Fig. 2

HIF-1α pathway activation is responsible for acquired LR and increased cancer stemness in HCC. a Top 156 upregulated DEGs of HuH-7-LR versus HuH-7 and SNU-387-LR versus SNU-387 were extracted from RNA-seq data. b Upon KEGG analysis show, it was revealed that the 156 upregulated DEGs were involved in associated with the HIF-1α pathway. c The expression of HIF-1α and HIF-2α was detected in WT and HCC LR cells via western blot analysis. d Changes in relative luciferase activity in WT and HCC LR cells were determined by luciferase reporter assay. e The ECAR of HCC LR cells and WT cells was identified using the Seahorse system. The means and SDs are represented on the column graphs. f qRT-PCR was used to measure the expression of glycolysis driver genes in WT and HCC LR cells. The effects of HIF-1α knockdown (shHIF-1α) or not (shNC) on HuH-7-LR and SNU-387-LR cell stemness were shown according to migration (g), in vitro self-renewal (h), and mRNA expression of stemness markers (i). j The WT and HCC LR cells with or without HIF-1α knockdown (shHIF-1α or shNC) were exposed to the indicated concentrations of lenvatinib for 48 hours. Trypan blue staining-based cell counting was used to detect lenvatinib sensitivity. The data were presented as mean ± SD of three individual experiments. The Student’s t test was used for comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 μm. DEGs differentially expressed genes, KEGG Kyoto Encyclopedia of Genes and Genomes, WT wild type, LR lenvatinib resistant, HIF-1α hypoxia-inducible factor-1α, HIF-2α hypoxia-inducible factor-2α, HCC hepatocellular carcinoma, ECAR extracellular acidification rate