Antibodies and reagents
The following antibodies were used in this study: RIPK1 (Cell Signaling Technology, catalogue no. 3493); p-hRIPK1 (Cell Signaling Technology, catalogue no. 65746); hRIPK3 (Cell Signaling Technology, catalogue no.13526); p-hRIPK3 (Cell Signaling Technology, catalogue no. 93654); hMLKL (Cell Signaling Technology, catalogue no. 14993; Abcam, ab183770; Thermo Fisher Scientific, PA5-34733); p-hMLKL (Cell Signaling Technology, catalogue no. 91689); anti-SIGLEC12 (Boster, A10550-1; Invitrogen, PA5-110369; Invitrogen, PA5-31457); TMPRSS4 (Cell Signaling Technology, catalogue no. 84382); anti-HMGB1 (Cell Signaling Technology, catalogue no. 6893); anti-β-actin (Sigma-Aldrich, A5316); anti-FLAG (Sigma-Aldrich, F1804); anti-FLAG–HRP (Cell Signaling Technology, catalogue no. 86861); anti-mouse IgG (Cell Signaling Technology, catalogue no. 7074); anti-rabbit IgG (Cell Signaling Technology, catalogue no. 7076), anti-mouse IgG (H + L) and F(ab′)2 Fragment (Alexa Fluor 488 Conjugate) (Cell Signaling Technology, catalogue no. 4408). All primary antibodies were used at 1:2,000 dilution. All secondary antibodies were used at 1:5,000 dilution.
The following reagents were used in this study: SM-164 (S7089), Nec-1s (S8641), GSK′872 (S8465), NSA (S8251), emricasan (S7775) and erastin (S7242) from SelleckChem; etoposide (E1383), α-ketoglutarate (349631), lipopolysaccharide (L4391), luminol (A8511), p-coumaric acid (C9008) and anti-FLAG M2 agarose beads (M8823) from Sigma-Aldrich; nigericin (11437) from the Cayman Chemical Company; Lipofectamine 3000 (L3000015), SuperSignal West Atto Ultimate Sensitivity Substrate (A38556) and Prolong Diamond Antifade Mountant with DAPI (P36966) from Thermo Fisher Scientific; polyethylenimine (PEI; 24765-100) from Kyfora Bio; polybrene (TR-1003) from EMD Millipore; and 10X Tris/Glycine/SDS Electrophoresis Buffer (1610772), Tween 20 (1610781), Stacking Gel Buffer for PAGE (1610799), Resolving Gel Buffer for PAGE (1610798), Precision Plus Protein Dual Color Standards (1610394) and nitrocellulose membrane (1620115) from Bio-Rad. Autoradiography films were from MTC Bio (A8815).
Cell lines and growth media
The 293T, HEK293-FlpIn-TREx, HeLa and MEF cells were grown in DMEM (Cytiva, SH30243.FS), with l-glutamine, 4.5 g l−1 glucose and pyruvate; HT-29 cells were grown in McCoy’s 5A medium (Gibco, 16600-082, with l-glutamine); and A549, Jurkat and THP-1 cells were grown in RPMI 1640 medium (Fisher Scientific, SH30255.01, with HEPES and l-glutamine). All media were supplemented with 10% fetal bovine serum (GeminiBio), 1× non-essential amino acids (Cytiva, SH30238.01) and 1× antibiotic/antimycotic solution (Sigma-Aldrich, A5955). All cell lines were regularly tested for mycoplasma contamination using Lonza’s MycoAlert Kit (catalogue no. LT07-318) and tested negative for mycoplasma.
Human tumour and normal tissue samples
Deidentified human benign (normal) lung and colon and lung and colon adenocarcinoma tumour specimens were obtained from the University of Texas Southwestern Tissue Management Shared Resource. Patients were enrolled and consented to a protocol approved by the institutional review board. Formalin-fixed tissues were processed as in the ‘Immunohistochemistry of tissue slices’ section.
Molecular cloning and plasmids
Molecular cloning was performed using New England Biolabs restriction enzymes and T4 DNA ligase. Plasmids were transformed into chemically competent NEB Stable (for lentiviral plasmids, at 30 °C) and DH5α Escherichia coli cells (for non-lentiviral plasmids at 37 °C). Plasmid purification and extraction were performed using a QIAprep Spin Miniprep Kit (Qiagen, 27106) and QIAquick Gel Extraction Kit (Qiagen, 28704). sgRNAs targeting SIGLEC12 were cloned into pSpCas9(BB)-2A-GFP (pX458) (Addgene, catalogue no. 48138). Cloned plasmids were amplified and purified using a ZymoPURE II Plasmid Midiprep Kit (Zymo Research, D4201).
Generation of knockout cell lines using CRISPR–Cas9
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