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Philip Johns
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1 Supplement Figure A 4 h control + h B 4 h control + h

2 Supplement Figure A-H BV6/TNF-α + ctrl A B 9 h C D h E F 8 h G H

3 Supplement Figure I-P + ctrl I J 8 h K L 4 h M N h O P

Supplement Figure 3 A Survival (% control) BV6 - + + + Necr.

4 Supplement Figure 3 A Survival (% control) BV Necr D F H I sirna RIP ctrl RIP sirna ctrl RIP3 RIP3 sirna ctrl MLKL MLKL RIP RIP3 mrna levels(% control) * *non-specific band (h) phax bc RIP-IP - - TNFR RelA B Survival (% control) 7 kda kda E Survival (% control) lysate kda BV6 sirna ctrl RIP RIP3 MLKL kda kda control sirna 5 kda kda J TNFR RelA BV6 BV6 + G (h) RIP Caspase 8 sirna ctrl TNFR sirna ctrl RelA ctrl RIP RIP3 MLKL bc - - kda 7 kda kda Survival (% control) C mrna levels (% control) control sirna RIP RIP3 Necr. GSK'87 NSA Caspase 8-IP K TNFR surface expression (%control) bc lysate ** ** (h) kda 35 kda kda

5 Supplement Figure 4 A nucleus (h) TNF-α RelA p5 PARP Caspase 3 cytoplasm kda kda 35 kda B D mrna (x-fold increase) IĸBα (h) pikbα HCT6 XIAP (h) pikbα cfliplong HT9 ciap C Survival (% control) (h) pikbα MLN TNF-α SW kda kda E mrna (x-fold change) HCT HT SW TNF-α IL-8 CCL TNF-α IL-8 CCL TNF-α IL-8 CCL F TNF-α (pg/ml) HCT6 HT9 SW4 HCT6 HT9 SW4 + G RIP RIP3 Caspase 8 MLKL * HCT6 HT9 SW4 * non-specific band 7 kda kda

6 Supplement Figure 5 A mrna (x-fold increase) IDN-734 B TNF-α IL-8 CCL C TNF-α (pg/ml) IDN-734 D mrna (x-fold change) Z-DEVD Z-VEID Z-IETD Survival (% control) + IDN Necr TNF-α IL-8 CCL

Supplement Figure 6 A mrna (x-fold increase) B C mrna (x-fold

siedd 3h E G mrna (x-fold increase) 5 3 - + - TNF-α IL-8 CCL

- - - - + siedd + + - - - - + bc lysate 4h - - Survival (%

siedd F 5 3 - + + - - + sijnk TNF-α IL-8 3 CCL 8 sicasp8

7 Supplement Figure 6 A mrna (x-fold increase) B C mrna (x-fold change) EDD Survival (% control) RIP bc RIP-IP 4h - - sirip3 siedd 3h E G mrna (x-fold increase) TNF-α IL-8 CCL + + Necr siedd bc lysate 4h - - Survival (% control) 3h 3 kda 7 kda TNF-α IL-8 CCL 3 sirip3 sirip3 siedd siedd F sijnk TNF-α IL-8 3 CCL 8 sicasp8 sicasp8 sicasp H TNF-α (pg/ml) 3 sicasp8 D TNF-α (pg/ml) I Proliferation (%) IDN-734 sirip3 siedd 5 3

8 Supplement Figure 7 A cleaved Caspase 3 (IHC-Score) Apoptosis-like Necroptosis-like Resistant n.s n.s * p =.4 p =.8 p =.7 p =.3 p = n.s p =. + B nuclear RelA (IHC-Score) µm + * + µm Apoptosis-like Necroptosis-like Resistant n.s ** * p =.58 n.s p = µm p =. p = p =.5 n.s p =.3 + µm µm µm C Tissue # RIP Apoptosis -like Necroptosis -like Resistant kda RIP3 Caspase 8 MLKL kda Ponceau S 7 kda

9 Supplement Figures Supplement Figure : Discrimination of apoptotic and necrotic cell death in HCT6 and HT9. A: HCT6 were pre-treated with (5 µm) for hours, followed by treatment with (5 µg/ml) for the indicated durations. Cellular size (forward scatter, FSC) and granularity (side scatter, SSC) versus cell permeability (PI-positivity, red dots) was analyzed via flow cytometry to discriminate apoptotic from necrotic cell death (according to Vanden Berghe et al.). After 4 hours of treatment, apoptotic particles (small PI-negative dots in the box of the lower left corner) and plasma membrane blebbing (spreading of the dots) were detected. After hours this was followed by the occurrence of an increased subpopulation of PI-positive cells (box in the center) indicative for plasma membrane permeability in the stage of secondary necrosis. Addition of suppressed induced apoptosis. Data shown of one out of three representative experiments. B: In HT9, which were treated as explained in Supplement Figure A, co-treatment of and caused primary necrotic death after hours of treatment as indicated by a PI-positive sub-population of enlarged celluar size (box shifted to right) in the absence of previous apoptotic changes. Data shown of one out of three representative experiments. Supplement Figure : Ultrastructural analysis of necroptosis. HT9 were pre-treated with for hours and incubated with BV6 (.6 µm) plus TNF-α (5 ng/ml) (A-H), or (5 µg/ml) (I-P). Morphological changes were analyzed via electron microscopy after the indicated durations. Cytoplasm of untreated HT9 cells (A, B, I, J) was rich of rough endoplasmic reticulum (ER) and

10 normal sized mitochondria with regular structure of cristae. After 9 hours of BV6/TNFα/ (C, D), or 8 hours of / treatment (K, L), discrete vacuolization of the ER was visible as a sign of cellular stress. Dehiscence of cristae and an edematous matrix in mitochondria of normal size indicated for early mitochondrial degeneration. After hours (E, F) or 4 hours (M, N), respectively, prominent enlargement of mitochondria was visible with partial loss of cristae and vacuolization. After 8 hours (G, H) or hours (O, P), dramatic vacuolization and disruption of cell organelles and rupture of plasma membranes was observed. Note that the structure of the nucleus remained intact until late stages of necroptosis. Scale bars: µm. Supplement Figure 3: RIP, RIP3 and MLKL are required for necroptosis A: BV6/TNF-α and induced necroptosis in HT9. Necroptosis was induced with (5 µm) and BV6 (.6µM) in the presence of TNF-α (5 ng/ml) for 48 hours. Mean percentage of treated cells obtained from three independent experiments with three technical replicates each (± SD, two-sided Student s t-test, P <.). B: Knockdown of RIP, RIP3 or MLKL had no significant effects on, BV6 or 5- FU treatment. HT9 were transfected with sirnas and treated with, BV6 (.6 µm) or (5 µg/ml) for 48 hours. Mean percentage of control (ctrl, scrambled sirna and ), obtained from three independent experiments with three technical replicates each (± SD). C: Knockdown efficiencies of indicated sirnas on mrna level as quantified via qrt- PCR after 48 hours. Mean percentage of control (ctrl, scrambled sirna and ), obtained from three independent experiments with two technical replicates each (± SD, two-sided Student s t-test, P <.).

11 D: Knockdown efficiencies of indicated sirnas on protein levels as analyzed by Western blot after 48 hours. Asterisk: non-specific band. : loading control. E: Effect of chemical RIP, RIP3 and MLKL inhibitors on necroptosis. HT9 cells were pre-treated with, Necrostatin- (Necr., µm), GSK 87 (5 µm) or Necrosulfonamide (NSA, µm) for hours, followed by addition of BV6 (.6 µm) or (5 µg/ml) for 48 hours. Mean percentage of control (), obtained from three independent experiments with three technical replicates each (± SD, two-sided Student s t-test, P <.). F: phax levels after treatment. HT9 cells were treated with (5 µg/ml) for the indicated durations and levels of phax were quantified via Western blot. G: Immunoprecipitation of caspase 8 after treatment with and. HT9 were treated with (5 µm) and (5 µg/ml) for the indicated durations. Co-immunopreciptitation was performed on lysates using a specific antibody against caspase 8 and samples were analyzed via Western blot. Bc: bead control. H: Immunoprecipitation of RIP after treatment with and. HT9 cells were treated with (5 µm) and (5 µg/ml) for 3 hours. Coimmunoprecipitation was performed on lysates using a specific antibody against RIP and samples were analyzed for RIP and RIP3 via Western blot. Bc: bead control. I: Knockdown efficiency of sitnfr and sirela on mrna level as quantified via qrt- PCR after 48 hours. Mean percentage of control (ctrl, scrambled sirna and ), obtained from three independent experiments with two technical replicates each (± SD, two-sided Student s t-test, P <.). 3

12 J: Knockdown efficiency of sitnfr and sirela on protein level as analyzed by Western blot after 48 hours. : loading control. K: Elevated TNFR cell surface expression after treatment. HT9 were treated with (5 µg/ml) for the indicated durations and receptor cell surface expression was analyzed via flow cytometry. Mean percentage of treated cells obtained from three independent experiments (± SD, two-sided Student s t-test, **P <.). Supplement Figure 4: Activation of NF-κB by and A: and induced nuclear translocation of RelA and p5. HT9 cells were treated with (5 µm) and (5 µg/ml) for the indicated durations. Nuclear and cytosolic compartments were separately analyzed for the presence of RelA and p5 via Western blot. TNF-α (5 ng/ml) treated samples served as a positive control. PARP: loading control for nuclear fraction. Caspase 3: loading control for cytosolic fraction. B: Necroptosis was accompanied by increased transcription of NF-κB target genes as quantified by qrt-pcr. HT9 cells were treated with and (as described in B) for 4 hours. Mean fold increase compared to control (), obtained from three independent experiments with two technical replicates each (± SD, two-sided Student s t-test, P <.). C: Effect of MLN-494 on necroptosis. HT9 were treated with and (as indicated in B) for 48 hours, in the presence or absence of MLN-494 ( µm) and/or TNF-α (5 ng/ml). Mean percentage of control (), obtained from three independent experiments with three technical replicates each (± SD, two-sided Student s t-test, P <.). 4

13 D: piκbα levels after and treatment was only detected in HT9, but not in HCT6 or SW4. After treatment with (5 µm) and (5 µg/ml) for 4 hours lysates were analyzed via Western blot. : loading control. E: and treatment induced increased mrna levels of NF-κB dependent target genes in HT9, but not in HCT6 or SW4. Cells were treated with (5 µm) and (5 µg/ml) for 4 hours and target genes were quantified via qrt- PCR. Mean fold change compared to control () obtained from three independent experiments with two technical replicates each (± SD). F: TNF-α production by HCT6, HT9 and SW4 cells in response to and Z- VAD. Cells were treated as described in B, and after 4 hours TNF-α was quantified in supernatants via ELISA. Mean of one out of three representative experiments with two technical replicates each (± SD). G: Basal expression levels of components of the necroptosis pathway in HCT6, HT9 and SW4 cells as quantified via Western blot. Asterisk: non-specific band. : loading control. Supplement Figure 5: Effects of various caspase inhibitors A: Effect of combinatory treatment of and the pan-caspase inhibitor IDN-734 on NF-κB target gene transcription as quantified by qrt-pcr. HT9 cells were pretreated for hours with IDN-734 (5 µm), followed by addition of (5 µg/ml) for 4 hours. Mean fold increase compared to control (), obtained from three independent experiments with two technical replicates each (± SD, two-sided Student s t-test, P <.). B: Effect of and IDN-734 on autocrine TNF-α production. HT9 cells were treated as indicated in C. Mean fold increase compared to control (), obtained 5

14 from three independent experiments with two technical replicates each (± SD, twosided Student s t-test, P <.). C: and IDN-734 induced necroptosis. HT9 cells were pre-treated for hours with IDN-734 (5 µm) and/or Necrostatin ( µm), followed by addition of (5 µg/ml) for 48 hours. Percentage of cells treated with. Mean obtained from three independent biological replicates with three technical replicates each (± SD, two-sided Student s t-test, P <.). D: Effects of Z-DEVD, Z-VEID and Z-IETD on NF-κB dependent target genes. HT9 cells were pre-treated for hours with Z-DEVD, Z-VEID or Z-IETD (5 µm each), followed by stimulation with (5 µg/ml) for 4 hours. mrna levels were quantified by qrt-pcr. Mean fold change compared to control (), obtained from three independent experiments with two technical replicates each (± SD). Supplement Figure 6: Robust activation of NF-κB in response to and Z- VAD requires caspase 8 A: Effects of Necrostatin- on NF-κB dependent target gene expression as quantified by qrt-pcr. HT9 were treated with (5 µm), Necrostatin- ( µm) and 5- FU (5 µg/ml) for 4 hours. Mean fold change compared to control (), obtained from three independent experiments with two technical replicates each (± SD, twosided Student s t-test, P <.). B: Immunoprecipitation of RIP after treatment with and. HT9 cells were treated with (5 µm) and (5 µg/ml) for 4 or 3 hours. Coimmunopreciptitation was performed on lysates using a specific antibody against RIP and samples were analyzed for EDD and RIP via Western blot. Bc: bead control. 6

15 C: Effects of RIP3 or EDD knockdown on NF-κB dependent target gene expression as quantified by qrt-pcr. HT9 were transfected with indicated sirnas and treated with (5 µm) and (5 µg/ml) for 4 hours. Mean fold change compared to control (, scrambled sirna and ), obtained from three independent experiments with two technical replicates each (± SD, two-sided Student s t-test, P <.). D: Effects of RIP3 or EDD knockdown on TNF-α secretion. Cell culture supernatants from Supplement Figure 6B were analyzed via ELISA for TNF-α levels. Means of one of three representative experiments with two technical replicates each (± SD, twosided Student s t-test, P <.). E: Effects of EDD knockdown on necroptosis. HT9 cells were transfected with the indicated sirnas and treated with (5 µm) and (5 µg/ml) for 48 hours. Mean percentage of control (, scrambled sirna, and ), obtained from three independent experiments with three technical replicates each (± SD). F: Effects of JNK knockdown on necroptosis. HT9 cells were transfected and treated as described in Supplement Figure 6D. Mean percentage of control (, scrambled sirna, and ), obtained from three independent experiments with three technical replicates each (± SD). G: Effects of caspase 8 knockdown on NF-κB dependent target gene expression as quantified by qrt-pcr. HT9 cells were transfected and treated as described in Supplement Figure 6B. Mean fold increase compared to control (, scrambled sirna and ), obtained from three independent experiments with two technical replicates each (± SD, two-sided Student s t-test, P <.). 7

16 H: Effects of caspase 8 knockdown on TNF-α secretion. Cell culture supernatants from Supplement Figure 6F were analyzed via ELISA for TNF-α levels. Means of one of three representative experiments with two technical replicates each (± SD, twosided Student s t-test, P <.). I: Immunohistochemical staining of Ki-67/MIB was performed on sections of xenografts and the fraction of positive nuclei was quantified via computer-assisted image analysis using Aperio Image Scope software. Mean percentage of positive nuclei obtained from representative vital tumor regions (> 5 nuclei), ± SD, twosided Student s t-test: not significant, P >.5. Supplement Figure 7: Analysis of CRC tumor specimens A: Immunohistochemical staining of cleaved caspase 3 was performed on sections of treated tissue slices and intensity of staining was quantified using the following immunohistochemical (IHC) score: = negative, = weakly positive, = moderately positive, 3 = strongly positive. Boxplots show IHC scores obtained from apoptosislike (n = 4, tissue #-4), necroptosis-like (n = 5, tissue #5-9) and resistant (n = 4, tissue #-3) cases (± SD, two-sided Student s t-test: *P <.5, P <., n.s.: not significant). B: Immunohistochemical staining of RelA was performed on sections of treated tissue slices and intensity of staining was quantified using the following immunohistochemical (IHC) score: percentage of positive nuclei ( = -9%, = - 9%, 3 = >%) x intensity of staining ( = negative, = weakly positive, = moderately positive, 3 = strongly positive). Boxplots show IHC scores obtained from the subgroups as explained in A (± SD, two-sided Student s t-test: *P <.5, **P <., n.s.: not significant). Asterisk: maximum outlier. 8

17 C: D: Basal expression levels of components of the necroptosis pathway in all human samples as quantified via Western blot using µg of protein lysate of each sample. and Ponceau S: loading control. 9

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3 P p25. p43 p41 28 FADD. cflips. PE-Cy5 [Fluorescence intensity] L S p4 3 D3 76 N S L Ve ct or p4 3 D3 76 N S L Ve ct or A aspase 8 FADD TRAF2 D95-R - + Vector D95L TL I S L D376N T RAIL-R1 T RAIL-R2 D95-R E-y5 [Fluorescence intensity] Supplemental Fig. 1 Different