DDC-induced ROS formation in C3H and C57BL hepatocytes is augmented by GAPDH and NDPK knockdown. (A) Biochemical analysis of protein expression of GAPDH, NDPK-B, and actin (loading control) 24 h after transfection of the isolated primary hepatocytes with the indicated siRNA. (B) Primary hepatocytes were transfected with the indicated siRNA for 24 h followed by the addition of 100 µM DDC for an additional 24 h. Representative differential interference contrast fluorescence images of ROS levels (green) in C3H and C57BL hepatocytes with DAPI counterstain (blue) are shown. Bar, 20 µm. (C) Quantification of hepatocyte ROS levels upon GAPDH and NDPK knockdown and DDC treatment. **, P < 0.01; ***, P < 0.001 using a one-way analysis of variance and relative to the respective control group. ROS levels in control-transfected DDC-treated C57BL hepatocytes were significantly higher compared with control-transfected DDC-treated C3H hepatocytes (P = 0.0009 using an unpaired t test). Results are represented as the mean and the SD (n = 15 cells/group), and the data for each tested group are representative of three independent hepatocyte isolations.