3 P p25. p43 p41 28 FADD. cflips. PE-Cy5 [Fluorescence intensity]

Transcription

1 L S p4 3 D3 76 N S L Ve ct or p4 3 D3 76 N S L Ve ct or A aspase 8 FADD TRAF2 D95-R - + Vector D95L TL I S L D376N T RAIL-R1 T RAIL-R2 D95-R E-y5 [Fluorescence intensity] Supplemental Fig. 1 Different isoforms modulate the composition of the D95 DIS. Differential composition of the D95 DIS A) in keratinocytes expressing different isoforms or mutants of L-expression. DIS analysis (I) was performed from a total of 5x16 cells. recipitates of non-stimulated cells served as specificity controls for ligand affinity precipitates and were assayed for comparable immune precipitation of D95 as a control. ells were characterized for, caspase-8, FADD, RI-1, and TRAF2 recruitment by western blotting. Total cellular lysates (Lysates) were analyzed in parallel from all samples. ) -R1 - -R4 and D95 cell surface expression of isoforms/mutants overexpressing keratinocytes were determined by FAS analysis. ultured cells were stained with -R1 (HS11), -R2 (HS21), -R3 (HS31), -R4 (HS42), and D95 (AO-1 IgG1) primary Abs as well as isotype-matched control Abs. Filled curves indicate receptor specific staining as compared to isotype-matched control staining (open curves). Kavuri et al, Supplemental Fig. 1

2 p38 pi -α p38 p- I -α R2-Fc TNF-R2-Fc QVD pi -α Short Expo. aspase QVD p p-p p R2-Fc TNF-R- Fc R2-Fc TNF-R- Fc Supplemental Fig. 2 -induced non-apoptotic signalling pathways are independent of an autocrine loop of TNF activated by. A) keratinocytes were pre-incubated with either or -R2-Fc or TNF-R2-Fc, or the combination of caspase inhibitor and receptor fusion proteins as indicated for 1hr. ells were subsequently stimulated with (.1µg/ml) for the indicated time points. ellular lysates were analysed for pi α and I -α degradation. served as loading control. Asterisk indicates non-specific band. ) keratinocytes were pre-incubated with either (1 µm) or QVD (1 µm) for 1hr and stimulated with (.1µg/ml) for the indicated time points. ellular lysates were analysed for pi -α, p-p38, p-, respectively., p38, or served as loading controls. ) keratinocytes were pre-incubated with either -R2-Fc (2µg/ml) or TNF-R2-Fc (2µg/ml) for 1hr and stimulated with (.5µg/ml) for the indicated time points. ellular lysates were analysed for p- (left), or p-p38 (right) activation. p38, and served as loading controls. Kavuri et al, Supplemental Fig. 2

3 L S aspase 8 aspase-3 p33 AR1 -tubulin p2 p17 p95 p % rystal violet optical density [% Surviving attached cells] [ng/ml] 5 pi -α Long expo Supplemental Fig. 3 Knockdown of proteins leads to increased sensitivity to -mediated apoptosis and NF- activation in A5RT3 keratinocytes. A5RT3 were infected with -specific sirna using recombinant retroviruses as explained earlier in the methods section. ells were subsequently stimulated with 5ng of recombinant for indicated time points and analysed for A), caspase-8, and caspase-3 expression and cleavage. Arrows indicate molecular weights of protein cleavage fragments. Western blotting of the caspase substrate AR-1 and detection of the 85kDa fragment served as marker for caspase activity under those conditions. Analysis of protein expression served as loading control. ) Knockdown of sensitizes to -induced cell death. A5RT3 generated as described in A) were treated with the indicated concentrations of for 24 hours. Viability was subsequently examined by crystal violet assay. Shown are mean SD of two independent experiments. ) Down regulation of leads to enhanced pi -α activation, Transduced cells were incubated with for 5ng for indicated time points and subsequently analysed for the activation of pi -α. -tubulin served as a loading control. Kavuri et al, Supplemental Fig. 3

4 L S aspase-8 Long Expo % rystal violet optical density [ % Suviving attached cells] [ng/ml] -tubulin pi -α ositive ontrol Actin Supplemental Fig. 4 Knockdown of proteins leads to increased sensitivity to -mediated apoptosis and but NF- activation is unchanged in IGR melanoma cells. IGR were infected with -specific sirna using recombinant retroviruses as explained earlier in the methods section. ells were subsequently stimulated with (4 ng/ml) for indicated time points and analysed for A), caspase-8 expression and cleavage. Arrows indicate molecular weights of protein cleavage fragments. Analysis of protein expression served as loading control. ) Knockdown of sensitizes to -induced cell death. IGR were generated as described in (A) and were treated with the indicated concentrations of for 24 hours. Viability was subsequently examined by crystal violet assay. Shown are mean SD of two independent experiments ) Downregulation of does not allow for activation of NF- activation as determined by phosphorylation of I -α. Transduced cells were incubated with (4ng/ml) for the indicated time points. Kavuri et al, Supplemental Fig. 4

5 [ng/ml] aspase Vector S p Time [12mins] Supplemental Fig. 5 confers resistance to - induced apoptosis and completely blocks induced caspase-8 and activation in keratinocytes. A) ontrol cells or S -expressing cells were preincubated with caspase inhibitor () or left untreated. ells were then treated with (,5 µg/ml; left panel) Subsequently, 3µg of total cellular lysates were analyzed by western blotting for caspase-8, activation. served as a control for comparable loading of cellular protein. Kavuri et al, Supplemental Fig. 5