Figure 1.

Hypoxia-induced response to radiotherapy in three head and neck squamous cell carcinoma cell lines. The LK0858, LK0863 and UT-SCC-14 cell lines were cultured in normoxic (21% O₂) and hypoxic (1% O₂) conditions. (A) Western blot analysis of HIF-1α and CA9 expression in cell lines cultured in normoxic and hypoxic conditions. β-actin was used as the loading control. (B) LK0858, LK0863 and UT-SCC-14 cells were irradiated (2, 4 and 6 Gy) at 48 h after seeding and subsequently returned to normoxic or hypoxic conditions. The hypoxic cells were exposed to hypoxia for 24 h prior to irradiation. After 9 days, the cytotoxic/cytostatic effect on cell proliferation was determined by a crystal violet assay. (C) UT-SCC-14 cells transiently transfected with either non-targeting siRNA or HIF-1α-targeting siRNA were exposed to hypoxia for 24 h prior to transfection and placed back under hypoxic conditions after transfection. The efficiency of HIF-1α downregulation was assessed via western blotting at 48 h post-transfection. In parallel experimental settings, the UT-SCC-14 cells were irradiated at 2, 4 and 6 Gy 24 h post-transfection with the respective siRNAs, followed by re-exposure to hypoxic conditions (1% O₂). After 9 days, the cytotoxic/cytostatic effect on cell proliferation was determined by a crystal violet assay. Cell proliferation is presented as the percentage of the untreated controls, and data are presented as the mean ± SD from three experiments performed in triplicate. *P<0.05. The data was analyzed using an unpaired Student's t-test. HIF, hypoxia-inducible factor; CA9, carbonic anhydrase 9; siRNA, small interfering RNA; N, normoxia; H, hypoxia; ns, not significant.