Figure 2.

Exploration of mechanisms for increased level of HIF-1α protein. For in vitro experiments, RCC4⁺ VHL were exposed to 70% Xe balanced with O₂ for 2 h and harvested at 0 to 24 h after this. (A) Exposure to Xe increases HIF-1α expression whether the VHL ubiquitylation process is present (RCC4⁺ cells) or absent (RCC4⁻ cells) (an example of three independent experiments). (B) In vivo effects of HIF prolyl hydroxylase domain–containing 2 (PHD2) enzyme. Exposure to Xe does not increase expression of the enzyme HIF PHD2 in RCC4⁺ VHL cells (n = 4). (C) Reverse transcription–PCR revealed that exposure to Xe did not alter the level of HIF-1α mRNA (n = 4). (D) Xe exposure significantly increased expression of the protein mTOR after 24 h (n = 4). C, control. *P < 0.05, **P < 0.01, ***P < 0.001 versus control. (E) Rapamycin (R), an mTOR inhibitor, attenuates Xe-induced HIF-1α expression 24 h after exposure. *P < 0.05, **P < 0.01 versus Xe (n = 4). (F) Xe exposure caused a time-dependent increase expression of mTOR in the mouse kidney (an example of three independent experiments). (G) In vivo, pretreatment with R 1.5 mg/kg intraperitoneally prevented enhancement of HIF-1α expression (an example of three independent experiments). Data are means ± SD.