Fig. 7.

1H3 affects CLK1 and CLK2 function and expression. a Effect of compound 1H3 on SRSF2 subcellular distribution upon overexpression of CLKs in HeLa B2 cell line. Cells were transfected with indicated GFP-CLK expression vectors and 48 h post transfection treated with DMSO or 200 nM 1H3 for 24 h, fixed and processed for immunofluorescence. Cells were stained with anti-SRSF2 antibody, a marker for nuclear speckles, and nuclei stained with DAPI. Shown are the representative images of the localization patterns of SRSF2 upon overexpression of CLK1, CLK2, or CLK3. Red arrows indicate loss of nuclear speckles due to CLK overexpression in DMSO or 1H3-treated cells upon CLK3 overexpression. Yellow arrows indicate restoration of nuclear speckles in 1H3-treated cells upon CLK1 or CLK2 overexpression. Images are representative of n = 3 independent experiments. b, c Healthy donor CD4+ T cells were infected with HIV-1 89.6 then treated with DMSO, 10 µM TG003, or 300 nM 1H3 for 3 d. Cells were harvested and the effect of individual treatments on CLK1, CLK2, CLK3, or SRPK1 b protein or c mRNA expression determined. Shown on the left in b is a representative western blot and, on the right, a summary of assays. Data shown corresponds to results from n > 6 individual patient samples from 2 independent experiments. d, e 1C8 inhibits HIV-1 gene expression and alters CLK expression. CEM-HIV* cells were treated with 10 µM 1C8 or equivalent volume of DMSO. HIV-1 expression was induced by addition of DOX + prostratin for 24 h. Cells were subsequently harvested, and the lysates analyzed by western blot for d HIV-1 Gag and Env expression or e expression of CLK1, CLK2, or CLK3. Shown are the representative western blots and below is a graphical summary of the blots across n = 3 independent assays. Band intensity was quantified relative to Dox induced DMSO control and normalized to total protein load using Bio-Rad ImageLab software. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001