Fig. 6

PKM2 regulates HIF-1α transcription via activation of NF-κB signaling pathway. a pancreatic cancer cells were transiently transfected with a p65 expression plasmid (YFP-p65). In parallel, cells transfected with control empty vector (GFP-empty) were cultivated in normoxia or hypoxia for 8 h. Lysates were subjected to SDS-PAGE and membranes were incubated with HIF-1α and p65 antibodies. β-actin was used as loading control. b PaTu2 cells were co-transfected with p65 and HIF-1α reporter. After incubation for 24 h in low oxygen atmosphere, lysates were prepared and luciferase was measured. c cancer cells with abrogated p65 were transfected 3xHRE-luc and then incubated in low oxygen atmosphere. Luciferase was measured 28 h after transfection. Bars are the means +/- SEM of at least three independent experiments. d PaTu2 cells featuring p65 knock-down were co-transfected with PKM2 expression plasmid and 3xHRE-luc and then grown in an atmosphere deprived of oxygen. Luciferase was scored 24 h later. Bars are the means +/- SEM of at least three independent experiments. e PaTu2 and Capan1 cancer cells with depleted PKM2 were co-transfected with a p65 expression plasmid and 3xHRE-luc and then grown in an atmosphere deprived of oxygen. Luciferase was scored 24 h later. Bars are the means +/- SEM of at least three independent experiments. f PaTu2 cells with abrogated PKM2 were co-transfected with a HIF-1α expression plasmid and a 3xκB-luc reporter and then grown in hypoxia. Luciferase was scored 24 h later. Bars are the means +/- SEM of at least three independent experiments. For all reporter assays TK-Renilla was used as internal control