File S1 - Suppression of Foxo1 Activity and Down-Modulation of CD62L (L-Selectin) in HIV-1 Infected Resting CD4 T Cells

File S1 includes the following: Figure S1. CD62L expression is maintained in the presence of common gamma chain cytokines. Resting CD4+ T cells were cultured with no cytokine (Ø) or the indicated cytokine for 2 days then analyzed for CD45RA and CD62L expression by flow cytometry. Neg. control was with an isotype-matched non-specific antibody. IL-2 (50 u/ml), IL-4 (25 ng/ml), IL-7 (2 ng/ml), IL-15 (10 ng/ml). Data are representative of >3 independent experiments. Figure S2. CD62L is down-modulated in HIV-1 expressing cells in both central memory and effector memory CD4+ T cells, and CCR7 is modestly down-modulated. IL-7-treated resting CD4+ T cells were infected with a single round GFP virus and then analyzed as in Figure 1, with additional staining with CCR7 to distinguish central memory (TCM) and effector memory (TEM) cells. Data are representative of >3 independent experiments. A. CD62L is down-modulated on naïve (CD45RA+), central memory (TCM, CD45RA-CCR7+) and effector memory (TEM, CD45RA-CCR7−) resting CD4+ T cells. A subset of TEM naturally lacks CD62L expression. B. CCR7 is slightly down-modulated on naïve and memory CD4+ T cells expressing HIV-1. Data are representative of >5 experiments which consistently show 20%–30% loss of CCR7 mean fluorescence intensity (MFI) in the HIV-1 expressing (GFP+) cells. Grey: isotype-matched IgG control antibody staining. Other histograms are color coded to match the legend font color. C. HIV-1 expression is highest in effector memory T cells as measured by GFP MFI. Figure S3. Foxo1 inhibitor AS1842856 applied to productively infected GFP+ cells. IL-7 treated resting CD4+ T cells were infected with a single round env-pseudotyped HIV-1 GFP reporter virus and sorted for GFP+ cells. On day 17 after infection, AS1842856 was applied and GFP expression was analyzed 3 days later. Cell viability was 14–20%. Table S1. TaqMan primer and probe sets for quantification of cellular RNA in Figure 3. Table S2. Values for RNA expression graphed in Figure 3. HIV-1 FS is fully spliced Rev-independent early viral RNA. HIV-1 FL is full length Rev-dependent late viral RNA. (PDF)