels were analyzed by immunoblot. d HepG2 cells were pre-treated with indicated concentrations of fatostatin (Fato) for 3 h, subsequently HG treatment was given for 6 h. Whole cell lysates were resolved by SDS-PAGE. PCSK9 and β-actin protein levels were analyzed by immunoblot. e Tumor tissue homogenates of three representative samples from group III and group IV were resolved on SDS-PAGE, and SREBP-1 protein level was analyzed by Western blot. f Whole cell lysates were prepared from HepG2 cells treated with LG and HG for 12 h and 24 h, respectively, or membrane and cytosolic fractions were prepared from HepG2 cells treated with LG and HG for 24 h by replenishing respective medium after 12 h. Expression of PCSK9 and LDLR were analyzed by immunoblot. Transferring receptor (TfR) and β-actin were included in the Western blot as loading controls for membrane and cytosolic fractions. g, h HepG2 cells were incubated in LG and HG medium for 12 h and processed for DiI-LDL uptake assay either by FACS or confocal staining. All bar graphs represent means ± SEM; **p < 0.01 denotes significant differences between the groups; ns non-significant