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Supplemental Figure 1Verifaction of Fas, TRAIL and TNFa Blocking Antibody Efficacy
(a, b) Jurkat cells were pulsed with [3H]-TdR and then treated with 10 mg/mL of Fas-agonistic APO-1 antibody (a) or 50ng/mL soluble His-tagged TRAIL (“Killer” TRAIL) (b), in the presence or absence of 10 mg/mL of Fas-blocking antibody (NOK-1) or 10 mg/mL TRAIL-blocking antibody (RIK-2), respectively, or 10 mg/mL isotype-matched control antibody. Apoptosis was assessed by the JAM test of DNA fragmentation: cells were harvested after 48 h onto filter mats and the amount of retained (intact) DNA was estimated by liquid scintillation spectrometry. Results are expressed as [3H]-TdR retention (in cpm), an inverse correlate of DNA fragmentation and hence apoptotic cell death. Bars represent means of 12-16 replicates and are representative of 2 independent experiments. Error bars are ±SD.
(c) EJ cells were cultured in the presence or absence of 800Units/mL TNFa, with or without TNFa-blocking antibody or isotype-martched control (10 mg/mL). After 48 h, cells were harvested and expression of ICAM-1 (CD54) was assessed by flow cytometry using anti-ICAM-1 primary and FITC-conjugated secondary antibodies. Results are plotted as median fluorescence intensity (MFI) in the FL-1 channel for triplicate samples and are representative of at least two experiments. Error bars are ± SD.
Supplemental Figure 2Functional blocking assays to assess the involvement of Fas/FasL, TRAIL-R/TRAIL and TNFR/TNFa in CD40-mediated death in RT4 cells
CD40-mediated apoptosis was assessed by the JAM test of DNA fragmentation. RT4 cells were pulsed with [3H]-TdR and cultured with 3T3 fibroblasts. Co-cultures were performed in the presence of 10 mg/mL NOK-1 (a), RIK-2 (b) and anti-TNFa (c) blocking antibodies, or control Ig. Cells were harvested at 48 h onto filter mats to estimate intact DNA by scintillation spectrometry. % DNA fragmentation was calculated as (S-E x 100)/S, where S = cpm recovered from control cultures (urothelial cells alone) and E = cpm recovered from test cultures, after background subtraction. Bars are means of 12-16 replicates and are representative of 2 independent experiments. Error bars are ± SD.
Supplemental Figure 3 Effects of pharmacological inhibitors of intracellular signaling pathways on CD40-mediated death in RT4 cells
Co-cultures of RT4 cells with 3T3neo and 3T3CD40L were performed in the presence of AP-1 inhibitor NDGA (a), JNK inhibitor SP600125 (b), and NF-kB inhibitor PDTC (c). Solvent-only controls were included in each case (Control). After 48 h of co-culture, apoptosis was assessed by the JAM test (see Supplemental Figure 2). Bars show mean % DNA fragmentation values of 12-16 replicate wells and are representative of 2 experiments. Error bars are ± SD.
Supplemental Figure 4Engineered CD40 expression renders HT1376 and VM-CUB-1 tumor cells susceptible to CD40-mediated apoptosis
Stable populations of CD40-transduced HT1376 (HT1376-CD40) and VM-CUB-1 (VM-CUB-1-CD40) as well as control cells (HT1376-neo and VM-CUB-1-neo, respectively) were established as described for RT112 cells (see Figure 6a and Materials and Methods for details) and CD40 expression was assessed by flow cytometry (not shown). HT1376-CD40 and VM-CUB-1-CD40 and their control counterparts were pulsed with [3H]-TdR and cultured alone or with 3T3neo and 3T3CD40L fibroblasts. Apoptosis was assessed by the JAM test at 48 h (as described in Supplemental Figure 2). Bars represent means of 16-20 replicate wells and are representative of 3 independent experiments. Error bars are ± SD.
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