Supplementary Figure S1: Transient knockdown of components along the RIPK1–RIPK3–MLKL–PGAM5–Drp1 axis in L929sahFas cells. L929sahFas cells were transfected with the indicated siRNAs, and 72 h later they were stimulated in triplicate with TNF (10 000 U/ml). Cell death was determined by fluorometry as described in Materials and Methods. Data are expressed as percentage of control ± StDev of one experiment, and is representative of three independent experiments (n=3).

Supplementary Figure S2: Stable knockdown of Pink1 does not affect TNF-induced necroptosis in L929 cells. After transduction of L929 cells with a lentiviral vector expressing either a control short hairpin (scramble knockdown) or a short hairpin targeting the 3’ untranslated region (3’UTR) of murine Pink1 (seq1 or seq2, specified in Material and Methods), cells were positively selected with puromycin. (A) Validation of endogenous mRNA levels of mPink1 by RT-PCR, with actin as a control. RNA was isolated from L929 cells expressing scramble hairpin or a hairpin against 3’UTR of Pink1 (seq 1 or seq 2). (B) They were next stimulated in triplicate with TNF (10 000 U/ml), and cell death was monitored by fluorometry after 2, 4 and 6 h. Data are expressed as percentage of cell death ± SEM (n=3).

Supplementary Figure S3: Inducible overexpression of siRNA-resistant Pink1 cannot rescue the protective effect of siRNAs targeting murine Pink1 from TNF-driven necroptosis. (A) During transient knockdown using control siRNAs (si scr) or siRNAs targeting murine Pink1, L929 cells were stimulated with doxycycline (10 µg/ml), promoting Dox-inducible expression of luciferase or Dox-inducible expression of siPink1-resistant Pink1 (Pink1#). Next, cells were stimulated in triplicate with TNF (10 000 U/ml) or agonistic anti-Fas Ab (250 ng/ml), and cell death and caspase activity were determined as described in Material and Methods. Data are expressed as percentage of cell death ± SEM (n=3). *, p <0.05. (B) Following knockdown with control siRNAs (si scr) or siRNA targeting Pink1 and simultaneous stimulation with Doxycylin, RNA was isolated and mRNA levels of Pink1, RIPK3 and actin were determined by RT-PCR.

Supplementary Figure S4: TNF-induced cell death is normal in Pink1 KO MEFs. Primary, non-immortalized Pink1 KO or littermate control MEFs were pretreated for 1 h with the pan-caspase inhibitor zVADfmk (Z, 10 µM), the TAK1-inhibitor NP-009245 (Ti, 1 µM) and/or necrostatin-1 (N, 10 µM). Subsequently, MEFs were stimulated in triplicate with TNF (10 000 U/ml). Data of the 9-h time point are expressed as percentage of cell death and caspase activity (AU, Arbitrary Units) ± SEM (n=3).

Supplementary Figure S5: Mitochondrial fission is observed during TNF-necroptosis in L929 cells. Cells expressing mitochondrially targeted GFP (mito-GFP) were pre-treated with 50 nM TMRM and 50 µM SytoxRed and then stimulated with TNF (10 000 U/ml). Cells were monitored every 5 min by live-cell imaging, as described in Materials and Methods, for four parameters: morphology using differential interface contrast (DIC), mitochondrial membrane potential using TMRM, mitochondrial network using mito-GFP, and plasma membrane permeability using SytoxRed. One to three hours after TNF stimulation (10 000 U/ml), but before plasma membrane rupture, the tubular mitochondrial network disintegrated in a diffuse or punctate pattern, in terms of both mito-GFP and TMRM fluorescence. Analysis was performed in a humidified atmosphere containing 5% CO2 at 37°C. Scale bars indicate 20 µm.

Supplementary Data D1: Design of the sequence modifications (in red) of murine Pink1 using smartpool siRNAs (Dharmacon Fisher) targeting murine Pink1 to generate a Pink1 form that is resistant to knockdown. Since three out of four siRNAs in the smartpool mix target the ORF of Pink1, only those three sequences had to be adjusted (in red). The protein sequence of the siRNA-resitant Pink1 is identical to the murine Pink1 sequence isolated from murine L929sahFas cells (data not shown), which corresponds entirely to the murine Pink1 CDS found on the NCBI website.

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