Fig. 4.
Fig. 4. Transcriptional repression of CD11b by overexpression of ZBP-89 in differentiating U937 cells and in. / Drosophila SL2 cells. (A) Dose-dependent repression of CD11b by ZBP-89 in stable U937 cells in whichCD11b-luciferase gene is incorporated in the genome. The various concentrations of pPacZBP-89 are indicated. Luciferase activity is normalized against β-galactosidase activity, and each histogram represents the mean ± SD of 3 independent experiments. In a typical experiment, the CD11b luciferase activity without ZBP-89 was 41 771 relative light units (RLU; normalized to 100%), compared to 22 RLU in stable cell lines lacking the exogenousCD11b promoter. (B) Histograms comparing the transrepression activity of ZBP-89 on CD11b-wt and mutantCD11b-M4a gene expression. Each transfection was performed 6 times. Luciferase activity is normalized against β-galactosidase activity, and the SD from the mean is presented with each histogram. The fold above background (conferred by the negative control plasmid pATLuc) of CD11b-wt luciferase activity without ZBP-89 is 29.32 ± 2.36 (n = 6), and this was considered as 100%. (C) Derepression of CD11b promoter activity by blocking endogenous ZBP-89 binding. U937 cells were transfected with pALbWt or pALbM4a then treated for 16 hours with PMA prior to harvesting. The level of Wt luciferase reporter gene activity, normalized against β-galactosidase activity to correct transfection efficiency, is expressed relative to that conferred by the negative control plasmid pATLuc. It represented 143.85 ± 8.5 (n = 3) fold above background and equated to 100%. (D) Transfection assays in DrosophilaSL2 cells were performed using 1 μg of the reporter construct pALbWt, 0.2 μg pPacSp1 expressing Sp1, 0.1 μg pIELacZ expressing β-galactosidase, and an increasing amount of pPacZBP-89 (expressing ZBP-89) from 0 to 1 μg. The various concentrations of pPacZBP-89 are indicated. The final DNA concentration in all transfections was adjusted to 2.6 μg by adding the empty vector pPacO. Sp1-driven promoter activity was expressed as a ratio of Sp1-driven CD11b-wt promoter activity divided by that obtained in the absence of Sp1. In the absence of ZBP-89 expression, the calculated level of Sp1-driven transcription (81.1 ± 12.2 above background, n = 3) was considered as 100%. (E) The same experiment was performed as in panel C using pALbM4a instead of pALbWt (actual values of Sp-1–driven transcription were 91 ± 10.68, n = 3, above background).

Transcriptional repression of CD11b by overexpression of ZBP-89 in differentiating U937 cells and in

Drosophila SL2 cells. (A) Dose-dependent repression of CD11b by ZBP-89 in stable U937 cells in whichCD11b-luciferase gene is incorporated in the genome. The various concentrations of pPacZBP-89 are indicated. Luciferase activity is normalized against β-galactosidase activity, and each histogram represents the mean ± SD of 3 independent experiments. In a typical experiment, the CD11b luciferase activity without ZBP-89 was 41 771 relative light units (RLU; normalized to 100%), compared to 22 RLU in stable cell lines lacking the exogenousCD11b promoter. (B) Histograms comparing the transrepression activity of ZBP-89 on CD11b-wt and mutantCD11b-M4a gene expression. Each transfection was performed 6 times. Luciferase activity is normalized against β-galactosidase activity, and the SD from the mean is presented with each histogram. The fold above background (conferred by the negative control plasmid pATLuc) of CD11b-wt luciferase activity without ZBP-89 is 29.32 ± 2.36 (n = 6), and this was considered as 100%. (C) Derepression of CD11b promoter activity by blocking endogenous ZBP-89 binding. U937 cells were transfected with pALbWt or pALbM4a then treated for 16 hours with PMA prior to harvesting. The level of Wt luciferase reporter gene activity, normalized against β-galactosidase activity to correct transfection efficiency, is expressed relative to that conferred by the negative control plasmid pATLuc. It represented 143.85 ± 8.5 (n = 3) fold above background and equated to 100%. (D) Transfection assays in DrosophilaSL2 cells were performed using 1 μg of the reporter construct pALbWt, 0.2 μg pPacSp1 expressing Sp1, 0.1 μg pIELacZ expressing β-galactosidase, and an increasing amount of pPacZBP-89 (expressing ZBP-89) from 0 to 1 μg. The various concentrations of pPacZBP-89 are indicated. The final DNA concentration in all transfections was adjusted to 2.6 μg by adding the empty vector pPacO. Sp1-driven promoter activity was expressed as a ratio of Sp1-driven CD11b-wt promoter activity divided by that obtained in the absence of Sp1. In the absence of ZBP-89 expression, the calculated level of Sp1-driven transcription (81.1 ± 12.2 above background, n = 3) was considered as 100%. (E) The same experiment was performed as in panel C using pALbM4a instead of pALbWt (actual values of Sp-1–driven transcription were 91 ± 10.68, n = 3, above background).

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