Figure 5
The effect of loss of HIF-1α expression in human metastatic melanoma WM9 cells on anchorage-independent growth and invasion. (A) WM9 cells were treated with either 100 nM HIF-1α siRNA or 100 nM control non-targeting siRNA (Dharmacon, Inc.) using the RNAifect® transfection reagent (Qiagen, Inc.). Knock down of HIF-1α was confirmed by western blot at 72 h post transfection. (B) At 48 h post transfection Ctrl siRNA or HIF-1α siRNA treated WM9 cells, were assayed for anchorage independent growth using the CytoSelect 96 well Cell Transformation Assay® (Cell Biolabs, Inc.). Briefly, the cells were seeded at 8.0 × 103 cells/well into a 0.4% agar layer poured over a 0.6% agar layer of a 96 well plate. Wells lacking cells served as a blank control. On day 5 after seeding, agar layers were solubilized, cells were lysed, and nucleic acid stained with CyQuant dye. Fluorescence intensity in each well was determined by a plate reader set at 485/520 nm. Results are expressed as Relative Fluorescent Units. (C) Photomicrograph of a representative field of colonies form in the Cell Transformation Assay by control siRNA transfected cells (left) vs. HIF-1α siRNA transfected cells. (D) Matrigel invasion assay. At 48 h post transfection, the Ctrl siRNA or HIF-1α siRNA transfected WM9 cells were seeded into 6-well Matrigel (+) chambers, and as a control, 6-well Matrigel (-) chambers (BD Biosciences) at 7.0 × 104 cells per well. The method and counting of invading cells was done as described in "Experimental Procedures". Results are expressed as % invasion of HIF-1α siRNA treated-WM9 cells relative to invasion by Control siRNA treated-WM9 cells corrected for invasion by similarly treated cells seeded in Matrigel (-) chambers. (E) Cell viability assay was done in WM9 cells transfected with control siRNA or HIF-1α siRNA at 48, 72 and 96 h following transfection by the trypan blue exclusion method. Data is expressed as the mean ± SEM of triplicate samples. The entire experiment was repeated twice with similar results. Student paired t test was used to analyse the data and all P values ≤ 0.01 or 0.05 are relative to control siRNA treated cells.