Induction of functional CD39 by hypoxia. (a) Confluent T84 monolayers were exposed to normoxia (pO₂ 147 torr, 18 hours) or hypoxia (pO₂ 20 torr, 18 hours). Total RNA was isolated, and CD39 mRNA levels were determined by RT-PCR using semiquantitative analysis (increasing cycle numbers, as indicated). As shown, β-actin transcript was determined in parallel and used as an internal standard. (b) More quantitative real-time PCR was employed to directly compare hypoxia-inducibility of CD39 and CD73. Data were calculated relative to internal control genes (β-actin) and are expressed as fold increase over normoxia ± SEM at each indicated time. Results are derived from two experiments in each condition. (c) Epithelial monolayers were exposed to indicated periods of hypoxia and washed, and surface CD39 activity was determined by HPLC analysis of E-ATP conversion to E-AMP. Data are derived from five to seven monolayers in each condition, and results are expressed as mean percent E-AMP conversion ± SEM.