TRAILshort-KO clone Jurkat-I14 demonstrated enhanced death in response to exogenous TRAIL (P<0.0001, Figure 3D) compared to parental WT cells. Open in a separate window Fig 3. TRAILshort Knockout Increases TRAIL Sensitivity and T Cell Apoptosis Following Acute HIV Infection HIV Infection.(A) Primary CD4 T cells were mock infected or infected with HIV-IIIB, and treated with anti-TRAILshort antibody (clone 2.2) or isotype control for 6 days. assessed using either the Cytotox Red reagent or Active Caspase 3/7-Green reagent (Essen Bioscience, Ann Arbor, MI). Live cell imaging data was analyzed using IncuCyte Zoom software (v 2016B). Western Blot and Immunoprecipitation Assays Jurkat cells were harvested from culture, centrifuged at 200g for 5 minutes, washed with cold PBS, centrifuged again at 200g for 5 minutes, then placed on ice and resuspended in cold lysis buffer (20 mM Tris/HCl pH 7.2, 150 mM NaCl, 0.1% NP40, 0.1% CHAPS, plus protease inhibitors aprotinin, leupeptin, pepstatin, and PMSF) for 5 to10 minutes. The lysate was then centrifuged at 400g for 5 minutes to pellet nuclei. The supernatant was PDGFRA transferred to a new tube. For Western blotting, the following antibodies were used: mouse anti-TRAILs clone 2 (Mayo Hybridoma Core); mouse anti-human CD253 (TRAIL) (BD Pharmingen, San Jose, CA); rabbit anti-DR5 (D4E9, Cell Signaling Technology, Danvers, MA); goat anti-Actin (I-19, HRP, Santa Cruz Biotechnology, San Jose, CA). 400 g of cytosol was used for each immunoprecipitation reaction (2 g anti-caspase 8, Millipore, Burlington, MA) or pull down (2 g DR5-Fc, R&D systems, Minneapolis, MN) with 10 L Gammabind Plus protein A/G agarose in 200 L of lysis buffer. Reactions proceeded overnight at 4C. The agarose beads were centrifuged and washed 2 with cold lysis buffer. Wash solution was aspirated, 10 L of 2 Laemmli sample loading buffer was added, and beads were heated at 90C for 3 minutes to release bound proteins. The proteins were harvested and run on PAGE before transferring to DNA2 inhibitor C5 PVDF membrane for Western blotting. QUANTIFICATION AND STATISTICAL ANALYSIS Descriptive statistics are generally presented as means +/? standard deviation (SD) unless otherwise noted. Parametric or non-parametric statistical tests were used DNA2 inhibitor C5 as appropriate and are listed in the respective figure legends. Statistical significance was accepted when P<0.05. Statistical analysis was performed using DNA2 inhibitor C5 GraphPad Prism 6 (GraphPad, Inc). RESULTS TRAILshort Knockdown Enhances TRAIL Sensitivity and Alters T Cell Viability Following Acute HIV Infection HIV infection, we used Jurkat T cells transfected with lentiviral shRNA constructs to inhibit the expression of TRAILshort. The mRNA encoding TRAILshort consists of exon 1 and exon 2 of the TRAIL gene, along with a 30 nucleotide sequence from exon 5 (30). Because of the sequence overlap between full-length TRAIL and TRAILshort, we specifically targeted shRNA against the splice junction to achieve specific knockdown of TRAILshort expression as against full-length TRAIL. We identified a construct that reduced TRAILshort protein expression by about 60%, as estimated by densitometry, yet minimally affected expression of full-length TRAIL protein (Figure 1A). In addition the TRAILshort knockdown (TRAILshort KD) did not affect expression of TRAIL, nor TRAIL receptors 1 and 2 (Figure 1B). The resulting TRAILshort KD cells displayed enhanced susceptibility to TRAIL-mediated killing compared to cells expressing the control shRNA (Control LV), as evidenced by TUNEL staining and reduced ATP content upon DNA2 inhibitor C5 treatment with 2ng/ml SuperKiller? TRAIL (skTRAIL, a recombinant, soluble human TRAIL oligomer, Enzo Life Sciences) (Figure 1C and ?andD).D). However, we observed no altered DNA2 inhibitor C5 susceptibility to TRAIL receptor-independent death when Jurkat T cells were exposed to hydrogen peroxide, confirming the specificity of TRAILshort in TRAIL-receptor mediated cell death (Figure 1E). Open in a separate window Fig 1. TRAILshort Knockdown Enhances TRAIL Sensitivity and Alters T Cell Viability Following Acute HIV Infection Infection Since genetic approaches to inhibit protein production can incur off-target effects, we extended the results obtained with shRNA-based approach by neutralizing TRAILshort using a monoclonal antibody specific for the C-terminus of TRAILshort (30, 31). We then assessed cell death using Incucyte real time live cell imaging and a GFP labeled HIV pseudovirus (see Methods). In this assay, cells are monitored every.
