FIG. 4.

Maintenance of H-Ras^V12-induced proliferation in 3T3 fibroblasts is dependent on pRB expression. (A) NIH 3T3 cells were transfected with a vector control (vector), a pRB-expressing plasmid (pRB), an H-Ras^V12-expressing vector (ras^V12), or the combination of H-Ras^V12 and pRB (Ras^V12 plus pRB), and colonies were counted 2 to 3 weeks postconfluence. The bar graphs are based on the results of two independent experiments. The microscopy images on the right illustrate the increased size of foci seen following transfection with a combination H-Ras^V12 and pRB, as indicated. (B) Western blot analysis of pRB protein levels in pools of NIH 3T3 cells transfected with vector or H-Ras^V12 as indicated in the left panel of the figure. The right panel shows the level of pRB protein in confluent H-Ras^V12-transfected NIH 3T3 cells that have been treated with the dimethyl sulfoxide control or PD89059 for 15 h. Confluent cells were used to minimize cell cycle effects. (C) Western blot analysis of levels of total pRB protein, cyclin D, as well as the levels of pRB protein phosphorylated on various sites using phospho-specific antibodies as indicated in the figure, in pools of NIH 3T3 cells transfected with vector or H-Ras^V12. (D) The proliferation of NIH 3T3, NIH 3T3 plus H-Ras^V12, and pRB^−/− plus H-Ras^V12 cells transfected with Rb siRNA oligonucleotides (RB-i) and control siRNA oligonucleotides (Control-i) 2 days posttransfection. Illustrated in the figure is the relative proliferation of cells transfected with Rb-siRNA oligonucleotides compared to cells transfected with control RNAi oligonucleotides. The results are based on three independent experiments. The microscopy images show representative examples of the reduced proliferation seen in H-Ras^V12-transformed NIH 3T3 cells treated with Rb-siRNA oligonucleotides or PD98059 2 days posttransfection or addition of drug. The bottom panel illustrates the decrease in pRB levels following transfection of pRB siRNA oligonucleotides (RB-i) or control RNAi (Control-i), as indicated. The extract from cells transfected with the control siRNA oligonucleotide was titrated to better illustrate the loss of pRB in the Rb-siRNA-treated samples, using total p44/42 as a loading control.