Supplementary MaterialsS1 Table: Transcriptome analysis of PBT exposed to AS1842856 treatment. results +/- SE with cells from 3 different donors are shown.(PDF) ppat.1007669.s002.pdf (86K) GUID:?0AD88B6C-C6E3-4A05-9501-61A7508773CB S2 Fig: AS1842856 induces significant T-cell size increase in all T cell subsets. (A) FSC of PBT treated with AS1842856 (500nM) or vehicle only were analyzed by FACS at different time points during 7 days of culture. Mean results +/- SE from 5 independent donors are shown. (B) PBT were cultured for 7 days with various concentrations of AS1842856 or the corresponding dilution of vehicle. (C) After 7 days of treatment with AS1842856 (500nM) or vehicle only, a total cell count of the viable cells in the culture was performed (mean results +/- SE with cells from five different donors). (D) PBT were cultured for 7 days with 500nM of AS1842856 or vehicle only; FSC of CD45RA-positive (na?ve) and CD45RA-negative (memory) sub-populations was then measured by FACS after labeling with CD4, CD8 and CD45RA-specific antibodies. Mean results +/- SE from 6 independent donors are shown.(PDF) ppat.1007669.s003.pdf (95K) GUID:?392876E5-9623-44DD-A4ED-5C431F20C3DA S3 Fig: AS1842856 does not initiate proliferation of PBT. PBT were cultured for 7 days with AS1842856 (500 nM) or vehicle only, then stained with CFSE and stimulated or not for 48 hrs with anti-CD3/CD28 coated beads. Cell fluorescence was analyzed by FACS. Result obtained with one representative donor (upper panel) and mean results +/- SE with T cells from 3 independent donors (lower panel) are shown.(PDF) ppat.1007669.s004.pdf (107K) GUID:?8CBF10D6-F5F7-4BDE-8072-A5C292DA8835 S4 Fig: Both AS1842856 and TCR stimulation lead to SAMHD1 phosphorylation. PBT were cultured for 7 days with AS1842856 (500nM) or vehicle only. A parallel stimulation with anti-CD3/CD28 coated beads was also performed as indicated. Cells were then collected, lysed and immunoblotted using specific antibodies directed to the phosphorylated form of SAMHD1 and -actin as a control (upper panel). Blot quantification of SAMHD1 phosphorylation, +/- SE, with cells from two different donors are shown in the lower panel. Data were normalized for values obtained with -actin blots.(PDF) ppat.1007669.s005.pdf (103K) GUID:?8B2F900E-E37A-48FC-A444-E196589D398A S5 Fig: Infection of AS1842856-treated PBT correlates with SAMHD1 phosphorylation levels. PBT from heathy donors were cultured with AS1842856 (500nM) or vehicle only NH2-Ph-C4-acid-NH2-Me for 7 days and infected with the HIV-1 strain NL4.3. After 3 days of infection, SAMHD1 phosphorylation was measured by FACS in the GAG positive (infected) and GAG negative (non-infected)-gated cells populations. Results obtained with one representative donor are shown in the left panel and mean results, +/- SE, with cells from three different donors in the right panel.(PDF) ppat.1007669.s006.pdf (97K) GUID:?1A6B54ED-FD8F-4F9C-A0BB-172B0F693999 S6 Fig: IB protein levels are not affected by AS1842856. PBT were cultured for 7 days with AS1842856 (500nM) or vehicle only and then stimulated or not with PMA NH2-Ph-C4-acid-NH2-Me plus ionomycin as indicated. After 30 min of stimulation, cells were collected, lysed and immunoblotted using specific antibodies against IB and -actin as a control (upper panel). Results of blot quantification, +/- SE, with cells from two different donors are shown in the lower panel. Data were normalized Rabbit Polyclonal to PARP (Cleaved-Gly215) for values obtained with -actin blots.(PDF) ppat.1007669.s007.pdf (115K) GUID:?A27D4812-2FB9-4944-BD85-4C9BC7855D26 S7 Fig: AS1842856 potentiates calcium responses. PBT were cultured in the presence of AS1842856 (500nM) or vehicle only for 7 days. Levels of intracellular calcium were measured by spectrofluorometry using the calcium fluorescent indicator Fura-2 at the steady state (A) or after ionomycin (500nM) stimulation (B). Mean results +/- SE of calcium responses obtained from 6 and 3 independent NH2-Ph-C4-acid-NH2-Me donors are shown in A and B, respectively.(PDF) ppat.1007669.s008.pdf (119K) GUID:?7CC421A7-35C6-4AC0-BD48-4FC67CF8023E S8 Fig: AS1842856 inhibits FOXO1 transcriptional activity in the Jurkat T cell model. (A) The promoter activity of the Forkhead responsive element (FRE) was measured using a dual luciferase assay in Jurkat NH2-Ph-C4-acid-NH2-Me JTag cells transfected with vectors encoding either GFP or a constitutively active form of FOXO1 (FOXO1TM GFP) together with luciferase reporter plasmids (FRE-Firefly luciferase and NH2-Ph-C4-acid-NH2-Me CMV-Renilla luciferase), and then treated for 18 hrs with various concentrations of AS1842856 or vehicle only. Mean results +/- SE from 3 independent experiments are.