Fig. 6. ROS enhance GBM adaptation to hypoxia via the HIF-1α‍-SERPINE1 pathway. (a, b) qPCR (a) and western blotting (b) were conducted to examine the efficiency of SERPINE1 knockdown in U87 and U251 cells. ^*** P<0.001, vs. siNC group. (c) The expression levels of SERPINE1 were measured in U87 and U251 cells, where SERPINE1 was knocked down or/and HIF-1α was overexpressed. (d) The role of SERPINE1 in cell migration was assessed by wound healing assay under hypoxia for 24 h (scale bar=200 μm). (e, f) The role of SERPINE1 in cell invasion was detected by matrigel invasion assay under hypoxia for 24 h (scale bar=100 μm). ^* P<0.05, ^** P<0.01, ^*** P<0.001, vs. hypoxia group; ^# P<0.05, ^## P<0.01, ^### P<0.001, vs. hypoxia+HIF-1α group. (g) GSEA analysis was performed to determine EMT enrichment in SERPINE1-high GBM tissues. (h) Western blotting was used to examine the levels of EMT-related proteins under hypoxia for 24 h. Cells were transfected with siRNA targeting SERPINE1 or/and plasmid expressing HIF-1α for 24 h, and then subjected to hypoxia for 24 h. The normoxia group was used as negative control. Data are expressed as mean±SEM (n=3). ROS: reactive oxygen species; GBM: glioblastoma; HIF-1α: hypoxia-inducible factor-1α; SERPINE1: serine protease inhibitor family E member 1; qPCR: quantitative real-time polymerase chain reaction; NC: negative control; GSEA: Gene Set Enrichment Analysis; EMT: epithelial-mesenchymal transition; siRNA: small interfering RNA; si: siRNA; SEM: standard error of the mean; mRNA: messenger RNA; TCGA: The Cancer Genome Atlas; NES: normalized enrichment score.