FIGURE 4.

ING2 RNAi suppresses TGF-β-induced transcription. A, ING2, GFP, and actin immunoblotting of lysates of 293T cells coexpressing Myc-tagged ING2 together with a control or an ING2 RNAi plasmid. Quantitative analysis of GFP or actin-normalized ING2 protein density indicates that ING2 RNAi led to a 60-70% reduction in ING2 protein level. The asterisk indicates nonspecific Myc-immunoreactive band. B, effect of ING2 RNAi on TGF-β-induced transcription in Mv1Lu cells. Cells transfected with 3TP-luciferase and CMV-β-galactosidase reporter plasmids together with the control or the ING2 RNAi vector were incubated in the absence or presence of TGF-β. C, the ING2 rescue construct reverses ING2 RNAi-mediated reduction of TGF-β-dependent transcription. The cells were transfected and incubated in the absence or presence of TGF-β as described for B, except for the inclusion of a control mammalian expression vector (-) or one encoding an ING2 RNAi-resistant ING2 protein as indicated. Luciferase activity in the lysates of cells described for B and C were determined as described for Fig. 1D. The data shown in each of B and C represent the means (±S.D.) of triplicate determinations from a representative experiment that was repeated three times.