|
|
Blood, Vol. 107, Issue 2, 651-654, January 15, 2006
Alkylamines cause V 9V 2 T-cell activation and proliferation by inhibiting the mevalonate pathway
Blood Thompson et al.
107: 651
Supplemental materials for: Thompson et al
Files in this Data Supplement:
- Figure S1. Effect of alkylamines on γ,δ–T-cell activation and proliferation (JPG, 40.5 KB)
-
(A) PBMCs were cultured for 7 days with 0.5mM BA, IBA, IPA or SBA, or 1µM ZOL, in the presence of rhIL-2, prior to dual-staining with anti-CD3-FITC and anti-pan- , -TCR-PC5 antibodies, prior to FACS analysis of the T cell gated population. Data shown are of one experiment and representative of two further experiments from independent donors. (B) PBMCs were treated with 0.5mM BA, IPA, IBA or SBA, or 1µM ZOL, in the presence of rhIL-2, for 48h. Conditioned media was harvested and IFN levels measured by ELISA. Data shown are the mean +/- S.D. from one experiment and are representative of three further experiments from independent donors.
- Figure S2. GGOH, but not FOH, rescues the inhibition of protein prenylation by alkylamines in J774 cells (JPG, 70.2 KB)
-
J774 cells were treated with (A) 1, 5 or 10mM BA, IBA, SBA or IPA for 24hr, (B) 10mM IBA, IPA or SBA ± 10µM FOH or GGOH for 24h, or (C) 100µM ALN or ZOL ± 10µM FOH or GGOH for 4h. Lysates were prepared and equal amounts of protein (30µg) from total cell lysates were analyzed by immunoblotting for unprenylated Rap1A and  -actin. Data shown are representative of three independent experiments. (D) J774 cells were metabolically-labelled with [14C]-mevalonolactone plus 5µM MEV for 4h to deplete cells of endogenous mevalonate. Cells were then further cultured for 20h with 1 or 10mM BA, IBA, SBA or IPA plus [14C]-mevalonolactone and 5µM MEV. Equal amounts of protein (50µg) were electrophoresed by SDS-PAGE and radiolabelled, prenylated proteins were detected by phosphor-imaging. Data shown are representative of two independent experiments.
- Figure S3. The inhibitory effect of mevastatin on alkylamine-induced Vγ9Vδ2 T cell activation and proliferation can be overcome by replenishing cells with mevalonate (JPG, 38 KB)
-
(A) PBMCs were cultured for 7 days with 0.5mM SBA and/or 1µM IPP ± 1µM MEV and/or 100µM mevalonate (MVA), in the presence of rhIL-2, prior to dual-staining with anti-V2-FITC and anti-CD3-PerCP antibodies and FACS analysis of the T cell gated population. Data shown are from one experiment and representative of one further experiment with independent donors. (B) PBMCs were treated with 0.5mM SBA and/or 1µM IPP ± 1µM MEV and/or 100µM mevalonate (MVA), in the presence of rhIL-2, for 48h. Conditioned media was harvested and IFN levels measured by ELISA. Data shown are the mean +/- S.D. from one experiment and are representative of one further experiment with independent donors.
- Figure S4. The inhibitory effect of statins on alkylamine-induced Vγ9Vδ2 T cell activation and proliferation is due to inhibition of HMG-CoA reductase (JPG, 33.2 KB)
-
(A) PBMCs were cultured for 7 days with 0.5mM SBA ± 1µM lovastatin (LOV) or 1µM desoxolovastatin (desoxoLOV), in the presence of rhIL-2, prior to dual-staining with anti-V2-FITC and anti-CD3-PerCP antibodies and FACS analysis of the T cell gated population. Data shown are from one experiment and representative of one further experiment with independent donors. (B) PBMCs were treated with 0.5mM SBA ± 1µM lovastatin (LOV) or 1µM desoxolovastatin (desoxoLOV), in the presence of rhIL-2, for 48h. Conditioned media was harvested and IFN levels measured by ELISA. Data shown are the mean +/- S.D. from one experiment and are representative of one further experiment with independent donors.
| |
