Fig. 2
Plk1 enhances PPP and biosynthesis in cancer cells. a Using [U-13C]-labeled glucose, M + 5-labeled R5P produced from PPP was detected by LC-MS. HeLa cells were synchronized by double HU block. When the cells were treated for the second round of HU block for 11 h, i.e., 1 h before releasing, U-13C glucose was added into the medium and further culture for 1 h and then harvested for G1 phase cell analysis. For cell samples of S and G2/M phases, after the regular second round of HU block for 12 h, cells were released into fresh HU-free medium and further cultured for 4 and 9 h, respectively, followed by supplementation of U-13C glucose and 1 h culture before harvesting cells for analysis. Cells were harvested and 13C-incorporated R5P was detected by LC-MS. The calculation of % of the m + 5 isotopologue of ribose is % of the sum of isotopologues. b–f The PPP metabolites were detected by NMR. Cells were cultured in medium containing [2-13C]- or [U-13C]-labeled glucose. The graphic description shows that by using the [2-13C]-labeled glucose, NMR measurement could distinguish the lactate produced from PPP and that derived from the general glycolysis (b). Lactate derived from the oxidative PPP was calculated by the total lactate multiplied with the ratio of 3-13C lactate detected by NMR in Plk1 overexpression (c) or knockdown cells (d). Using the [U-13C]-labeled glucose, R5P and some downstream nucleotides generated from the PPP flux were detected by NMR in Plk1 overexpression (e) or knockdown cell lines (f). To induce Plk1 shRNA expression, cells were grown in the presence of 0.1 μg/ml doxycycline for 72 h. The calculation of isotopologue of ribose is total intensity of 13C-labeled ribose moiety. g U-13C glucose metabolic flux assays were performed in Plk1-overexpressing HeLa cells with G6PD knockdown by NMR. The results were normalized to cell numbers or cell wet weight. Data were represented as the mean ± s.d. or s.e.m. *P < 0.05 as compared to indicated group by two-sided Student’s t-test
