Fig. 2

TGF-β promotes glycolysis of NSCLC cells under hypoxia by increasing the PKM2/PKM1 ratio via canonical TGF–β/SMAD signaling. a, b, Following the addition or loss of TGF-β in A549 and H1299 cells, total proteins were extracted and western blot analysis with the indicated antibodies was performed. c, The PKM2/PKM1 ratio was increased dramatically in hypoxia while decreased in normoxia when treated with TGF-β in A549 and H1299 cells. d, Following the culture of A549 and H1299 cells with and without TGF-β (5 ng/ml) in normoxic and hypoxic environments, the expression of HIF-1α was detected by western blotting. e, When Smad2/3 was knocked down, the relative RNA expression of PKM1 and PKM2 in A549 and H1299 cells (Treated with TGF-β) showed opposite changes in normoxic and hypoxic conditions. This trend of change was more significant after knocking down HIF-1α at the same time. f, g, Total proteins were extracted from A549 and H1299 cells with or without Smad2/3 and HIF-1α knockdown and western blot analysis was performed with the indicated antibodies. h, ECAR and OCR reflect the influence of p-Smad2/3 and oxygen concentration on metabolic reprogramming, as examined by Seahorse Extracellular Flux Analyzer XFe96 assay. The concentration of all TGF-β used in this part of the experiment was 5 ng/ml