Nature Protocols: doi: /nprot µM Sytox Green. IMR32 - Apoptosis *** Effect PI on DEVD-amc intensity. IMR32 - Apoptosis ***

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1 PI Intensity % Permeabilized cells (PI positivity) Intensity (caspase activation, AV) PI intensity PI intensity cleavage (caspase activation, AV) Amc intensity Sytox Green Intensity Sytox Green Intensity % Permeabilized cells (Sytox Green) a b 3 Sensor saturation 2 1 1µM Sytox Green R² = Number of permeabilized cells/well Gain 14 Gain 125 Gain 11 Gain 1 Gain 9 Gain IMR32 - Apoptosis STS c IMR32 - Apoptosis d Effect PI on intensity STS Anti-Fas µM propidium Iodide e f 25 2 Effect on PI intensity Anti-Fas 25 2 Propidium iodide 1µM Fluorobrite, no phenol red, regular g 1µM Propidium Iodide h Number of permeabilized cells/well L929 - Apoptosis L929 - Apoptosis Fas Fas Nature Protocols: doi:1.138/nprot

2 Supplementary Figure 1 Validation of the FAN assay a, The linear range for SYTOX Green decreases at lower concentrations (< 5μM) even if signal intensity is below the saturation level of the detector. b- c, Cell membrane permeabilization kinetics (b) and caspase activation (c) in IMR-32 cells treated with 2 μm Staurosporin (STS). d- e The effect of on propidium iodide (PI) intensity and vice versa. Fas-overexpressing L929 cells were treated with 25 ng/ml anti-fas antibody in the presence or absence of 2 μm or 1μM PI, and amc intensity (d) and PI intensity (e) was measured 3 h later. f, The effect of phenol red on PI intensity is minimal for permeabilized L929 cells. g- h, Cell membrane permeabilization kinetics measured with 1μM PI (g) and caspase activation (h) in L929-Fas cells treated with anti-fas Ab (25 ng/ml). The same plate was measured at 6 and 3 hours after stimulation, and returned to the tissue culture incubator in the interval time. Error bars = standard error of the mean, obtained from three independent experiments (n=3), each measured in triplicate., p.1. Non-significant differences are not indicated. AV, arbitrary values. Nature Protocols: doi:1.138/nprot

3 Serial dilution of cells (1:2) NC A B Serial dilution of C SYTOX Green D (1:3) E F NC G H Figure legend empty well µm SYTOX Green 1 µm SYTOX Green 1,5 µm SYTOX Green 2,2 µm SYTOX Green 3,3 µm SYTOX Green 5 µm SYTOX Green ###### number of cells per well Supplementary Figure 2 Example of a plate lay-out to determine to optimal combination of gain setting, SYTOX Green concentration and cell number. Nature Protocols: doi:1.138/nprot

4 Supplementary Table 1: Inhibitors used to dissect non-apoptotic signaling pathways Inhibitor target Cell death type Sub-stock 96 well (µm) Final (µm) zvad-fmk pan caspase apoptosis Nec-1s RIPK1 necroptosis GSK84 RIPK3 necroptosis 18 1 PJ-34 PARP parthanathos 18 1 Ferrostatin Unknown, ferroptosis inhibits lipid peroxidation Reference Supplementary References 1 Thornberry, N. A. et al. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 356, (1992). 2 Degterev, A. et al. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol 4, (28). 3 Dondelinger, Y. et al. MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Rep 7, (214). 4 Mazzone, G. L. & Nistri, A. Effect of the PARP-1 inhibitor PJ 34 on excitotoxic damage evoked by kainate on rat spinal cord organotypic slices. Cell Mol Neurobiol 31, (211). 5 Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, (212). Nature Protocols: doi:1.138/nprot

5 Supplementary Note 1 "Genscript file used to calculate means, standard error of means and statistical significance for the data presented in graph 2 b and c" "set TIME variate and calculate LOG-transformed values of background corrected means" VARIATE [VAL = 1...1] TIME CALC LOGT,LOGT1, LOGT2, LOGT3, LOGT4, LOGT5,LOGT6,LOGT7,LOGT8,LOGT9,LOGT1 = LOG1(ABS(NEWT[])) "Form Data structures for Repeated Measurements - Multiple Variates Model" DELETE [REDEFINE=yes] _subject, _Data, %_Time, %_NEWEXP, %_NEWINHIB, %_NEWSTIM FACTOR [LEV=!(#TIME)] %_Time;!(18(#TIME)) FACTOR [LEVELS=18] %_subject;!((1...18)1) APPEND [NEW=_Data] LOGT1,LOGT2,LOGT3,LOGT4,LOGT5,LOGT6,LOGT7,LOGT8,LOGT9,LOGT1 APPEND [NEW=%_NEWEXP] 1(NEWEXP) APPEND [NEW=%_NEWINHIB] 1(NEWINHIB) APPEND [NEW=%_NEWSTIM] 1(NEWSTIM) "Fit Repeated Measurements - Multiple Variates Model" VCOMP [FIXED=(%_NEWEXP+%_NEWINHIB*%_NEWSTIM)*%_Time; FACTORIAL=9] %_subject/%_time;\ CONSTRAIN=positive VSTRUCTURE [%_subject.%_time] FACTOR=%_Time; MODEL=ar; ORDER=1; HETEROGENEITY=none REML [PRINT=model,components,deviance,waldTests; MAXCYCLE=2; FMETHOD=automatic;\ MVINCLUDE=explanatory,yvariate] _Data VAIC [PRINT=aic,sic; INCLUDE=*] VPLOT [RMETHOD=all] fittedvalues,normal,halfnormal,histogram VGRAPH [METHOD=lines; PSE=allmeans] %_Time; GROUPS=%_NEWINHIB; TRELLISGROUPS=%_NEWSTIM DELETE [REDEFINE=yes] pred_means1, pred_means2 VKEEP TERMS=%_NEWSTIM.%_NEWINHIB; MEANS=pred_means1 VKEEP TERMS=%_NEWSTIM.%_NEWINHIB*%_Time; MEANS=pred_means2 PRINT pred_means1 V2TABLE pred_means1;var_means1 PRINT var_means1 PRINT pred_means2 Nature Protocols: doi:1.138/nprot

6 "Define contrasts of calculated proportions of Triton" VARIATE [VALUES = -1,,1,,,,1,,-1] Clev1 "contrast between proportion zvad and proportion for FAS" VARIATE [VALUES =,,,-1,1,,1,-1,] Clev2 "contrast between proportion Nec-1 and proportion for TNF" TEXT Title[1...2]; VALUES=\ 'COMP[1] = contrast between proportion zvad and proportion for FAS',\ 'COMP[2] = contrast between proportion Nec-1 and proportion for mtnf' "test comparisons irrespective time" TABLE [CLASSIFICATION = %_NEWSTIM,%_NEWINHIB] Comp[1...2]; VALUES = Clev1, Clev2 PRINT Comp[1...2] VTCOMPARISON [PRESENTCOMBINATIONS = %_NEWSTIM,%_NEWINHIB] Comp[] " test comparisons across time" FOR [NTIMES=2; INDEX=i] CAPTION!t('Testing the interaction between',#title[i],'and time'); STYLE=meta VTCOMPARISON [GROUPS = %_Time; PRINT = wald]comp[i] PRINT Comp[i] ENDFOR "Define proportions of Triton" VARIATE [VALUES =,,1,,,,,,-1] Clev3 "proportion zvad for FAS" VARIATE [VALUES = 1,,,,,,-1,,] Clev4 "proportion for FAS" VARIATE [VALUES =,,,,1,,,-1,] Clev5 "propr Nec-1 for TNF" VARIATE [VALUES =,,,1,,,-1,,] Clev6 "proportion for TNF" TEXT Title[1...4]; VALUES= 'proportion zvad for FAS','proportion for FAS','proportion Nec-1 for TNF','proportion for TNF' "test proportions irrespective time" TABLE [CLASSIFICATION = %_NEWSTIM,%_NEWINHIB] Compi[1...4]; VALUES = Clev3, Clev4, Clev5, Clev6 PRINT Compi[1...4] VTCOMPARISON [PRESENTCOMBINATIONS = %_NEWSTIM,%_NEWINHIB] Compi[] " define variates to save proportion values and se's at each time point" VARIATE [NVAL = 1] estim[1...4],se[1...4] Nature Protocols: doi:1.138/nprot

7 " test proportions across time" FOR [NTIMES=4; INDEX=i] CAPTION!t('Testing the interaction between',#title[i],'and time'); STYLE=meta VTCOMPARISON [GROUPS = %_Time; PRINT = wald]compi[i];estimate = estim[i]; SE = se[i] PRINT Compi[i] PRINT estim[i],se[i] ENDFOR " Plot proportions +/- se across time series" VARIATE [NVAL = 2] stack_mean_propsforfas,stack_mean_propsfortnf,stack_se_propsforfas, stack_se_propsfortnf STACK STACKED=stack_mean_propsforFAS; V1=!p(estim[1],estim[2]) STACK STACKED=stack_se_propsforFAS;V1=!p(se[1],se[2]) STACK STACKED=stack_mean_propsforTNF; V1=!p(estim[3],estim[4]) STACK STACKED=stack_se_propsforTNF;V1=!p(se[3],se[4]) FACTOR [LABELS=!T(male,female)] Sex;VALUES=!T(4(male,female)) FACTOR [labels=!t(nec1,)] NECROSIS; VALUES =!T(Nec1,) FACTOR [labels=!t(zvad,)] APOPTOSIS; VALUES =!T(zVAD, ) APPEND [NEW=%_NEWNECRO] 1(NECROSIS) APPEND [NEW=%_NEWAPOPT] 1(APOPTOSIS) TABLE [CLASSIFICATION = %_NEWNECRO,%_Time]propTNF_means;VALUES = stack_mean_propsfortnf TABLE [CLASSIFICATION = %_NEWNECRO,%_Time]propTNF_se;VALUES = stack_se_propsfortnf TABLE [CLASSIFICATION = %_NEWAPOPT,%_Time]propFAS_means;VALUES = stack_mean_propsforfas TABLE [CLASSIFICATION = %_NEWAPOPT,%_Time]propFAS_se;VALUES = stack_se_propsforfas DTABLE [METHOD=line;YTRANSFORM = exp1] proptnf_means; BAR=propTNF_se DTABLE [METHOD=line; YTRANSFORM=exp1] propfas_means; BAR=propFAS_se Nature Protocols: doi:1.138/nprot