Fig. 4.

PKM2 regulates hypoxia-induced HIF-1α accumulation and VEGF-A secretion. a, b PaTu2 cancer cells were transfected with either a GFP-PKM2 construct or GFP empty vector and incubated in normoxic (No) or hypoxic atmosphere (Hy). Forty hours after transfection cells were subjected to immunofluorescence microscopy. c immunoprecipitation of endogenous PKM2 was performed with lysates of PaTu2 cultivated in normoxic or hypoxic condition. Membranes were incubated with HIF-1α and PKM2 antibodies. d pancreatic cancer cells transduced with a non-targeting control shRNA or PKM2-specific shRNAs were incubated under hypoxia or normoxia for 8 h and HIF-1α levels were determined using western blot analysis. β-actin was used as loading control. e pancreatic cancer cells were transiently transfected with 3xHRE-luc and pTK-Renilla and then incubated under normoxic or hypoxic conditions as indicated. Cell lysates were subjected to luciferase assay. Bars are the means +/- SEM of at least three independent experiments. f PaTu2 and Capan1 cancer cell lines with abrogated PKM2 were transiently transfected with VEGF-A-luc and pTK-Renilla. Four hours after transfection cells were incubated under normoxic or hypoxic conditions and then cell lysates were subjected to luciferase assay. Bars are the means +/- SEM of at least two independent experiments performed in duplicate. g supernatants of pancreatic cancer cells transduced with PKM2-specific shRNAs or a non-coding shRNA incubated in low oxygen were subjected to VEGF-A-specific ELISA. Bars are the means +/- SEM of at least two independent experiments conducted in duplicate