Fig. 3

Interaction of the TGF–β/SMAD signaling pathway and HIF-1α regulates the expression of PKM2 and PKM1 under hypoxia by promoting c-Myc expression. a, Changes in protein expression of c-Myc and its downstream genes in A549 cells with or without Smad2/3 and HIF-1α knockdown under normoxia or hypoxia. b, PKM1 and PKM2 coexisted in cancer cells. PKM2 (Green) was highly expressed in lung adenocarcinoma tissues, while PKM1 (Red) expression was relatively low. The expression of PKM1/2 in adjacent tissues was opposite. c, Changes in mRNA expression of downstream genes of c-Myc in A549 and H1299 cells (Treated with TGF-β) with or without Smad2/3 and HIF-1α knockdown under normoxia or hypoxia. d, Western blot analysis showing that decreased protein expression of PKM2 and increased PKM1 caused by knockdown of Smad2/3 and HIF-1α can be reversed by overexpression of c-Myc. e, Under normoxia, the non-degradable HIF-1α mutants and TGF-β synergistically promoted glycolysis and proliferation of NSCLC cell lines. f, ECAR and OCR reflect the effects of the non-degradable HIF-1α mutants and TGF-β on metabolic reprogramming, as examined by Seahorse Extracellular Flux Analyzer XFe96 assay. g, Western blot results showed that the non-degradable HIF-1α mutants and TGF-β could synergistically promote the alternative splicing of PKM and significantly increased the ratio of PKM2/PKM1 under normoxia. The concentration of all TGF-β used in this part of the experiment was 5 ng/ml