Figure 7.

Influence of hypoxia inducible factor (HIF)‐1α expression on β‐tubulin conformation. (A) β‐tubulin protein expression in the PC14PE6 and NCI‐H441 cells. The cells were transfected with plasmids encoding enhanced green fluorescent protein (EGFP) and siRNA specific to HIF‐1α (+) or empty vector (–) to PC14PE6 or HIF‐1α (+) or empty vector (–) to NCI‐H441 and cultured under normoxic (N) or hypoxic (H) conditions for 48 h with selection medium. Cells were harvested and lyzed with western lysis buffer under normoxic or hypoxia conditions, then subjected to immunoblotting with anti‐β‐tubulin and anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as described in Materials and Methods. (B) Immunofluorescence analysis of polymerized β‐tubulin protein expression in PC14PE6 and NCI‐H441 cell lines. After transient transfection and culture for 48 h with the selection medium, cells were seeded on chamber slides and incubated overnight. Cells were treated in the annormoxic chamber for 24 h and immunofluorescence analysis was carried out on human lung cancer cell lines, PC14PE6 and NCI‐H441, with the combination of polymerized β‐tubulin antibody as described in Materials and Methods. Representative results from three independent experiments are shown. Scar bar = 10 µm.