Hypoxia induces HIF1α protein levels and CA9 and VEGFA transcription in soft tissue sarcoma cell lines. (A) HIF1α protein levels are stabilized and increase during hypoxia. Cell lines were cultured under hypoxia (H) or normoxia (N), after the indicated times cell lysates were prepared and analysed by Western blotting for HIF1α expression. HIF1α levels peak at 6 hours of hypoxia. Upon prolonged hypoxia (24 and 48 hours) HIF1α protein levels decrease, but remain increased compared to the HIF1α levels detected in cells cultured under normoxic conditions. β-Actin was used as a loading control. (B,C) mRNA levels of HIF1 target genes CA9 (B) and VEGFA (C) are upregulated during hypoxia (grey bars) compared to normoxia (black bars). CA9 and VEGFA expression was determined by RT-PCR and normalized to the expression of HPRT. Bars indicate average fold change of mRNA expression ± S.D.(n = 2) compared to the levels detected at 0 h which are arbitrarily set at 1. Statistical significance between CA9 and VEGF expression in normoxic and hypoxic samples at the various time points was determined by two-sample t-tests: * = p < 0.05, ** = p < 0.005, *** = p < 0.0005. The complete panel of 12 soft tissue sarcoma cell lines was examined, the results obtained with five representative cell lines are shown.