DOX HIF-1αP402A/P564A/N803A transgene induction and target gene up-regulation. (A) Real-time reverse-transcribed polymerase chain reaction analysis of rapid and sustained elevations of transgene and “canonical” HIF-1 target gene mRNAs represented by glucose transporter-1 (Glut1) and CAIX in TetON-HIF-1 transgenic (denoted as “DTG” double transgenic) compared with NTG mice during continuous DOX induction. Prior analysis demonstrated no difference between NTG and DOX day 0 TetON-HIF-1 controls. (B) Transgene protein expression from Western blots of ear nuclear extracts on DOX day 14 in DTG, single TRE-HIF-1αP402/P464A/N803A transgenic mice (NTGTG), or NTG controls. Each dot in scatter plots represents one mouse. Horizontal bars represent the mean. Gene expression measurements for HIF-1α, Glut1, and CAIX were from the same samples. DTG data at each DOX day were compared with NTG or TetON-HIF-1 day 0 data (data not shown), using unpaired Student t test: **P < .01; ***P < .001. (C) Representative HIF-1α transgene and dual VEGF-keratin-14 immunofluorescence, showing expression localization in the interfollicular and hair follicle basal cell compartment after 3 and 14 days of DOX induction. Arrowheads indicate the basolateral VEGF localization within the basal cells. Bar represents 50 μm.
