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. 2015 Dec 7;10(12):e0144229. doi: 10.1371/journal.pone.0144229

Fig 1. The effect of DYRK1A knockdown or inhibition on HIV-1 replication.

Fig 1

(A) To test the shRNA targeting against DYRK1A, HEK293T cells were transfected with either 6.25 ng, 12.5 ng or 25 ng of shDYRK1A or shControl and 48-hours after transfection DYRK1A protein expression was determined by Western blot. The results shown are representative for 3 independent experiments. The ratio is calculated by dividing the intensity of the DYRK1A bands by the intensity of the β-actin band as determined with ImageJ. (B) The effect of DYRK1A downregulation on HIV-1 replication was tested by transfecting HEK293T cells in 96-wells plates with either 6.25 ng, 12.5 ng or 25 ng of shDYRK1A or corresponding concentrations of the shControl. Forty-eight hours after DYRK1A downregulation cells were inoculated at a MOI of 0.01 with a VSV-G-pseudotyped single-round luciferase virus. Luciferase activity was determined 48 hours post infection as a measure for viral replication and expressed relative to the corresponding shControl. Data is shown as mean and SD of three independent experiments. (C) The effect of DYRK1A inhibition on HIV-1 replication in HEK293T was analyzed by infection with a VSV-G-pseudotyped single-round luciferase virus. Twenty-four hours post infection cells were treated with 24 μM, 48 μM or 120 μM of either INDY or DMSO control and after an additional 24-hours luciferase activity was determined as a measure for viral replication and expressed relative to DMSO control (No Drug). Data is shown as mean and SD of three independent experiments. (D) The effect of DYRK1A inhibition on HIV-1 replication in PBMC was analyzed by infecting PHA-stimulated PBMC from four healthy blood donors with a VSV-G-pseudotyped single-round luciferase virus at a MOI of 0.1. Twenty-four hours post infection, cells were treated with either 24 μM or 48 μM of INDY or DMSO control and after another 24-hours luciferase activity was determined as a measure for viral replication and expressed relative to the DMSO control (No drug). Data is shown as mean and SD of 4 independent donors. Significance was determined with an unpaired student’s T test. *p<0.05, **p<0.01, ***p<0.001.