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The receptor-interacting protein kinase 1 (RIP1) functions as a key regulator for NF-κB activation, apoptosis and necroptosis induced by tumor necrosis factor (TNF-α).1,

Materials and Methods

Reagents

TNF-α and z-VAD were purchased from R&D (Minneapolis, MN, USA) and Calbiochem (Anaheim, CA, USA), respectively. The Smac mimetic, Cycloheximide and LPS were obtained from Sigma. Nec-1 was from Enzo Life Science (Alexis, USA). The following antibodies were used for western blotting: p-IkBa (Cell Signaling), p-ERK (Cell Signaling Technology, Danvers, MA, USA), ERK (Cell Signaling), p-P38 (Cell Signaling), P38 (Cell Signaling), p-P65 (Cell Signaling), p-JNK (Cell Signaling), JNK (Cell Signaling), PARP (Cell Signaling), Caspase-3 (Cell Signaling), HA (Cell Signaling), Caspase-8 (Enzo Life Science), RIP1 (BD Biosciences, Franklin Lakes, NJ, USA), p-RIP1(S166) (Cell Signaling) mouse RIP3 (ProSci, San Diego, CA, USA), MLKL (Abcam, Cambridge, UK), p-MLKL (Abcam), β-actin, α-Tubulin and anti-flag-HRP (Sigma). Anti-phospho-RIP3 antibody (mouse) was generated in our lab and immunoaffinity-purified. Cell viability was determined by measuring ATP levels using Cell Titer-Glo kit (Promega, Madison, WI, USA).

Mice

Mice were housed in a specific pathogen-free facility, which belongs to Institute for Nutritional Sciences. Fadd−/− mice (C57BL/6) were gifted by Dr. Jianke Zhang (Thomas Jefferson University, Philadelphia, USA), and Rip3−/− mice (C57BL/6) were provided by Dr. **aodong Wang (NIBS, Bei**g, China). Animals were subsequently backcrossed on a C57BL/6 background for at least 10 generations. A novel mutant, RIP1Δ, was obtained from the experiment with two amino acids G26F27 in the P-loop of RIP1 deleted. To generate Rip1Δ/Δ, Rip1K45A/K45A, Rip1−/− mice by crispr-cas9 mutation system (Bioray Laboratories Inc., Shanghai, China), different sgRNA were designed to target RIP1 kinase domain. (Rip1Δ/Δmutant with gRNA: 5′-GACCTAGACAGCGGAGGCTT-3′; Rip1K45A/K45A mutant with gRNA: 5′-GCCCTGTGTATACTTTTTTC-3′; Rip1−/− with gRNA: 5′-GATGGCATCCAGTGACCTGC-3′). Additional information is provided upon request. All mutant mice and WT mice used in these studies shared a common genetic C57BL/6 background. Animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (CAS), University of Chinese Academy of Sciences.

Cerulein-induced acute pancreatitis

Male WT, Rip3−/− and Rip1Δ/Δ mice littermates at 8 weeks of age (n=7) were treated every hour for 12 consecutive hours with cerulein (50 μg/kg, Sigma) intraperitoneal injection. Animals were assayed 24 h after the first injection. Serum amylase activity was assayed by amylase activity assay kit (Sigma).

Isolation and culture of MDFs, MEFs, BMDMs and thymocytes

MDFs were separated from the skin of newborn mice, and MEFs were isolated from E13.5 to E14.5 embryos. MDFs and MEFs were cultured in DMEM medium supplemented with 10% FBS and penicillin/streptomycin. BMDMs from isolated bone marrow cells collected from mouse femurs and tibias were induced to differentiate in vitro. Bone marrow cells were cultured for 7 days in RPMI medium containing 10% FBS, penicillin/streptomycin and 50 ng/ml M-CSF, and medium was changed every 2 days. Thymocytes were isolated from thymus and cultured in RPMI medium containing 10% FBS, penicillin/streptomycin and 50 μM β-ME.

Immunoblotting and immunoprecipitation

Cells were harvested at different time points, washed with PBS and lysates with 1 × SDS sample buffer containing 100 mM DTT and boiled for 5 min at 95 °C for reducing gel. For mouse tissue protein extraction, E14.5 fetal liver and other tissues were ground by pestle and mortar with liquid N2, and the protein was extracted with RIPA lysis buffer. The lysates were cleared by centrifugation for 20 min at 13 200 × g, quantified by BCA kit (Thermo Scientific, Rockford, IL, USA) then mixed with SDS sample buffer and boiled at 95 °C for 5 min. The samples were separated using SDS-PAGE, transferred to PVDF membrane (Millipore, Darmstadt, Germany) with 100 v for 2 h. The proteins were detected by using a chemiluminescent substrate (Thermo Scientific). To immunoprecipitate RIP1, cell extract protein was incubated for 3 h with 5 μl of RIP1 antibody (BD Biosciences). After mixing end over end for overnight (4 °C) with 30 μl of G-Agarose beads, the agarose was collected and washed three times with cell lysis buffer (Tris-HCl 20 mmol/l (pH 7.5), NaCl 150 mM, EDTA 1 mM, EGTA 1 mM, Triton X-100 1%, Sodium pyrophosphate 2.5 mM, β-Glycerrophosphate 1 mM, NaVO4 1 mM, Leupeptin 1 μg/ml.). Immunoprecipitates were denatured in SDS, subjected to SDS-PAGE, and immunoblotted.

RIP3 and MLKL oligomerization detection

The cells were cultured in six-well plates and treated with indicated stimuli. Cells were harvested at different time points and lysed with 2 × DTT-free sample buffer (Tris-Cl (PH 6.8) 125 mM, SDS 4%, Glycerol 20%, Bromophenol blue 0.02%) immediately. Total cell lysates were separated using SDS-PAGE, and transferred to PVDF membrane (Millipore), and western blotting was performed with RIP3 or MLKL antibodies.

Flow cytometry

Antibodies against mouse CD3, CD4, CD8, CD19, Mac-1 and Gr-1 from eBioscience (Carlsbad, CA, USA) were fluorescence-conjugated and were used for flow cytometry analysis in this study. We prepared single-cell suspension from lymph nodes, spleen and thymus, respectively, and stained them with fluorescence-conjugated antibodies for half an hour in staining buffer. After staining, cells were immediately analyzed by flow cytometry (FACSAria III, BD Biosciences).

Immunofluorescence

MDFs were plated overnight on coverslips before various stimulations. After stimulation, cells were washed with PBS and fixed with 4% PFA in PBS for 15 min. Next, the cells were blocked with 0.3% Triton X-100 and 5% normal donkey serum (Jackson immunoResearch, Baltimore Pike, West Grove, PA, USA) for 1 h at room temperature, then followed by first antibody incubation at 4 °C for overnight. Signals were developed with Alexa fluorescence antibodies (Invitrogen). Finally, the cells were stained with DAPI for 10 min. Confocal microscopy analysis was performed using a Zeiss 710 laser-scanning microscope (Zeiss, Thornwood, NY, USA).

RT-PCR

Total RNA was extracted using Trizol reagent (Life Technologies), according to the manufacturer’s instructions. After quantification, 2 μg total RNA was reverse transcribed to complementary DNA (Takara, Dalian, China). Transcript levels of indicated cytokines were quantified by quantitative RT-PCR on an ABI 7500 real-time PCR instrument with SYBR Green. Relative expression was calculated using LC32 as an internal control as indicated. Primers used were as follows: mIL-1b: 5′-CCCAACTGGTACATCAGCAC-3′ and 5′-TCTGCTCATTCACGAAAAGG-3′; mTNF: 5′-CCCACTCTGACCCCTTTACT-3′ and 5′-TTTGAGTCCTTGATGGTGGT-3′; mIL-6: 5′-CGGAGAGGAGACTTCACAGA-3′ and 5′-CCAGTTTGGTAGCATCCATC-3′; mCXCL-1: 5′-CTGGGATTCACCTCAAGAACATC-3′ and 5′-CAGGGTCAAGGCAAGCCTC-3′; mMCP-1: 5′-TTAAAAACCTGGATCGGAACCAA-3′ and 5′-GCATTAGCTTCAGATTTACGGGT-3′; mCCL-5:5′-GCTGCTTTGCCTACCTCTCC-3′ and 5′-TCGAGTGACAAACACGACTGC-3′.