Fig 2: The ligand of DR3, TL1A, is required for DR3-mediated apoptosis. a The cell line stably expressing Flag-tagged DR3 was established in HT29 cells. Dose response curves of HT29 cells and HT29-DR3 cells to diazonamide were plotted. Cell viability was measured after 48 h of treatment. b–d HT29 or HT29-DR3 tumor-bearing mice were treated with vehicle (b), 7.5 mg/kg taxol (c), or 20 mg/kg taxol (d) for the indicated time. Tumor growth was monitored. Error bars, SEM. e Identification of TL1A as the only TNF family death ligand bound to DR3. A SILAC experiment was carried out to quantify the DR3 binding proteins as described in the Materials and Methods. HT29-DR3 cells growing in the light medium were treated with 100 nM diazonamide for 12 h. Untreated cells were grown in the heavy medium. Equally combined cell lysates were subjected to immunoprecipitation with an anti-Flag antibody. Proteins in the whole elution with 3× Flag peptide were quantified and the Light/Heavy ratios were calculated. SD (geo) stands for geometric s.d. The closer the SD (geo) is to 1, the better the protein quantification is. The median from peptide ratios was used as the protein ratio. (f) HT29-DR3 cells were treated with 100 nM diazonamide for 12 h. The DR3/TL1A complex was analyzed by Flag immunoprecipitation followed by western blotting with antibodies against Flag, TL1A, and TRADD. g HT29-DR3 cells were treated as in f. The DISC complex was analyzed by caspase-8 immunoprecipitation followed by western blotting with antibodies against caspase-8 and FADD. h The dose response curves of wild-type MEFs, FADD-/- MEFs, and TRADD-/- MEFs to diazonamide. i Knockdown of TL1A blocked anti-mitotics-induced apoptosis. HeLa cells were transfected with four TL1A siRNAs for three rounds before being treated with 100 nM diazonamide for 48 h. Cell viability was determined (lower panel). Protein levels of TL1A in the knockdown cells were determined by western blotting with an anti-TL1A antibody (upper panel). j, k Exogenous expression of TL1A sensitizes PANC-1 cells to taxol. j The relative TL1A mRNA levels in HeLa and PANC-1 cells were determined by qRT-PCR (lower panel). TL1A protein levels in HeLa or PANC-1 cells were determined by western blotting with anti-TL1A and anti-Actin antibodies (upper panel). k Flag-tagged TL1A was stably expressed in PANC-1 cells. Dose response curves to taxol were plotted for parental PANC-1 cells and PANC-1-TL1A cells. Cell viability was measured after 48 h treatment (left panel). Cell lysates from PANC-1 and PANC-1-TL1A cells were subjected to western blotting with anti-Flag, anti-TL1A and anti-Actin antibodies (right panel)

Fig 3: PARP-1 is activated with distinct kinetics by TNF and MNNG. In (a–f), L929Ts cells were treated with 100 ng/ml TNF for the indicated times. a Cells were analyzed for PARP-1 by immunoblotting with antibody 9542. The same lysates were additionally analyzed on separate gels for TRADD, FADD, caspase-8, and RIP1. Arrows indicate the positions of the full-length proteins, which were additionally verified for TRADD, FADD, caspase-8, and RIP1 by parallel separation of lysates from 293T cells overexpressing the respective protein (293T+). Untransfected 293T cells (293T-) were included for control. Shifted signals for TRADD and RIP1 in these lanes represent the cross-reactive endogenous human proteins that differ in size from the endogenous murine proteins of L929Ts cells. b Western blot with antibody 9542 that detects all cleavage fragments of PARP-1. Arrows indicate the position of the full-length protein and the 89-kDa caspase-3 cleavage product present only in apoptotic control lysates (Co, extracts from cells treated with 100 ng/ml TNF and 2 µg/ml of the protein biosynthesis inhibitor CHX for 1 h). A longer exposure is shown for the lower part of the blot to increase the visibility of the reactive bands. c Cells were additionally treated with the proteasome inhibitor MG-132 (52.6 µM) as indicated. PARP-1 was detected with antibody 556362. d The expression levels of PARP-1 mRNA were determined by qRT-PCR in parallel to protein levels (shown below, PARP-1 was detected with antibody 9542). The expression of PARP-1 is shown relative to untreated cells. Comparable results were obtained with a different primer pair. e Following detection of PARP-1 (antibody 9542), the membrane was reprobed with an antibody for PAR to reveal the presence of poly(ADP-ribosyl)ated proteins. f Lysates from cells treated with TNF for the indicated times were additionally incubated for the indicated times in the presence of 10 mM ß-mercaptoethanol and 0.05 U PARG before PARP-1 was detected with antibody 9542 and reprobing of the membrane with an antibody for PAR. g L929Ts, L929ATCC, and L929sA cells were left untreated, treated with TNF as above, or treated with 0.5 mM MNNG for 15 min and further incubated with fresh medium without MNNG for the indicated times before PARP-1 was detected with antibody 9542. h Lysates from L929Ts cells treated with MNNG as in (g) for the indicated times were incubated with PARG and analyzed as in (f). For all Western blot panels, detection of actin served as a loading control. For all figures, representative data from one out of at least two or more experiments with similar results are shown (n = 2) and error bars indicate the standard deviations (SD) from at least triplicate determinations (n = 3)