Fig. 5
From: PKM2 promotes tumor angiogenesis by regulating HIF-1α through NF-κB activation
PKM2 mediates hypoxia-triggered VEGF-A secretion by activation of NF-κB/p65 subunit. a immunoprecipitation of endogenous p65 was performed with lysates of PaTu2 and BxPC3 cells. Membranes were incubated with PKM2 and reprobed with p65 antibodies. b, c PaTu2 and Capan1 cancer cell lines with abrogated p65/Rel (shp65 #G3 or shp65 #F12) were transiently transfected with VEGF-A-luc and pTK-Renilla. Four hours later cells were subjected to normoxic or hypoxic conditions. After 24 h cell lysates were prepared and subjected to luciferase assay. Bars are the means +/- SEM of at least three independent experiments. d supernatants of PaTu2 or Capan1 cells transduced with p65/RelA-specific shRNA (shp65) 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. e PaTu2 and Capan1 cancer cell lines with abrogated PKM2 were transiently transfected with 3xκB-luc and pTK-Renilla. After incubation under normoxic or hypoxic conditions cell lysates were prepared and reporter activity assayed. Bars are the means +/- SEM of at least three independent experiments. f, g pancreatic Capan1 cells with abrogated PKM2 were transiently co-transfected with p65 and VEGF-A-luc reporter before incubation in low oxygen atmosphere. Luciferase was measured after 24 h. h supernatants of Capan1 cells with deleted PKM2 and overexpressing p65 cultivated in O2-deprived atmosphere or normoxia were subjected to VEGF-A-specific ELISA. Bars are the means +/- SEM of at least two independent experiments conducted in duplicate (No – normoxia; Hy – hypoxia)