Fig. 3

Glucose-dependent regulation of PCSK9 in HCC cells. a HepG2 cells were cultured in LG, HG, and LG + Mannitol (Mann) for 12 h. Expression of PCSK9 protein was examined by immunoblot in three independent sets of experiments. Band intensities were measured by densitometry and normalized with β-actin. b HepG2 cells were cultured in LG and HG for 12 h. PCSK9 mRNA level was quantified by qRT-PCR. c HepG2 cells were cultured in LG and HG for 12 h and 24 h. Immunoblot analysis was performed with total protein lysates. PCSK9 protein band intensities were quantified by densitometry and normalized to β-actin. Extracellular PCSK9 in culture supernatant was quantified by ELISA. d HepG2 cells were seeded in 35-mm plates and allowed to adhere. LG medium was added in all the plates for 24 h. Then, LG medium was removed and cells were incubated in HG medium for 12 h and 24 h, respectively. HG medium was replaced with fresh HG medium after 12 h, and the plate was incubated for further 12 h (lane 4). Whole cell lysates were subjected for immunoblotting to analyze expression of PCSK9. Band intensities were quantified by densitometry and normalized to β-actin. Glucose remaining in the culture supernatant was estimated at each time point. e HepG2 cells were treated with indicated concentrations of glucose (Glu), glutamine (Glt), pyruvate (pyr), lactate (lact) 2-deoxyglucose (2-DG), and cytochalasin B (CytB) for 12 h and expression of PCSK9 was analyzed by Western blot. Bar graphs represent mean ± SEM; n = 3; *p < 0.05, **p < 0.01 denote significant differences in the groups; ns non-significant