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Blood, Vol. 112, Issue 3, 690-698, August 1, 2008
Up-regulation of c-FLIPshort by NFAT contributes to apoptosis resistance of short-term activated T cells
Blood Ueffing et al.
112: 690
Supplemental materials for: Ueffing et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 832 KB)
- Figure S1. Effect of different signaling pathway inhibitors on c-FLIP expression (JPG, 52.8 KB)
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(A) Human peripheral T cells were treated for 16 h with 5 µg/ml PHA-L in the presence or absence of 20 µM PD098059 (MEK1 inhibitor), 1 µM SB 203580 (p38 inhibitor), or 1 µM cyclosporine A (CsA) (d1 T cells). Subsequently, cells were analyzed by Western blotting. Freshly prepared T cells (d0 cells) served as a control. (B) Inhibition of mitogen-activated protein kinases (MAPK), phosphatidylinositol 3-kinase (PI3K), mammalian target of rapamycin (mTOR) or glycogen synthase kinase 3 beta (GSK3β) does not interfere with induction of c-FLIPshort in primary T cells. Human peripheral T cells were immediately lysed (d0) after isolation or stimulated for 16 h (d1) with either 5 µg/ml PHA-L or PHA-L plus 1 µM cyclosporine A (CsA), 25 µM roscovitine (Ros, Cdk2 inhibitor), 1 or 100 nM rapamycin (mTOR inhibitor), 3 µM SB415286 (GSK3β inhibitor), 50 nM PD098059 (MEK1 inhibitor), 50 nM SB203580 (p38 inhibitor), 1 µM JNK-II, or 10 nM wortmannin (WM, PI3K inhibitor). c-FLIP expression was analyzed by Western blotting. Tubulin or β¬actin served as a loading control. Note that roscovitine inhibited not only c-FLIPshort expression but also T cell activation, as revealed by a strong downregulation of the activation markers CD25 and CD69 (data not shown).
- Figure S2 (JPG, 50.7 KB)
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(A) Regulation of c-FLIP expression in CEM cells. Cells were stimulated for the indicated time with 20 ng/ml PMA and 1 µM ionomycin in the absence or presence of 1 µM CsA or 10 µg/ml cycloheximide (CHX) and c-FLIP expression was analyzed by Western blot. Tubulin served as a loading control. (B) Controls for the ChIP experiments in CEM cells. The panel shows a ChIP with rabbit control IgG and anti-acetylated histone-3 (Ac-H3) that served as negative and positive control for the ChIP procedure shown in Figure 5C. Here, the GAPDH promoter was analyzed as a constitutively transcribed gene. Genomic DNA (input) and water were used as positive and negative controls for PCR analysis.
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