Ab187091

MLKL^−/− HT29 human adenocarcinoma cell lines and MLKL^−/− U937 human monocytic cell lines were generated in house using CRISPR-Cas9 technology^{39 (link)}. Recombinant hTNF was produced in-house and used at a final concentration of 100 ng/mL. Smac mimetic (Compound A), and the caspase inhibitor IDN-6556 were provided by TetraLogic (Malvern, PA, USA). Rat-anti hRIPK3 1H2 (1:1000)^{24 (link),57} and rat anti-MLKL 3H1 (1:2000)^{16 (link)} (biotinylated and non-biotinylated forms) were produced in-house (3H1 available from Millipore as catalog number MABC604). Anti-human MLKL pS358 (1:1000, ab187091) and anti-Actin (1:10,000, ab5694) were purchased from Abcam. Horseradish peroxidase (HRP) conjugated goat anti-rat IgG (Southern Biotech 3010-05), HRP-conjugated goat anti-mouse IgG (Southern Biotech 1010-05), HRP-conjugated goat anti-rabbit IgG (Southern Biotech 4010-05) and HRP-conjugated streptavidin (Millipore SA202) were used for the secondary detection of primary antibodies (all 1:10,000).

MCF7 cells grown on glass coverslips and in co-culture with primary NK cells for 3 h were fixed in 3.7% paraformaldehyde (PFA)/phosphate-buffered saline (PBS) for 15 min, permeabilized with 0.5% Triton-X100 for 30 min, washed and blocked in 5% BSA/0.1% Triton-X100/PBS for 1 h, and then incubated with the primary antibody against phospho-MLKL (S358) (#ab187091, Abcam) and the fluorescently conjugated Alexa-Fluor secondary antibody (Invitrogen), as well as stained with Hoechst (Invitrogen) and Phalloidin (Alexa Fluor 647, Invitrogen). All procedures were conducted at room temperature, except for phospho-MLKL primary antibody binding (overnight at 4 °C). The coverslips were mounted on glass slides with ProLong Gold antifade reagent (Invitrogen) and imaged using the Andor Dragonfly spinning disc confocal microscope with a  60x objective.

Western blotting analysis was performed as previously reported^{62 (link)}. An equal amount (40–80 μg) of protein samples were detected. Primary antibodies against p-MLKL (Rabbit, 1:500, ab187091; Abcam, UK), MLKL (Rabbit 1:1000, ab194699; Abcam, UK), p-RIP1 (Rabbit, 1:500, 65746; Cell Signaling Technology, USA), RIP1 (Mouse, 1:500, ab72139; Abcam, UK), caspase 8 (Rabbit, 1:1000, A0215; ABclonal, China), caspase 3 (Rabbit, 1:1000, A11319; ABclonal, China), TNF-α (Rabbit, 1:500, ab6671; Abcam, UK), IL-6 (Mouse, 1:500, ab9324; Abcam, UK), iNOS (Rabbit, 1:500, ab15323; Abcam, UK), IL-1β (Rabbit, 1:500, ab2105; Abcam, UK), NF-kB p65 (Rabbit, 1:1000; ab16502, Abcam, UK), IkBα (Mouse, 1:1000, 4814; Cell Signaling Technology, USA), p-IkBα (Rabbit, 1:1000, 2859; Cell Signaling Technology, USA); H3 (Rabbit, 1:1000, AF0009; Beyotime, China) and β-actin (Rabbit, 1:3000, AC026; ABclonal, China) were used. Protein expression levels were analyzed using ImageJ software and normalized to β-actin (National Institutes of Health, Bethesda, MD, USA). Phosphorylated protein expression was evaluated compared to total protein expression.

Tissue samples were homogenized in NP-40 lysis buffer using a tissue grind pestle (Kimble/Chase) or with a bead ruptor 12 (Omni International) to obtain protein lysates. These were separated by SDS–PAGE, transferred to polyvinylidene difluoride membrane and analysed by immunoblotting. Membranes were probed with the following antibodies: anti-p-AKT (#4060), anti-AKT (#4685), anti-p-GSK-3 (#8566), anti-GSK-3 (#5676), anti-p-ERK (#4377) and anti-ERK (#4695; Cell Signaling), anti-Caspase-8 (Enzo #ALX-804-447), anti-RIPK3 mouse (IMGENEX #IMG-5523), anti-RIPK3 human (#ab56164), anti-phospho-MLKL human (#ab187091) and mouse (#ab196436; Abcam) and anti-GAPDH (ABD Serotec #MCA4739). All primary antibodies were used at the dilution 1:2,000. As secondary antibodies, anti-rabbit-horseradish peroxidase (HRP; #NA934V) and anti-mouse-HRP (#NA931V; Amersham) and anti-rat-HRP (Santa Cruz #sa2956) were used. All secondary antibodies were used at the dilution 1:5,000. Unedited scans of all western blot images are shown in Supplementary Fig. 11.

Protein extracts from mouse muscles, C2C12 myoblasts, and MEFs (strain CF-1, Millipore) were analysed using the following primary antibodies: rabbit antibody to RIPK3 clone H-43 (Santa Cruz, sc-135170, 1:300), rabbit antibody to RIPK3 (Sigma, PRS2283, 1:1000), mouse antibody to GAPDH (Abcam, 9484, 1:500). Secondary antibodies were: IRDye 680RD conjugated goat anti-rabbit IgG (LI-COR, 925-68071, 1:15,000) and IRDye 800CW conjugated goat anti-mouse IgG (LI-COR, 926-32210, 1:15,000). Detection was performed using a LI-COR Odyssey instrument (fluorescent detection) and the quantification of images was performed using ImageJ. Uncropped scans are shown in the Supplementary Figure 6.
For immunofluorescence, mouse antibody to human Laminin α2 (clone 5H2, MAB1922, 1:1000), rabbit to RIPK3 clone H-43 (Santa Cruz, sc-135170, 1:50), rabbit antibody to human phospho-MLKL (Abcam, Ab-187091, 1:80), rat antibody to CD68 (clone FA-11, 137001, 1:50), rabbit antibody to mouse pan-Laminin (Sigma, L9393, 1:1000), rabbit antibody to Collagen VI (Abcam, Ab6588, 1:150), and neonatal myosin heavy chain antibody (Clone BF34; Developmental Studies Hybridoma Bank, 1:50) were used.

Histones were extracted using a histone extraction kit (Abcam Cambridge, MA) according to manufacturer protocol. To induce necroptosis and serve as a positive control, cells were treated with pan-caspase inhibitor Q-VD-OPh (25 μM) and BV6 (7.5 μM) for 30 min followed by incubation with TNF-α (50 ng/ml). Western blot was performed using methods previously described^{16 (link)}. Antibodies against p27 (3686), RIP1 (4926), acetylated H3K9 (9649S), and H3K14 (7627S) were obtained from Cell Signaling Technology (Danvers, MA). Antibodies against phosphorylated MLKL (ab187091), MLKL (ab184718), and RIP3 (ab56164) were obtained from Abcam (Cambridge, MA). Antibody against H3 (06–755) was purchased from Upstate (Lake Placid, NY) and tubulin (CP-06) from Calbiochem (San Diego, CA). HRP conjugated secondary antibodies were obtained from Sigma Aldrich (St. Louis, MO).