Figure 1

Effect of Hif1a deletion upon growth and invasion. (A) Wild-type (WT) mammary tumor epithelial cells (MTECs) grown to 80% confluence were subjected to hypoxic culture for the indicated times up to 24 hours, or cells continued to be cultured under normoxic conditions such that the time t = 0 sample was harvested on the same day as the time t = 24 hours hypoxic condition sample. High-salt enriched whole-cell extracts were resolved on 3% to 8% Tris-acetate gels and blotted onto polyvinylidene fluoride membrane, which was divided horizontally at approximately 60 kDa. The top half of the blot was used to detect HIF-1α, and the lower portion was used to detect lamin (loading control) to avoid the need to strip and reprobe the blot. (B) Growth curve of WT and knockout (KO) MTECs cultured at normoxia (Nor) or hypoxia (Hyp) in growth medium supplemented with 5% fetal bovine serum (FBS) + epidermal growth factor (EGF) (left) or with 2% FBS (right). For cells grown in 5% FBS + EGF, a representative graph is shown in which the mean ± SEM of cell number per time point of quadruplicate wells per genotype/oxygen tension is plotted per time point. For cells grown in 2% FBS, the grand mean ± SEM of cell number is presented, which was calculated as an average of the mean cell number observed per replicates per time point as observed in three replicate experiments. All data were analyzed by two-way analysis of variance (ANOVA, *P < 0.05). (C) The mean fold change (FC) in invasion was normalized to the invasion index observed for WT cells cultured at normoxia (FC = 1.0). Data represent the mean FC in invasion observed in three independent experiments. All columns were compared using one-way ANOVA with a Bonferroni posttest. *P < 0.05.