Fig 2: TNF-induced necroptosis is not blocked by inhibition of the PARP pathway. a L929Ts, L929ATCC, and L929sA cells were left untreated, treated with 100 ng/ml TNF for the indicated times, or treated with 1 mM MNNG for 15 min and further incubated for 16 h with fresh medium without MNNG in the presence or absence of the PARP inhibitor 3-AB (2.5 mM). Subsequently, activation/disappearance of PARP-1 was detected with antibody 9542. Detection of actin served as a loading control. b Cells were stimulated with TNF or with MNNG (0.5 mM) as in (a) before intracellular ATP levels were measured and loss of membrane integrity as a marker for cell death was determined by flow cytometry and PI staining. For clarity of presentation, the percentage of intact, large PI-negative cells with preserved membrane integrity is shown. c Cells were stimulated as in (a) with incubation for 14 h after MNNG treatment before purified cytosolic fractions were analyzed for translocation of AIF. The purity of the fractions was verified by detection of actin (cytosolic marker), COXIV (mitochondrial marker) and histone (nuclear marker). Equal loading was verified by staining with Ponceau S (Supplementary Figure S2b), since actin displayed a decrease in these highly purified fractions during cell death. At 48 h of TNF treatment, L929Ts cells additionally displayed a decrease of AIF, due to this very late stage of cell death. Nevertheless, 3-AB prevented both AIF translocation and actin decrease selectively in MNNG-, but not TNF-treated cells. d Left panels Necroptosis was induced in L929Ts, NIH3T3, Jurkat, and HT-29 cells in the absence or presence of the PARP1 inhibitors 3-AB (2.5 mM), olaparib (L929: 1 µM; NIH3T3, Jurkat: 50 nM; HT-29: 2 µM) or PJ-34 (20 µM) by treatment with 100 ng/ml TNF for 24 h (L929Ts, HT-29), 16 h (NIH3T3) or 20 h (Jurkat) with addition of 20 µM (NIH3T3, HT-29) or 50 µM (Jurkat) zVAD-fmk and 2 µg/ml (Jurkat) or 5 µg/ml (HT-29) CHX. In parallel, cells were treated with MNNG for 16 h as in (a) with or without addition of 3-AB, olaparib or PJ-34. Right panel to test whether or not zVAD-fmk inhibits MNNG-induced cell death, cells were treated with MNNG as in (a) in the absence or presence of 20 µM (Jurkat: 50 µM) zVAD-fmk. Subsequently, loss of membrane integrity was analyzed by flow cytometry. Lower panel To verify the efficacy of the inhibitors (in addition to their effect on MNNG-induced necrosis shown in the upper panels), activation/disappearance of PARP-1 in L929Ts cells treated with 100 ng/ml TNF for 24 h in the presence or absence of 2.5 mM 3-AB, 1 µM olaparib or 20 µM PJ-34 cells was detected with antibody 9542. Detection of actin served as a loading control. e L929Ts or Jurkat cells were nucleofected with an siRNA that does not elicit an RNA interference response (negative control) or with siRNA specific for murine or human PARP-1 (PARP-1 siRNA). Together with wild-type (WT) and PARP-1-deficient MEF, the cells were stimulated either with 0.5 mM MNNG for 15 min and further incubated for the indicated times with fresh medium without MNNG or with 100 ng/ml TNF in the absence (L929Ts) or presence (Jurkat, MEF) of 20 µM zVAD-fmk and 2 µg/ml (Jurkat) or 1 µg/ml (MEF) CHX for 24 h before loss of membrane integrity was measured by PI staining and flow cytometry. Control Western blots for PARP-1 were performed to verify successful downregulation of endogenous murine or human PARP-1 or to show presence or absence of murine PARP-1. Detection of actin served as a loading control. f Inhibition of necroptosis by the JNK-inhibitory peptide JNK-inhibitor III was assayed in L929Ts cells treated with 100 ng/ml TNF for 16 h or not and increasing concentrations of JNK-inhibitor III. g Left panel Primary lung fibroblasts (LF) from wild-type (WT), JNK1-(JNK1-/-) and JNK2-deficient (JNK2-/-) mice or immortalized MEF from JNK1/JNK2 double-deficient (JNK1/2-/-) mice were left untreated or incubated with 100 ng/ml TNF and 20 µM zVAD-fmk in the presence or absence of 2 µg/ml CHX to elicit necroptosis. After 18 h, loss of membrane integrity was analyzed by flow cytometry. Right panel Control Western blots were performed to verify presence or absence of endogenous JNK1 and/or JNK2 and equal loading (actin). h L929Ts cells were stimulated with 100 ng/ml TNF for the indicated times with or without the necroptosis inhibitor necrostatin-1 (Nec-1, 10 µM). Activation of JNK was analyzed by detection of phosphorylated p46 JNK1 and p54 JNK2 (arrows) in Western blots (left panel) using antibody AF1205. Detection of actin served as a loading control. To demonstrate the efficacy of the inhibitor, loss of membrane integrity of identically treated L929Ts cells was determined after 24 h by flow cytometry (right panel). i L929Ts cells were incubated with 100 ng/ml TNF for 5 h or with MNNG (0.5 mM) as in (a) with optional addition of 20 µM zVAD-fmk and/or a combination of 20 µM each of zFA-fmk, E-64, and CA-074 Me before loss of membrane integrity was measured by flow cytometry. j L929Ts, L929ATCC and L929sA cells were preincubated for 24 h or not with the cathepsin D inhibitor pepstatin A (PepA, 100 µM) followed by either addition of 100 ng/ml TNF or not for 24 h, or by treatment with MNNG (0.5 mM) as in (a) or not. Subsequently, loss of membrane integrity was measured by flow cytometry. k Wild-type (WT), cathepsin D-deficient (CD-/-), and CD-/- MEF stably transfected with the pro-cathepsin D cDNA (CD-/-CatD) were incubated for 14 h with 100 ng/ml TNF in combination with 20 µM zVAD-fmk and 5 µg/ml CHX or with 1 mM MNNG for 30 min and further incubated for 16 h with fresh medium without MNNG before loss of membrane integrity was determined