FIGURE 7.

MSK1 activity is required for anchorage-independent growth of Ras-transformed mouse fibroblasts. a, Ciras-3 cells were grown in soft agar with or without H-89. b, 20 μg of MSK1 knockdown or control cell lysates were resolved by 10% SDS-PAGE and immunoblotted with anti-MSK1 and anti-β-actin antibodies. c and d, equal numbers of Ciras-3 cells stably expressing the MSK1 knockdown-targeting vector mm1, mm2, or mm3 or the pGIPZ control and mock-transfected cells were grown in soft agar. The numbers of colonies were counted after 14 days and plotted for each cell line. Images of plates are shown for the Ciras-3 cells stably expressing the MSK1 knockdown-targeting vector mm3 and pGIPZ. Data are representative of three individual experiments. *, statistically significant difference as determined by a Student's t test comparison of mm1 and mm3 with the pGIPZ control (p < 0.01). e, Ciras-3 cells stably expressing the MSK1 knockdown-targeting vector mm3 or the pGIPZ control and mock-treated cells were seeded in triplicate in a 96-well plate and evaluated for cell proliferation using CellTiter 96® Aqueous One Solution reagent. The absorbance readings were measured at 490 nm, and measured values were plotted as described under “Experimental Procedures.” Data are representative of three individual experiments.