Figure 5. KHK-A promotes de novo nucleic acid synthesis and reduces ROS production.
(a) Hep3B and Huh7 cells with or without expression of hnRNPH1/2 shRNA were labeled with D-[6-14C]-glucose for 6 h. Influx of glucose into RNA extracted from the cells was measured. The data represent the mean ± s.d. from n = 3 independent experiments. A two-tailed Student’s t test was used. * represents P < 0.01 between the cells with or without hnRNPH1/2 depletion.
(b) Hep3B and Huh7 cells expressing KHK shRNA with or without reconstituted expression of the indicated rKHK proteins were labeled with D-[6-14C]-glucose for 6 h. Influx of glucose into RNA extracted from the cells was measured. The data represent the mean ± SD from n = 3 independent experiments. A two-tailed Student’s t test was used. * represents P < 0.01 between the cells with or without KHK depletion. # represents P < 0.01 between the KHK-depleted cells with reconstituted expression of WT rKHK-A and the KHK-depleted cells with reconstituted expression of rKHK-A L83A and WT rKHK-C.
(c) Hep3B and Huh7 cells expressing PRPS1 shRNA were reconstituted the expression of the indicated rPRPS1 proteins. Immunoblot analysis was performed with the indicated antibodies (left panel). These cells were labeled with D-[6-14C]-glucose for 6 h. Influx of glucose into RNA extracted from the cells was measured. The data represent the mean ± s.d. from n = 3 independent experiments (right panel). A two-tailed Student’s t test was used. * represents P < 0.01 between the cells with or without PRPS1 depletion. # represents P < 0.01 between the PRPS1-depleted cells with reconstituted expression of WT rPRPS1 and rPRPS1 T225A.
(d) Normal hepatocytes (left panel) and Hep3B cells (right panel) were labeled with D-[6-14C]-glucose for 1 h in the presence or absence of Pi (50 mM) for the indicated periods of time. Influx of glucose into RNA extracted from the cells was measured. The data represent the mean ± s.d. from n = 3 independent experiments.
(e) Hep3B cells with reconstituted expression of rKHK-C or rKHK-A were cultured in the presence or absence of the indicated dosage of fructose for 2 h. Levels of cellular ROS were measured. The data represent the mean ± s.d. from n = 3 independent experiments. A two-tailed Student’s t test was used. * represents P < 0.01 between the indicated cells and the counterparts without fructose treatment. In c, data represent 1 out of 3 experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 7.