Figure 4

Effect of HIF-1αFL and HIF-1α785 overexpression in radial growth phase SbCl2 cells on anchorage-independent growth and invasion. SbCl2 cells were transiently transfected at 80% confluence with either mock (no plasmid DNA), pLenti-V5-Lacz, pLenti-V5-D-TOPO-HIF-1αFL or pLenti-V5-D-TOPO-HIF-1α785 using FuGene 6 transfection reagent. (A) After 48 h nuclear protein was extracted and over-expression was confirmed by western blot using HRP-conjugated anti-V5 antibody and mouse monoclonal anti-HIF-1α antibody (1 μg/ml). LaminB1 was used as loading control. (B) SbCl2 cells 24 h post transfection with either mock (no plasmid DNA), Lacz, HIF-1αFL or HIF-1α785 were subjected to Matrigel invasion assay as described in "Material and Methods". Results are expressed as percent invasive cells corrected for invasion level of cells seeded in Matrigel (-) chambers. (C&D). SbCl2 cells transfected either with reagents alone (mock), Lacz, HIF-1αFL or HIF-1α785 were subjected to CytoSelect 96 well Cell Transformation Assay^® (Cell Biolabs, Inc.) as described in "Material and Methods" for 4 (C) and 5 days (D). Results are expressed as Relative Fluorescent Units. Data is expressed as the mean ± SEM of triplicate values. ANOVA with TUKEY for multiple pairwise comparison was used to analyze the data and all P values ≤ 0.05 are relative to Mock and Lacz overexpressing cells. The entire experiment was repeated two additional times with similar results.