TFII-I enhances gene expression from plasmid constructs containing CMV promoter.

<p>Plasmid constructs coding for human oncogene Mdm2, human deubiquitinase USP48, human tumor suppressor p14 (Arf), jellyfish green fluorescent protein (GFP), and bacterial β-galactosidase (β-Gal) under the control of the CMV promoter were transfected into U2OS cells alone or in combination with pcDNA4/TFII-I. Whole cell extracts were resolved by SDS-PAGE and protein expression was analysed by Western blotting. Alpha-tubulin (α-Tub) served as a loading control.</p

Mdm2 over-expression does not lead to TFII-I degradation.

<p>(A) U2OS cells were transfected with plasmids coding for TFII-I (pcDNA4/TFII-I) and Mdm2 (pCHDM1A) and changes in TFII-I protein levels 24 hours post-transfection were analyzed using SDS-PAGE and Western blotting. PCNA served as a loading control. (B) U2OS cells were transfected with plasmids encoding TFII-I (pcDNA4/TFII-I) and Mdm2 (pCHDM1A), irradiated with ionizing radiation (γ rays, 6Gy) 24 hours post-transfection, and lysed 6 hours later. Western blotting was used to compare TFII-I protein levels in cell lysates of unirradiated controls and irradiated samples, both in the absence and in the presence of Mdm2 over-expression.</p

Mdm2 interacts with TFII-I.

<p>(A) HEK293T cells were transfected with a plasmid coding for human Mdm2. Endogenous TFII-I was co-immunoprecipitated from cell lysates using anti-Mdm2 antibody. (B) U2OS cells were transfected with a plasmid coding for human Mdm2. The subcellular localization of endogenous TFII-I and of the ectopically expressed Mdm2 was determined by immunofluorescence. DAPI was used to label cell nuclei. (C) Endogenous TFII-I and Mdm2 were co-immunoprecipitated from H1299 cell extracts using anti-TFII-I and anti-Mdm2 mouse monoclonal antibodies. Anti-tubulin α antibody served as a negative control. (D) Plasmids coding for GST and GST-tagged TFII-I mutant lacking the nuclear localization signal (ΔNLS) were transfected into U2OS cells either alone or together with Mdm2. Subcellular localization of the GST protein tag, GST-TFII-IΔNLS, and Mdm2 was analyzed using immunofluorescence. DAPI staining was used to label cell nuclei.</p

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

<p>(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</p

Ectopic expression of TFII-I activates CMV promoter.

<p>(A) CMV promoter sequence with potential TFII-I binding sites. (B) Schematic representation of CMVdel luciferase vectors used in this study. (C) CMVdel series vectors were transfected into U2OS cells, either alone or in combination with pcDNA4/TFII-I and luciferase activity was measured in cell extracts 24 h post-transfection. The upper graph shows the relative changes in luciferase activity induced by TFII-I for each construct of the series individually. Data obtained in three independent experiments are presented as mean +/- standard deviation (* P<0.05, *** P<0.001). The obtained P values suggest significant differences in luciferase activity between cells co-transfected with TFII-I and cells transfected only with the respective luciferase construct. The lower graph shows the same set of results presented in relation to the full-length construct (activity in cells transfected with CMVdel1 was set as 1). Representative Western blot shows TFII-I protein levels in each sample (loaded in the same order as in the graphs). Endogenous PCNA served as a loading control.</p

Endogenous TFII-I contributes to CMV promoter activity in human cells.

<p>U2OS cells were transfected with a mixture of siRNAs targeting TFII-I (si TFII-I) or non-targeting control siRNAs (si ctrl). Twenty four hours later, the same cells were transfected again, this time with the CMVdel1 luciferase construct (A) or the β-galactosidase construct pcDNA3.1(-)/myc-His/LacZ (LacZ) (B). Cells were lysed 24 hours later. The graphs show the relative changes in luciferase activity or β-galactosidase activity, respectively, in three independent experiments (mean +/- standard deviation; * P<0.05, ** P<0.01). The representative results of Western blotting analysis illustrate the efficiency of TFII-I knock down in the presented experiments.</p