Figure 4

Depletion of PDK1 counteracts H19-mediated glycolysis and stemness. (a–b) MDA-MB-231 and MCF-7 cells expressing either NTC or shH19 were cultured under hypoxia for 12 h compared with NTC cells cultured in normoxia. PDK1 expression was detected by western blotting. (c–e) MDA-MB-231 cells expressing either NTC or shPDK1 were infected with lentivirus expressing H19 and empty vector (EV) for establishing stable cells. Intracellular glucose uptake (c), lactate production (d) and cellular ATP levels (e) were then measured and normalized based on protein concentration. (f) Mammosphere formation ability was analyzed, the scale bar represents 100 μm. (g and h) Immunodeficient mice (n=5) were subcutaneously inoculated with equal number of single cells (5 × 10⁵ cells per mice) (g) and tumor volume were monitored after 24 days (h). (i) In vivo limiting dilution assays performed by plating decreasing numbers of primary xenografted tumor cells (H19, shPDK1 and H19 plus shPDK1) into immunodeficient mice (n=5) calculated with extreme limiting dilution assay analysis (left); right panel: stem cell frequencies were estimated as the ratio 1/x with the upper and lower 95% confidence intervals, where 1=stem cell and x=all cells. (j) There was a significant correlation of PDK1 and H19 mRNA levels in breast patient samples (n=15, R²=0.7262, P<0.001; linear regression analysis). Data shown are mean±s.d. (n=3), *P<0.05, **P<0.01 and ***P<0.001, respectively.