FIGURE 3.

Clonogenic and anchorage-independent growth was reduced in HCT116 cells stably transfected with Ron shRNA. A, Ron knockdown clones and the control cells were lysed and subjected to Western blot analyses with an antibody to Ron. Actin was used as a loading control. B and D, cells were plated in 24-well plates at 300 cells/well. Cell colonies were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and visualized after 2 weeks of incubation (B). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide stain was dissolved in dimethyl sulfoxide. Relative cell numbers were then determined by the resultant absorbance at 595 nm. Values are the means of four replicates (D). Error bars indicate S.E. C, anchorage-independent colony formation in soft agarose by HCT116 control and Ron knockdown cells was determined as described under “Experimental Procedures.”