Fig. 5.

6PGD controls Ru-5-P level to regulate LKB1-AMPK signaling and subsequently ACC1 activity and lipogenesis. (a) 6PGD KD H1299 (left) and K562 (right) cells were assayed for general ROS levels in the absence and presence of NAC (1 and 3 mM) by measuring intracellular ROS-mediated DCFDA oxidation to fluorescent DCF by flow cytometry. The relative general ROS levels were normalized to the control vector cells without NAC treatment. (b) Cell proliferation rates were determined by cell counting in 6PGD KD H1299 cells treated with either or both NAC and Compound C (left) or 6PGD KD cells treated with either or both AMPK shRNA and NAC (right). (c-f) LKB1-deficient A549 cells with 6PGD knockdown (c) and control vector cells were assayed for intracellular Ru-5-P levels (d), phosphorylation levels of AMPK (e), and lipogenesis (f). (g-h) Cell lysates from 6PGD KD A549 cells were treated with increasing concentrations of Ru-5-P, followed by Western blot for phosphorylation levels of AMPK (g) or lipogenesis assay (h). Final levels (fold) of Ru-5-P were normalized to the control vector cells without treatment. (i-j) A549 vector and 6PGD knockdown cells were tested for cell proliferation rate by cell counting (i) and ROS level (j) in the presence or absence of NAC (3 mM). (k-n) Normal proliferating HaCaT vector and 6PGD knockdown cells were assayed for phosphorylation levels of AMPK (k), oxidative PPP flux rate (l), intracellular Ru-5-P levels (m), and lipogenesis (n). (a-n) Data are from a single experiment that is representative of 3 independent experiments for (a, c, e, g) and 2 independent experiments for (b, d, f, h-n). Source data for independent replications and experiments with sample size<5 are available in Supplementary Table 1. Uncropped Western blots are provided in Supplementary Figure 9.