Fig 7. Effects of TFII-I and Mdm2 on transcription are specific to CMV promoter.

(A) Over-expression of TFII-I and Mdm2 does not influence the activity of HIV 3’ LTR promoter. H1299 cells were transfected with pHIVlacZ (HIV-LacZ) alone or with HIV-LacZ plus pCEP4-Tat (TAT), together with plasmids coding for TFII-I (pcDNA4/TFII-I) and Mdm2 (pcHDM1A). The activity of β-galactosidase was measured 24 h later. Data obtained in three independent experiments are presented as mean +/- standard deviation. Representative Western blot shows TFII-I and Mdm2 protein levels in lysates of transfected cells. Endogenous PCNA served as a loading control. (B) Over-expression of TFII-I does not induce significant changes in cell proliferation or viability. U2OS cells were transfected with pEGFP-C2 to mark the transfected cells with GFP, together with either pcDNA4/LacZ (GFP+LacZ) or with pcDNA4/TFII-I (GFP+TFII-I). Flow cytometry was used 24 h post-transfection to determine the proportion of GFP-positive cells in the total cell population and cell viability in a PI exclusion assay. The experiment was performed in triplicates and the table shows the real counts of GFP-positive cells, their proportion in total cell population (mean) and the proportion of dead transfected cells (PI-positive, GFP-positive) in total cell population (mean). Untransfected U2OS cells served as a negative control. The Western blot shows GFP, TFII-I and Mdm2 expression in total cell lysates of transfected cells. (C) Sp1 does not require Mdm2 co-expression for inhibiting CMV promoter. H1299 cells were transfected with pcDNA4/LacZ alone, with pcDNA4/LacZ plus pcDNA4/TFII-I or pcDNA4/LacZ plus pEGFP-Sp1, together with plasmid coding for Mdm2 (pcHDM1A). The activity of β-galactosidase was measured 24 h later (right panel). Data obtained in three independent experiments are presented as mean +/- standard deviation