Sm 164

HT29 cells were purchased from American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) along with 1% penicillin and streptomycin (HyClone) at 37°C and 5% CO₂. HEK293T cells were purchased from ATCC and maintained in DMEM (Gibco) containing 10% FBS along with 1% penicillin and streptomycin at 37°C and 5% CO₂. To construct Gab1-knockdown HT29 cell line, shGab1 cDNA were constructed into the PLKO1-puro vector (Addgene plasmid 8453) and packaged into lentiviruses. HT29 cells were infected with shGab1 lentivirus and then screened by puromycin.
For stimulation, cells were plated on 6-well plates at 5 × 10⁵ cells per well overnight and then pretreated with RIPK1 inhibitor Nec-1s (10 μM; Selleck Chemicals) for 1 hour. After that, cells were stimulated with T/S/Z mix — TNF-α, 50 ng/mL (Novoprotein); SM-164, 50 nM (Selleck Chemicals); Z-VAD-FMK, 50 μM (Selleck Chemicals) — for the indicated time. Cell lysates and culture supernatant were further analyzed by Western blot.

zVAD, HG-9-91-01, and YKL were purchased from MedchemExpress. Necrostatin-1 was from Calbiochem. GSK’872 and SM-164 were from Selleck. Protein A/G Magnetic Beads were purchased from Pierce. Lipofectamine 2000 was from Life Technologies. LPS and poly I:C were purchased from InvivoGen. Mouse and human recombinant TNF were purchased from Novoprotein. Propidium iodide (PI) and cycloheximide (CHX) was from Sigma. Human recombinant RIPK1 and RIPK3 were from AtaGenix. Elisa kits for TNF and IL-1β were purchased from R&D. ADP-Glo kinase assay kit was from Promega. Immunoblotting was performed with following antibody: anti-RIPK1 (610459, BD Biosciences); anti-hRIPK1 S166 (44590, Cell Signaling); anti-mRIPK1 S166 (31122, Cell Signaling); anti-hRIPK3 (13526, Cell Signaling); anti-mRIPK3 (2283, ProSci); anti-hRIPK3 S227 (91702, Cell Signaling); anti-mRIPK3 T231/S232 (91702, Cell Signaling); anti-hMLKL (ab184718, Abcam); anti-mMLKL (AP14272b, ABGENT); anti-hMLKL S358 (ab187091, Abcam); anti-mMLKL S345 (ab196436, Abcam); anti-GAPDH (sc-32233, Santa Cruz); anti-cleaved Caspase-8 Asp387 (8592, Cell Signaling); anti-cleaved Caspase-3 (9661, Cell Signaling); anti-GSDME (ab215191, Abcam), anti-GSDMD (ab209845, Abcam), anti-Caspase-1 (AG-20B-0042-C100, AdipoGen), anti-Caspase-11 (NB120-10454, Novus).