Journal: Retrovirology

Article Title: Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency.

doi: 10.1186/s12977-022-00605-4

Figure Lengend Snippet: Fig. 1 Depletion of SR kinases has differential effects on HIV-1 protein levels. a Schematic of HIV-1 rtTAGagzipGFP provirus used to generate CEM-HIV* cell line. b, c CEM-HIV* cells were infected with shRNA lentivirus targeting CLK1, CLK2, CLK3, or SRPK1 and transduced cells were selected with puromycin for 72 h. Following puromycin selection, HIV-1 gene expression was induced with doxycycline (Dox, 4.5 µM) + prostratin (Pros,2.56 µM) and cells harvested for western blots after 24 h of induction. Shown are the representative western blots indicating expression levels of b the target kinase, c or HIV-1 Env, Gag, and Tat levels. Band intensity was quantified relative to Dox induced shRNA control and normalized to either total protein stain for Env and Gag blots or GAPDH for Tat blots using Bio-Rad ImageLab software. Data are indicated as mean ± SEM, n ≥ 4 independent experiments, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. Dotted vertical lines on the blots represent cropping of lanes on the same representative blot to show shcontrol lanes adjacent to shRNA target depletion lanes

Article Snippet: Recombinant human CLK1 (50 ng, C57-11G, SignalChem Biotech Inc. Canada) was incubated with DMSO or a range of serial dilutions of 1C8 in kinase buffer (40 mM Tris–HCl pH 7.5, 25 mM MgCl2, 0.1 mg/mL BSA and 0.25 mM DTT) containing 50 μM of ATP (Promega, Madison, WI, USA).

Techniques: Infection, shRNA, Selection, Gene Expression, Western Blot, Expressing, Control, Staining, Software

Journal: Retrovirology

Article Title: Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency.

doi: 10.1186/s12977-022-00605-4

Figure Lengend Snippet: Fig. 2 Effect of SR kinase depletion on HIV-1 RNA accumulation. a Schematic of HIV-1 provirus indicating the position of primers used to detect viral RNAs. b CEM-HIV* cells were depleted of CLK1, CLK2, CLK3, or SRPK1 by transduction with lentiviruses expressing shRNAs to these SR kinases. Following selection of transduced cells with puromycin for 72 h, HIV-1 gene expression was induced by addition of Dox (4.5 µM) + prostratin (2.56 µM). Cells were harvested for RNA extraction after 24 h of induction. HIV-1 unspliced (US), singly spliced (SS), multiply spliced (MS) RNA levels were determined by RT-qPCR. Viral mRNA levels were normalized to ß-actin and the mean mRNA levels expressed relative to sh control. Data are indicated as mean ± SEM, n = 3 independent experiments, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001

Article Snippet: Recombinant human CLK1 (50 ng, C57-11G, SignalChem Biotech Inc. Canada) was incubated with DMSO or a range of serial dilutions of 1C8 in kinase buffer (40 mM Tris–HCl pH 7.5, 25 mM MgCl2, 0.1 mg/mL BSA and 0.25 mM DTT) containing 50 μM of ATP (Promega, Madison, WI, USA).

Techniques: Transduction, Expressing, Selection, Gene Expression, RNA Extraction, Quantitative RT-PCR, Control

Journal: Retrovirology

Article Title: Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency.

doi: 10.1186/s12977-022-00605-4

Figure Lengend Snippet: 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−

Article Snippet: Recombinant human CLK1 (50 ng, C57-11G, SignalChem Biotech Inc. Canada) was incubated with DMSO or a range of serial dilutions of 1C8 in kinase buffer (40 mM Tris–HCl pH 7.5, 25 mM MgCl2, 0.1 mg/mL BSA and 0.25 mM DTT) containing 50 μM of ATP (Promega, Madison, WI, USA).

Techniques: shRNA, Quantitative Proteomics

Journal: Retrovirology

Article Title: Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency.

doi: 10.1186/s12977-022-00605-4

Figure Lengend Snippet: Fig. 4 Activation of primary CD4+ T cells selectively alters SR kinase and SR protein levels. Primary CD4+ T cells were isolated from healthy (HIV uninfected) human donors and untreated (control) or treated with activators (anti-CD3/CD28 and IL-2). Cells were harvested at different times (24 h, 48 h, 4 d, and 6 d) with or without activation for analyses by western blots or RT-qPCR to look for changes in the expression of SR kinases and SR proteins. a Top and bottom panels on the left are the representative western blots probed for CLK1, CLK2, CLK3, and SRPK1. Top and bottom panels on the right are the quantitation of blots for at least 3 donors (except for 4 d and 6 d post-activation for CLK2, CLK3, and SRPK1 expression levels where only one donor was used). b Quantification of CLK1 and SRPK1 mRNA levels in CD4+ T cells of 3 donors by RT-qPCR assay. mRNA levels were normalized to ß2-microglobulin and mean mRNA levels were expressed relative to untreated control. c Quantitation of western blots for SR protein expression levels in untreated versus treated/activated CD4+ T cell lysates (see Additional file 1: Fig. S4 for representative western blots) across at least 3 donors. For western blots, band intensity was quantified relative to untreated control and normalized to total protein load using Bio-Rad ImageLab software. Data are indicated as mean ± SD, n = 3 or 4 independent experiments, *p ≤ 0.05 and **p ≤ 0.01

Article Snippet: Recombinant human CLK1 (50 ng, C57-11G, SignalChem Biotech Inc. Canada) was incubated with DMSO or a range of serial dilutions of 1C8 in kinase buffer (40 mM Tris–HCl pH 7.5, 25 mM MgCl2, 0.1 mg/mL BSA and 0.25 mM DTT) containing 50 μM of ATP (Promega, Madison, WI, USA).

Techniques: Activation Assay, Isolation, Control, Western Blot, Quantitative RT-PCR, Expressing, Quantitation Assay, Software

Journal: Retrovirology

Article Title: Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency.

doi: 10.1186/s12977-022-00605-4

Figure Lengend Snippet: Fig. 8 CLK1 and CLK2 act at distinct steps to regulate HIV-1 gene expression. HIV-1 provirus integrated into the host genome is transcribed by the cellular RNA polymerase II (RNAP II). CLK1 acts to suppress the use of the HIV-1 promoter while CLK2 promotes steps in viral RNA synthesis/ processing post initiation (elongation, splicing, or 3′ end formation)

Article Snippet: Recombinant human CLK1 (50 ng, C57-11G, SignalChem Biotech Inc. Canada) was incubated with DMSO or a range of serial dilutions of 1C8 in kinase buffer (40 mM Tris–HCl pH 7.5, 25 mM MgCl2, 0.1 mg/mL BSA and 0.25 mM DTT) containing 50 μM of ATP (Promega, Madison, WI, USA).

Techniques: Gene Expression

Journal: Malaria Journal

Article Title: Tafenoquine and NPC-1161B require CYP 2D metabolism for anti-malarial activity: implications for the 8-aminoquinoline class of anti-malarial compounds

doi: 10.1186/1475-2875-13-2

Figure Lengend Snippet: Cure rates in C57BL/6 wild-type (WT), CYP 2D knock-out, and humanized/CYP 2D6 knock-in mice 31 days post-inoculation with Plasmodium berghei in the absence or presence of NPC-1161B and tafenoquine

Article Snippet: Heads and thoraxes were triturated with a mortar and pestle and suspended in medium RPMI 1640 containing 1% C57BL/6 mouse serum (Rockland Co, Gilbertsville, PA, USA).

Techniques: Knock-Out, Knock-In