Figure 1
From: NADPH oxidase 4 modulates hepatic responses to lipopolysaccharide mediated by Toll-like receptor-4
LPS stimulation increased TNF-α and Nox4 expression. Huh7 cells were either treated with LPS (1 µg/ml) or PBS for up to 48 hrs, and analyzed for TNF-α (A) and Nox4 (B) mRNA expression by qRT-PCR. Data was normalized with GAPDH mRNA and calculated by ΔΔCt method and expressed as fold increase from the control. (C) TNF-α and Nox4 protein levels in Huh7 cells after LPS treatment were determined by western blotting. (D) Nox enzyme activities were determined in Huh7 cells either untreated or pretreated with DPI (10 µM) for 30 minutes before 6 hrs of LPS stimulation by monitoring NADPH-dependent and SOD-inhibited reduction of cytochrome c, as described in materials and methods. # indicates statistically significant difference from DPI (0) LPS (−) (p < 0.05). (E) Huh7 cells either untreated or pretreated with DPI (10 µM) for 30 minutes before 3 hrs of LPS stimulation. Then, cells were labeled with 5 µM CM-H2DCFDA for 15 min and intracellular generation of H2O2 was monitored by confocal microscopic analysis of DCF fluorescence. (F,G) Nox4 and TNF-α mRNA and protein levels in Huh7 cells either untreated or treated with Actinomycin D (1 µM) for 30 minutes before 3 hrs of LPS stimulation. # indicates statistically significant difference from control LPS (−) (p < 0.05). * indicates statistically significant difference from the corresponding controls (p < 0.05). Lines with p values also indicate statistically significance (p < 0.05) between the groups.
