Fig. 1

Plk1 activates G6PD during cell cycle progression. a–c HeLa cells were synchronized into G1, S, and G2/M phases, and cells were harvested and subjected to western blot (a) and G6PD or 6PGD enzyme activity measurement (b), and NADPH and NADP⁺/NADPH ratio levels (c) at indicated time after release. n = 3 biologically independent replicates. Data were presented as mean ± s.d. *P < 0.05 as compared to G1 phase group by two-sided Student’s t-test. β-actin served as loading control. d–f HeLa cells were synchronized at G0/G1 phase with double HU block. The G6PD activity (d), and NADPH and NADP⁺/NADPH ratio levels (e) were measured in the presence or absence of 1 μM Plk1 inhibitor BI2536 at indicate hours after release. FACS analysis of cell cycle progression was shown in f. n = 3 biologically independent replicates. Data were presented as mean ± s.d. *P < 0.05 as compared to 0 h group, ^# P < 0.05 as compared to DMSO group by two-sided Student’s t-test. g, h HeLa cells were transfected with empty vector (EV) or gradual amount of Flag-Plk1 plasmids. Cells were harvested and subjected to western blot and G6PD activity measurement (g), and NADPH and NADP⁺/NADPH ratio levels (h). n = 3 biologically independent replicates. Data were presented as mean ± s.d. *P < 0.05 as compared to EV group by two-sided Student’s t-test. β-actin served as loading control. i, j Plk1 protein levels and G6PD enzyme activity (i), and NADPH levels and NADP⁺/NADPH ratio (j) were measured in HeLa cells stably expressing non-targeting control (NTC) or shPlk1. n = 3 biologically independent replicates. Data were presented as mean ± s.d. *P < 0.05 as compared to NTC group by two-sided Student’s t-test. β-actin served as loading control