Supplementary MaterialsS1 Table: Secreted IL2 levels from Jurkat-T cells co-incubated with control or Pom-treated BCBL-1 or JSC-1 cells. cultured in the absence or presence of indicated concentrations of Pom. After 48 hours (BCBL-1 and JSC-1) or 72 hours (BC-2), live/lifeless analysis was performed using trypan blue staining. Number of live cells and % viability (% of total cells that are alive) for BCBL-1 (A), JSC-1 (B), and BC-2 (C) were calculated and offered as % of control-treated cells.(TIF) ppat.1009091.s003.tif (169K) GUID:?C074EE81-A63E-4DA7-B38F-CE6F19BB5190 S3 Fig: Validation Nerolidol of ICAM-1 and B7-2 blocking antibodies. (A and B) BCBL-1 cells produced for 48 hours in the absence or presence of Pom were treated with blocking antibodies (isotype control, anti-ICAM-1 Ab, anti-B7-2 Ab, or both anti-ICAM-1 and anti-B7-2) at a 10ug/mL final concentration each for 30 minutes. The cells were then stained with PE-conjugated anti-ICAM-1 or anti-B7-2 antibodies and circulation cytometry was performed to measure the surface expression of ICAM-1 (A) and B7-2 (B). (C) Burkitts lymphoma cell collection Daudi was pretreated with isotype control or blocking antibodies like in (A) and (B) and then co-incubated for 6 MSH4 hours with IL2-Jurkat T cells in the presence of 2.5g/mL anti CD3 antibody. T-cell activation induced by Daudi cells is usually presented as relative light models (RLU).(TIF) ppat.1009091.s004.tif (652K) GUID:?C65D7BB6-41E0-4173-9725-58872F52CC64 S4 Fig: Pom does not induce CD80/B7-1 or PD-L1 surface expression in PEL cell lines or CD3 and CD28 in Jurkat T-cells. (A) Surface expression levels of CD80/B7-1 in BCBL-1 or JSC-1 cells treated with indicated concentrations of Pom for 48 hours as measured by circulation cytometry using PerCP/Cy5.5-conjugated anti-B7-1 antibody. (B) Surface expression of CD3 and CD28 on Jurkat T cells after 48 hours treatment with Pom measured by circulation cytometry using FITC-conjugated anti-CD3 or anti-CD28 antibodies.(TIF) ppat.1009091.s005.tif (641K) GUID:?A2E2EF5E-CE83-41BF-8BF9-491905930C74 S5 Fig: Pom-treatment of both PEL and T cells produces higher T-cell activation than treatment of either alone. (A) IL2-Jurkat cells were grown in the absence or presence of 1M Pom. After 48 hours, Pom was washed out and they were incubated for 6 hours with or without control PEL cells in the absence or presence of various concentrations of anti-CD3 antibody. Activations of ctrl or Pom-treated IL2-Jurkat cells in the absence or presence of ctrl BCBL-1 (left) or JSC-1 (right) cells are expressed as average RLU from 3 individual experiments. (B) Both PEL cells and IL2-Jurkat cells were cultured in the absence or presence of Pom. After 48 hours, cells were washed with PBS to remove Pom and Jurkat T-cell activation assay was performed with numerous concentrations of anti-CD3 Ab. Activations of ctrl or 1M Pom-treated Jurkat cells by ctrl or Pom-treated BCBL-1 (left) or JSC-1 (right) cells are expressed as average RLU from 3 individual experiments.(TIF) ppat.1009091.s006.tif (305K) GUID:?7240D480-575E-46B1-9C6F-D91736C6186B S6 Fig: Inhibition of IKZF1, IRF4, and cMyc is not sufficient for Pom-induced increases in surface markers. (A) Relative number of live BCBL-1 cells after 48 hours treatment with indicated concentrations of JQ-1 as measured by trypan blue exclusion method. (B) Protein levels of Ikaros, IRF4, cMyc, and control -actin in the nuclear lysates of BCBL-1 cells treated with numerous concentrations of JQ-1 for 48 hours. (C) Surface expression levels of ICAM-1 and B7-2 in BCBL-1 cells 48 hours after treatment with JQ-1. Cells were stained with FITC-conjugated IgG isotype control, anti-ICAM-1, or anti-B7-2 antibodies and analyzed using circulation cytometry.(TIF) ppat.1009091.s007.tif (565K) GUID:?5EE40588-FA7C-40B5-A7CF-28CFB528CFFA S7 Fig: Determining the role of IL-10 signaling in pom-induced increase in ICAM-1 and B7-2. (A) Relative IL-10 levels in the supernatant of Nerolidol BCBL-1 and JSC-1 cells as measured by ELISA Nerolidol 24 hours post treatment with Pom from 3 individual experiments. Average IL-10 produced was ~ 4 ng/mL and ~25 ng/mL in control BCBL-1 and JSC-1 cells respectively. (B) Relative mRNA levels of MARCH 8 and 1 in BCBL-1 cells and MARCH 8 in JSC-1 cells after 48 hours with or without Pom. MARCH 1 was measured but was undetectable in JSC-1 cells. mRNA levels are normalized to that of 18S RNA and expressed as fold switch over 0M-pom treated Nerolidol cells. Error bars represent standard deviation from 3 impartial experiments. (C) Surface expression level of CD83 48 hours post-treatment with DMSO ctrl or Pom. (D and E) BCBL-1 cells were pretreated with anti IL-10 Ab (10g/mL) or recombinant human IL-10 (rIL-10).