Figure 5

Real-time PCR analysis for the relative expression of HIF-1α and OS-9 among five SCC cell lines. Cells were plated in 6-well plates using a density of 5 × 10⁴ cells/well and were allowed to grow to 80% confluence. The cells were washed twice with cold PBS, lysed in RIPA buffer for 10 min and scraped. The extracts were centrifuged and equivalent amounts of protein (50 μg) were then separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h in blocking buffer, which was subsequently replaced by the primary antibody in blocking buffer, overnight at 4°C. After incubation, the membranes were washed three times in washing buffer. Primary antibody was detected using horseradish peroxidase-linked goat antimouse (Santa Cruz Biotechnology, Santa Cruz, CA) or goat antirabbit IgG antibody (Santa Cruz Biotechnology) and visualized with SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). The bands were scanned and quantified using NIH image software. The levels of HIF-1α were: SCC- 4a (1.0 ± 0), SCC- 9 (1.3 ± .2); SCC-15 (1.0 ± .3), SCC-25b (1.5 ± .5), and UMBSCC-11 (4.0 ± .7). The levels of OS-9 expression were:SCC-4a (1.0 ± 0), SCC- 9 (1.2 ± .3); SCC-15 (1.0 ± .3), SCC-25b (.5 ± .1), and UMBSCC-11 (7.6 ± .8). (*, P < 0.05)