Immunoblot analysis of proteins involved in isoflurane-induced hypoxia-inducible factor (HIF)-1α up-regulation after isoflurane exposure. Renal cell carcinoma cells were treated with or without 2% isoflurane in 21% oxygen and 5% carbon dioxide balanced with nitrogen for 2 h, and immunoblotting was performed at different time point (0, 2, 4, 8, and 24 h) postgas exposure. Upstream HIF-1α effector phosphorylated protein kinase B (p-Akt) was up-regulated in a time-dependent manner (A); amounts of the tumor suppressor and negative regulator of the PI3K pathway phosphorylated phosphatase and tensin homolog (p-PTEN) were not affected (B). (C) Prolyl hydroxylase (PHD)-2, a negative regulator of HIF-1α through the hypoxic pathway, was also unaffected. (D) Nuclear amounts of HIF-1β remained stable. (E) Furthermore, increased amounts of HIF-1α translocation to the nucleus activated the cascade of HIF downstream effectors as evident by the up-regulation of the proangiogenic factor vascular endothelial growth factor A (VEGFA). Data were presented as mean ± SD. One-way ANOVA with Tukey corrections; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 versus naive control (NC; n = 3–4). Iso = isoflurane; mTOR = mammalian target of rapamycin; PI3K = phosphatidylinositide 3-kinase.