Fig. 6
G6PD activity is vital for Plk1-regulated cell proliferation. a HeLa cells stably transfected with tet-inducible NTC or shG6PD (expressing RFP) were subjected to the living cell imaging station. To induce shRNA expression, cells were grown in the presence of 0.1 μg/ml doxycycline for 72 h. Then living cell imaging was done. The mitotic duration time of RFP positive cells was analyzed. n = 20 from 5 different fields. b HeLa cells stably expressing tet-inducible shG6PD (expressing RFP) were further transfected with vectors expressing eGFP-G6PD wild type and its mutants as indicated. Cells were treated with doxycycline in the presence or absence of NAC (2 mM) and Nuc mixtures (25 μM) in the medium, followed by living cell imaging. Images were recorded for 48 h. The mitotic duration time of GFP and RFP double-positive cells was statistically calculated. n = 20 from 5 different fields. c HeLa cells stably expressing NTC or shG6PD were further infected with viruses expressing empty vector (EV) or Flag-G6PD wild type or its mutants as indicated. Cells were treated with vehicle or Nuc mix (25 μM) and NAC (2 mM). Cell numbers were counted 4 days after treatment. Data were presented as mean ± s.d. *P < 0.05 as compared between indicated groups by two-sided Student’s t-test. d HeLa cells stably expressing NTC or tet-inducible shPlk1 were grown in the presence of 0.1 μg/ml doxycycline to induce Plk1 shRNA expression. After 48 h of treatment, 2 mM NAC and 25 μM nucleosides mix were added together with doxycycline for another 24 h. Cell cycle distribution was monitored by flow cytometry. Representative histogram data and statistical results were shown. e, f HeLa cells stably expressing tet-inducible shPlk1 (e) or HeLa cells stably overexpressing Plk1 with further G6PD knockdown by shRNAs (f) were cultured in the medium supplemented with both 2 mM NAC and 25 μM nucleosides mix for 96 h. To induce Plk1 shRNA expression, cells were grown in the presence of 0.1 μg/ml doxycycline. Cell numbers were determined by trypan blue counting. Data were presented as mean ± s.d. *P < 0.05 as compared between indicated groups by two-sided Student’s t-test
