This indeed is what we found, since NK cells have a decreased ability to lyse virus-infected cells lacking Vpr. for HIV-1 p24 antigen (Ag). Two-dimensional plots were derived following acquisition on a flow cytometer of 104 viable cells. Markers in ZM 336372 dot plots were positioned based on the staining controls. The figure is representative data from three separate experiments.(0.98 MB TIF) ppat.1000613.s002.tif (962K) GUID:?4132CF9A-F1E1-47EA-95C2-C3A9C5A719CC Figure S3: Effect of point mutations in postions 65 and 80 of Vpr on the cell cylcle of HIV-infected cells. Infected primary T-cell blasts and uninfected CD4pos T-cells were stained with TO-PRO-3 in order to obtain the (G2+M)/G1 ratio.(0.67 MB TIF) ppat.1000613.s003.tif (657K) GUID:?3D66DDA8-B86F-4FF5-A311-C2AC1DBCA1C4 DUSP10 Figure S4: Expression of DCAF1 is not affected by HIV-1 Vpr. The HeLa cell line was either treated with 10 M ZM 336372 aphidocolin (Aph), or infected with VSV-G pseudotyped HIV-1 with wild-type Vpr (Vpr) or HIV with Q65R and R80A mutations in Vpr (Vpr QR). Following treatment/infection cells lysates were made and western blotted. Western blots were probed with DCAF1 specific antibody.(0.56 MB TIF) ppat.1000613.s004.tif (542K) GUID:?80294EAF-C4BF-4EC6-9630-546FC5278354 Figure S5: Inhibition of ATR activity relieves Vpr-induced G2 arrest. Primary CD4pos T-cell blasts were exposed to 4 mM of the ATR inhibitor, caffeine (B and D) or vehicle (A or C) and either infected with HIV-1 (C and D) or left uninfected (A and B). Forty-eight hrs. following exposure to caffeine and HIV-1 infection the cell cycle profile of the uninfected and infected cells were detected by TO-PRO-3 staining.(1.50 MB TIF) ppat.1000613.s005.tif (1.4M) GUID:?B1434D32-9830-471B-88D7-E613E16507C4 Figure S6: Inhibition of ATM activity reduces NKG2D ligand expression on primary CD4pos T-cells treated with aphidocolin. Primary CD4pos T-cells were treated with 10 M aphidicolin in the presence of 10 M KU55933 (ATM-specific inhibitor) or 4 mM caffeine. As a negative control aphidicolin-treated cells were exposed to vehicles used to dissolve the inhibitor in solution. Following 48 h exposure to KU55933, caffeine or vehicle the cells were stained with a fusion ZM 336372 protein of human NKG2D and the Fc portion of human IgG1 and fluorochrome conjugated-goat anti-human IgG Fc specific antibody (blue line) or secondary antibody alone [staining control (red line)]. The histograms are gated for 104 viable CD4pos cells. This is a representative of two experiments.(0.71 MB TIF) ppat.1000613.s006.tif (689K) GUID:?C85B4E57-3390-4F76-869F-B7D0B0E67454 Figure S7: Ability of NK cells to lyse autologous T-cells infected with HIV-1 lacking Vpr. Primary CD4pos T-cell blasts were infected with HIV-1 that were deficient in expression of Vpr (Vpr). As a control, the same cells were infected with wild-type (WT) HIV-1. Following infection the infected cells were isolated, labeled with 51Cr and mixed with autologous NK cells at 2.51 (A and C) and 51 (B and D) effector cell to target cell ratios. Prior to the lytic assay some of the NK cells were exposed to blocking antibodies to NKG2D (C and D). At the end of the incubation period culture fluids were harvested and analyzed for the presence of 51Cr. Percent specific lysis was determined as described in the Materials and Methods section. Each point designates a sample from each group. Bars represent the mean percent specific lysis. This supplemental figure is a dot plot representation of Figure 10.(1.02 MB TIF) ppat.1000613.s007.tif (1000K) GUID:?8C37D2EE-82DB-4C2F-912B-6E9674FA5584 Figure S8: Expression of viral proteins by various HIV-1 mutants. HIV-1-infected CD4pos cells were lysed in cell lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.25% Na-deoxycholate, 1 mM EDTA. 1 mM PMSF, 1 mM Na3VO4, 0.1% SDS, and protease inhibitors), run on 15% SDS-PAGE gels, transferred to PVDF, and probed for the indicated proteins with specific antibodies. (A) Lysates from CD4pos cells infected with DHIVVpr, DHIVVif, DHIVVpr,Vif or DHIV containing Vpr with point mutations in specific residues. (B) Lyates from CD4pos T-cells infected with DHIVNef.(3.19 MB TIF) ppat.1000613.s008.tif (3.0M).