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Supplemental Table 1: Primer used in this study

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Supplemental Table 1: Primer used in this study

hMLKL_for CCCATGATCCCTCTGTGC

hMLKL_rev GACTCCTTGGTTTAGATTTTGATACAC

hIRF1_for ACA GCA CCA GTG ATC TGT ACA AC

hIRF1_rev TTC CCT TCC TCA TCC TCA TCT

h28S_for TTGAAAATCCGGGGGAGAG

h28S_rev hRIPK1_for hRIPK1_rev hRIPK3_for hRIPK3_rev

ACATTGTTCCAACATGCCAG

GTGTACAAGGGGCCCAACT

CGGCTGTGTCTCAGTCTGTT

GCCTCCACAGCCAGTGAC

TCGGTTGGCAACTCAACTT

(2)

Supplemental Table 2: gRNAs used in this study

IRF1 gRNA1 TTAATTCCAACCAAATCCCG

IRF1 gRNA2 TCTAGGCCGATACAAAGCAG

IRF1 gRNA3 GAACTCCCTGCCAGATATCG

(3)

Supplemental Table 3: Results for upregulation of MLKL in DNA microarrays using platform Interferome

Dataset ID

Fold Change

Interferon Type

Treatment

Time [h] Probe ID

In Interferome deposited publication

147 3.199128 I 4 A_24_P185044 PMID: 25536401

315 2.4995701 II 6 A_24_P185044 no publication deposited 318 2.497687 II 6 A_24_P185044 PMID: 23801603 125 3.4278443 I 3 A_23_P61050 no publication deposited

147 3.194414 I 4 A_23_P61050 PMID: 25536401

174 2.0067785 II 20 A_23_P61050 PMID: 27233968 226 2.5212712 I 6 A_23_P61050 no publication deposited 293 16.121225 II 24 A_23_P61050 PMID: 27770554 294 3.5354736 I 72 A_23_P61050 PMID: 25414701 315 2.9219253 II 6 A_23_P61050 no publication deposited 318 2.9206066 II 6 A_23_P61050 PMID: 23801603

44 2.54955 I 6 238025_at PMID: 20483728

67 5.379501 I 6 238025_at PMID: 22875473

144 5.7262244 II 24 238025_at PMID: 25168428

185 2.7878575 I 16 238025_at PMID: 24569457

300 2.4528027 I 6 238025_at PMID: 20805419

305 2.2880716 II 1.5 238025_at PMID: 22610275

306 2.308728 I 1.5 238025_at PMID: 22610275

75 2.02588 I 16 ILMN_1667825 PMID: 23626809

78 2.3977 I 12 ILMN_1667825 PMID: 23045702

81 2.18407 I 4 ILMN_1667825 PMID: 23045702

82 2.72911 II 12 ILMN_1667825 PMID: 23045702

83 2.69625 II 24 ILMN_1667825 PMID: 23045702

128 6.2672963 II 10 ILMN_1667825 PMID: 26424569 129 7.0557027 I 10 ILMN_1667825 PMID: 26424569 130 5.0771055 I 10 ILMN_1667825 PMID: 26424569 131 6.8568344 II 10 ILMN_1667825 PMID: 26424569

161 4.46232 I 6 ILMN_1667825 PMID: 24065129

162 4.98231 II 6 ILMN_1667825 PMID: 24065129

163 4.1562304 I 3 ILMN_1667825 PMID: 20956346

275 6.420122 I 8 ILMN_1667825 PMID: 22851595

276 5.1070356 I 2 ILMN_1667825 PMID: 22851595 277 5.8366957 I 8 ILMN_1667825 PMID: 22851595 278 4.5659633 I 2 ILMN_1667825 PMID: 22851595 280 2.1331036 I 48 ILMN_1667825 no publication deposited 281 2.233389 I 48 ILMN_1667825 no publication deposited 284 4.0358825 II 6 ILMN_1667825 PMID: 26030458 373 2.003676 I 24 ILMN_1667825 PMID: 23633948

(4)

Dataset ID

Fold Change

Interferon Type

Treatment

Time [h] Probe ID

In Interferome deposited publication 374 2.4460273 I 6 ILMN_1667825 PMID: 23633948 375 2.4212413 I 6 ILMN_1667825 PMID: 23633948

78 2.12785 I 12 ILMN_1715863 PMID: 23045702

82 2.19505 II 12 ILMN_1715863 PMID: 23045702 128 5.0299034 II 10 ILMN_1715863 PMID: 26424569

129 5.98513 I 10 ILMN_1715863 PMID: 26424569

130 4.0076976 I 10 ILMN_1715863 PMID: 26424569 131 5.810739 II 10 ILMN_1715863 PMID: 26424569 275 4.7557836 I 8 ILMN_1715863 PMID: 22851595

277 5.095506 I 8 ILMN_1715863 PMID: 22851595

278 3.021476 I 2 ILMN_1715863 PMID: 22851595

82 2.35299 II 12 ILMN_1737498 PMID: 23045702 83 2.37726 II 24 ILMN_1737498 PMID: 23045702 128 6.188601 II 10 ILMN_1737498 PMID: 26424569 129 7.521052 I 10 ILMN_1737498 PMID: 26424569 130 5.4541273 I 10 ILMN_1737498 PMID: 26424569 131 5.5040517 II 10 ILMN_1737498 PMID: 26424569

161 3.15909 I 6 ILMN_1737498 PMID: 24065129

162 3.31444 II 6 ILMN_1737498 PMID: 24065129

163 2.6113572 I 3 ILMN_1737498 PMID: 20956346 275 5.5661116 I 8 ILMN_1737498 PMID: 22851595 276 3.2793548 I 2 ILMN_1737498 PMID: 22851595 277 3.9632547 I 8 ILMN_1737498 PMID: 22851595

278 2.264143 I 2 ILMN_1737498 PMID: 22851595

143 4.3258543 I 6 8002778 PMID: 22005912

201 2.9556017 I 2 8002778 PMID: 24561305

202 2.20003 I 6 8002778 PMID: 24561305

301 3.5955815 I 6 8002778 PMID: 22677194

302 4.2909565 II 24 8002778 PMID: 22677194

303 2.6675231 I 24 8002778 PMID: 22677194

304 2.8380008 II 6 8002778 PMID: 22677194

307 5.3234553 II 48 8002778 PMID: 23150618

308 2.6459115 II 48 8002778 PMID: 23150618

Supplemental Table 3 continued

(5)

0 5 10 15

0 3 6 9 12

mRNA levels [fold change]

time [h]

MLKL RIPK1 RIPK3

Supplemental Figure 1

IFNγ

**

* *

(6)

Supplemental Figure 2

MLKL

IFNγ - + _________ EFM-192A

β-Actin

PC pMLKL

42 kDa 54 kDa 54 kDa

(7)

Supplemental Figure 3

0 20 40 60 80 100

0 1 3 10 30 100

PI positive cells [%]

IFNγ [ng/ml]

Ctrl zVAD

** ** ** ** **

** ** ** *

*

(8)

1 Supplemental figure legends

Suppl. Fig. 1: Effect of IFNγ on RIPK1 or RIPK3 expression.

EFM-192A cells were treated for indicated time points with 1.5 ng/ml IFNγ. mRNA levels of MLKL, RIPK1 and RIPK3 were quantified by qRT-PCR analysis and are shown as fold increase relative to untreated control cells with mean and SEM of at least three independent experiments performed in duplicate; *P < 0.05; **P < 0.01.

Suppl. Fig. 2: Effect of IFNγ on MLKL phosphorylation.

EFM-192A cells were treated with 1.5 ng/ml IFNγ for 24 hours. Protein expression of phospho-MLKL (pMLKL), MLKL and β-Actin was analyzed by Western blotting. As positive control (PC) for pMLKL, HT-29 cells were treated with 1 μM BV6 and 20 μM zVAD.fmk for 24 hours.

Suppl. Fig. 3: IFNγ induces cell death when caspases are inhibited.

EFM-192A cells were treated with indicated concentrations of IFNγ with or without 20 μM

zVAD.fmk for 72 hours. Cell death was determined by PI/HOECHST staining and

ImageXpress Micro XLS system. Mean and SD of at least three independent experiments

performed in duplicates are shown; *P < 0.05; **P < 0.01.

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