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. 2019 Jul 30;93(16):e00412-19. doi: 10.1128/JVI.00412-19

FIG 6.

FIG 6

Impact of BRG1 knockdown on HBZ inhibition of basal and Tax-dependent viral LTR transcription. (A) Effect of BRG1 knockdown on Tax-dependent viral promoter activity. HeLa-HTLV-1-LTR-Luc cells were cotransfected with 25 ng of HIS-pCAGGS-Tax, alone or together with 4 μg shRNA against BRG1 (ShBRG1). Luciferase activity was measured at 72 h posttransfection. LTR-dependent luciferase activity was normalized to the protein concentration in lysates and is expressed as fold activation. Error bars represent SD calculated from three independent experiments. * indicates significance at a P value of <0.05. (B) Effect of BRG1 knockdown on the ability of HBZ to suppress Tax-dependent viral promoter activity. HeLa-HTLV-1-LTR-Luc cells were cotransfected with 25 ng of HIS-pCAGGS-Tax together with 2 μg FLAG-HBZ plus shRNA against BRG1. Luciferase assays were performed as described above for panel A. (C) Effect of BRG1 knockdown on Tax-dependent viral promoter activity in Jurkat-U3-LTR-HTLV-1-Luc cells. Luciferase assays were performed on lysates from Jurkat-U3-LTR-HTLV-1-Luc cells cotransfected with 25 ng of HIS-pCAGGS-Tax, alone or together with 4 μg shRNA against BRG1 or the shRNA negative control. Luciferase activity was measured at 48 h posttransfection. LTR-dependent luciferase activity was normalized to the protein concentration in the lysates, and the means of data from three independent experiments are expressed as fold activation. The P value indicates no significance (NS). (D) Effect of BRG1 knockdown on the ability of HBZ to suppress Tax-dependent viral promoter activity. Luciferase assays were performed on lysates from Jurkat-U3-LTR-HTLV-1-Luc cells cotransfected with either 4 μg shRNA against BRG1 or the shRNA negative control together with 25 ng of HIS-pCAGGS-Tax, alone or together 1 μg FLAG-HBZ. Luciferase assays were performed as described above for panel C. (E) Effect of BRG1 knockdown on basal LTR activation. Luciferase assays were carried out on lysates from Jurkat-U3-LTR-HTLV-1-Luc cells cotransfected with either 4 μg shRNA against BRG1 or the shRNA negative control. Luciferase assays were performed as described above for panel C. (F) Effect of BRG1 knockdown on the ability of HBZ to suppress basal activation of the viral promoter. Luciferase assays were performed on lysates from Jurkat-U3-LTR-HTLV-1-Luc cells cotransfected with either 4 μg shRNA against BRG1 or the shRNA negative control together with 1 μg FLAG-HBZ. Luciferase assays were performed as described above for panel C. (G) Immunoblot analysis of BRG1 knockdown was performed on Jurkat-U3-LTR-HTLV-1-Luc cells. Cells were transfected with 4 μg of shRNA against BRG1 plasmids (lane 2) and the shRNA negative control (lane 1).