Fig. 3

CLK1 but not CLK2/3 depletion alters HIV-1 transcription initiation and enhances response to LRAs. a Quantification of TAR and R-U5-Gag RNA levels in CEM-HIV* cells depleted of individual CLK1, 2, 3, or SRPK1 by shRNA lentivirus. Relative quantification was performed using comparative cycle threshold (CT) values. PUM1 was used as a reference gene to normalize the CT value and the fold changes calculated using 2^−ΔΔCT method. b CEM-HIV* cells were depleted of CLK1 by transduction with shRNA lentivirus and transduced cells selected with puromycin for 72 h. Cells were induced by addition of Dox only or both Dox + prostratin. Following induction of provirus expression, cells were fixed and the frequency of GagzipGFP positive cells determined by flow cytometry. c CLK1 depletion enhances the ability of different LRAs to promote HIV-1 protein expression. CEM-HIV* cells were depleted of CLK1 by transduction with lentiviruses expressing shRNA. Following selection of transduced cells with puromycin for 72 h, HIV-1 gene expression was induced by addition of Dox (4.5 µM) alone, or with Dox and an LRA-prostratin (Pro, 2.56 µM), bryostratin (Bry, 25 nM), panobinostat (Pb, 40 nM), or JQ1 (2 µM). Following induction for 24 h, cells were harvested, and cell lysates analyzed for effects on GagzipGFP expression. d Effect of CLK1/2 single and double depletions on GagzipGFP expression in CEM-HIV* cells. Infection with shRNA viruses, selection and provirus induction are as previously detailed. Representative western blots are shown on the left and a graphical summary of n > 3 assays on the right. Data are indicated as mean ± SEM, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001