Figure 2
Comparison of HIF-1α mRNA expression and stability in LTLTCa and MCF-7Ca cells. A) LTLTCa and MCF-7Ca cells were plated and cultured in their respective passage media under normal cell culture (nonhypoxic) conditions. Total RNA was extracted and HER2, ERα, HIF-1α and 18S rRNA were analyzed by real-time RT-PCR analysis. Results shown are expressed as the mean fold-change in mRNA levels compared with MCF-7Ca cells after normalization to 18S rRNA (mean ± SD of n = 6 independent cell samples/group; *versus MCF-7Ca; effect of gene type P <0.0001, effect of cell type P = 0.1376, interaction between gene type and cell type P <0.0001; *MCF-7Ca versus LTLTCa for specific gene, P <0.0001; two-way ANOVA). B) LTLTCa and MCF-7Ca cells were treated with vehicle or 0.5 μg/ml actinomycin D for 0 to 16 hours. Total RNA was extracted and HIF-1α mRNA underwent real-time RT-PCR. Results are expressed as least square means of log transformed averages of mRNA expression at various timepoints (trend over time) after normalization to corresponding vehicle-treated samples and analysis by linear mixed effect model, adjusting for experiment, cell line and cell line*time interaction (means ± SD of n = 6 independent samples/group; P <0.001 for effect of cell line, time, their interaction and experiment). C) LTLTCa cells were treated with vehicle or 100 μM CoCl2 for three hours and then with 100 uM cycloheximide for 0 to 60 minutes. Whole cell protein was extracted and underwent Western blot for HIF-1α and AIB1 protein. Shown are representative blots and overall densitometry results of n = 6 independent cell samples/group. Densitometry results are expressed as mean fold-change in protein levels compared to vehicle-treated-0 minutes cycloheximide cells after normalization to AIB1 (mean ± SD of n = 6 independent cell samples/group; *versus no CoCl2-0 minutes CHX, P <0.0001, one-way ANOVA). ANOVA, analysis of variance; ERα, estrogen receptor alpha; HER2, human epidermal growth factor receptor 2; HIF-1α, hypoxia inducible factor 1 α subunit; n, number; SD, standard deviation.