Fig. 1. Mammary epithelial cells contain functional HIF1. (A) Upper panel: HIF1, a triplet present at 120 kDa, was detected in purified mammary epithelial cells (MEC) at normoxia (21% O₂) and was induced dramatically upon exposure to 0.5% oxygen. Lower panel: to demonstrate equivalent loading, total protein was stained with a reversible protein detection dye prior to blotting. (B) Purified MEC were cultured at either normoxia (white bars) or hypoxia (gray bars), harvested, and expression of target genes was normalized to 18S rRNA. After normalization, the relative expression of each gene was expressed as a percentage of that observed in wild-type cells at normoxia (mean±s.e.m.). No significant differences in gene expression were observed for any gene between Hif1a wild-type (WT, Hif1a^f+/f+ only) and null cells (Hif1a^f+/f+ cells infected with Adeno-Cre) cultured under normoxic conditions. At hypoxia, relative to the robust induction of Pgk, Glut1 and Vegf observed in wild type cells, induction of all of these mRNAs was decreased by at least 50%. (C) Genomic DNA was prepared from the mammary glands (mg) and ovaries (ov) of Hif1a^f+/f+ (Cre-negative) or Hif1a^f+/f+, MMTV-Cre-positive mice at either day 15 of gestation (15-P) or at mid-lactation and used for Southern blotting as described previously (Ryan et al., 1998). As a control for complete excision, DNA was prepared from primary mouse embryonic fibroblasts (MEF) previously infected with Adeno-Cre.