Caco-2 cells seeded on a 96-well plate were stimulated by inhibitors added as follows. After treatment Monmouthfor 48 h, the MTT experiment was carried out to determine the cell viability. The working concentrations of Vitamin E, Z-VAD-FMK, 3-Methyladenine, VX-765, GSK-872 (MedChemExpress, Monmouth, NJ, USA), Necrosulfonamide and Nec-1s (Selleck, Houston Texas USA) were 30, 10, 10, 5, 10, 0.5, and 10 μM, respectively. The inhibitors were dissolved in DMEM (1% BSA + 0.1% DMSO) and combined with the CLNAs at their IC50.
Gsk872
Manufactured by MedChemExpress
Sourced in United States, China
GSK872 is a laboratory compound manufactured by MedChemExpress. It is a chemical reagent intended for use in research applications. The core function of GSK872 is as a kinase inhibitor, specifically targeting the Mixed Lineage Kinase Domain-Like (MLKL) protein. Further details on the intended use or applications of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Nec 1, by Selleck Chemicals (2 mentions)
Z vad fmk, by MedChemExpress (2 mentions)
Necrosulfonamide, by Selleck Chemicals (1 mentions)
3 methyladenine, by MedChemExpress (1 mentions)
Vx 765, by MedChemExpress (1 mentions)
Vitamin e, by MedChemExpress (1 mentions)
Doxorubicin, by Cayman Chemical (1 mentions)
Staurosporine, by Cayman Chemical (1 mentions)
Pf 3644022, by Bio-Techne (1 mentions)
Gemcitabine, by Selleck Chemicals (1 mentions)
Nec 1, by Enzo Life Sciences (1 mentions)
Lps escherichia coli o55 b5, by Merck Group (1 mentions)
Etoposide, by Merck Group (1 mentions)
Bms345541, by Axon Medchem (1 mentions)
Zvad fmk, by Bachem (1 mentions)
α tubulin, by Merck Group (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Necrostatin, by Selleck Chemicals (1 mentions)
Vcam 1, by Santa Cruz Biotechnology (1 mentions)
3 methyladenine, by Merck Group (1 mentions)
11 protocols using gsk872
1
Caco-2 Cell Viability Assay
2
Kinase Inhibitor Library for Cell Treatments
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The kinase inhibitor library, composed of 149 selective or broad-spectrum kinase inhibitors dissolved in DMSO (10 mM stock concentration), was purchased from Cayman Chemicals (#10505). The following reagents were used for cell treatments at given concentrations: LPS (Escherichiacoli O55:B5, #L2880, Sigma-Aldrich, 100 ng ml−1), recombinant human TNFα (rHuTNF, #50435.50, Biomol, 10 ng/ml), Birinapant/SM (#HY-16591, MedChem Express, 1 µM), pan-caspase inhibitor zVAD-fmk (#4026865.0005, Bachem, 25 μM), MK2 inhibitor PF3644022 (#4279, Tocris, 5 μM), RIPK1 inhibitor Nec-1 (#BML-AP309-0020, Enzo Life Sciences, 50 µM), RIPK1 inhibitor Nec-1s (# 10-4544-5 mg, Tebu-Bio, 50 µM), RIPK3 inhibitor GSK872 (#HY-101872, MedChem Express, 5 µM), IKKβ inhibitor BMS345541 (#Axon 1731, Axon Medchem, 5 µM), TBK1/IKKε inhibitor BX795 (#T1830, Tebu-Bio), 5-Iodotubercidin (#HY-15424, MedChem Express, 2.5–10 µM), ABT-702 dihydrochloride (#HY-103161, MedChem Express, 5 µM), Staurosporine (#81590, Cayman, 5 µM), Etoposide (#E1383, Sigma, 5 µM), Doxorubicin (#15007, Cayman, 5 µM), Gemcitabine (#S1714, Selleckchem, 100 nM).
3
Zebrafish Inflammatory Model and RIPK3 Inhibition
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To establish the inflammatory model, 3 dpf zebrafish larvae were anesthetized with 0.02% tricaine and yolk-microinjected with 1 nL LPS (1 mg/mL) per larva on an agarose plate as reported previously [42 (link)]. PBS-injected larvae were used as the control group.
Four different Ripk3 inhibitors (GSK872, GSK840, GSK843, and RIPK3-IN-1) [43 (link), 44 (link)] were purchased from MedChemExpress, USA. The concentrations of these inhibitors used were listed in Table1 . When treated with Ripk3 inhibitors, WT larvae were soaked in egg water with inhibitors from 2 dpf. The larvae were injected with PBS or LPS at 3 dpf and further soaked in egg water with inhibitors until collected.
Four different Ripk3 inhibitors (GSK872, GSK840, GSK843, and RIPK3-IN-1) [43 (link), 44 (link)] were purchased from MedChemExpress, USA. The concentrations of these inhibitors used were listed in Table
The concentrations of RIPK3 inhibitors used.
| RIPK3 inhibitor | Teratogenic concentration | Experimental concentration |
|---|---|---|
| GSK872 | 100 μM | 30 μM |
| GSK840 | 20 μM | 10 μM |
| GSK843 | 5 μM | 2.5 μM |
| RIPK3-IN-1 | 30 μM | 20 μM |
The used concentrations of RIPK3 inhibitors were listed. The teratogenic concentrations represent the minimal concentrations that would induce larval deformity, while the experimental concentrations represent the final applied concentrations in this study.
4
Lyso-Gb3 and Cell Death Signaling
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Lyso-Gb3 (lyso-Ceramide trihexoside) was purchased from Matreya, LLC. Necrostatin (a RIP1 inhibitor) was purchased from Selleckchem. 3-methyladenine (3-MA, an autophagy inhibitor) and dimethyl sulfoxide were supplied by Sigma. GSK-872 (a RIP3 inhibitor) were obtained from MedChemExpress. Antibodies were purchased from the following vendors, as follows: VCAM-1, ICAM-1, and RIP3 (Santa Cruz Delaware); MLKL (Millipore); LC3 and beclin-1 (Cell Signaling Technology); RIP1 (Invitrogen); and α-tubulin (Sigma).
5
Investigating Cell Death Mechanisms
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Minocycline hydrochloride, Nec-1s, GSK-872, and Z-VAD-FMK were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Anti-HMGB1 antibody and anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor 488 Conjugate) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-ZO-1, anti-RIPK3 and anti-MLKL antibodies were purchased from Proteintech (Rosemont, IL, USA). Mitochondrial membrane potential assay kit with JC-1 and ROS assay kit were purchased from Beyotime Biotechnology (Shanghai, China). DMEM/F12 and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum was purchased from Gibco (Logan, UT, USA). Dead cell apoptosis kit with Annexin V Alexa Fluor 488 & Propidium Iodide (PI), goat anti-rabbit IgG (H+L) (Alexa Fluor 488), and goat anti-mouse IgG (H+L) (Alexa Fluor 568) were purchased from Invitrogen (Carlsbad, CA, USA).
6
Restoring Phenotype of Ddb1-Deficient CD4+ T Cells
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Primary CD4+ T cells were cultured in RPMI-1640 medium supplemented with 10% FBS, HEPES (10 mM), sodium pyruvate (1 mM), β-mercaptoethanol (50 μM), penicillin, and streptomycin. Naïve CD4+ T cells were isolated from spleens and lymph nodes of 8-week-old WT and Ddb1fl/fl; OX40-cre mice, and cells were co-cultured with Antigen Presenting Cells (APC cells) in 96-well U bottom plate with soluble 1 μg/ml anti-mouse CD3 and 1 μg/ml anti-mouse CD28 antibodies. CD4+ T cells were activated for 0–6 days and harvested for flow cytometry analysis or western blotting.
Experiment to restore the phenotype of Ddb1 deficient activated CD4+ T cells and CD4+ T cells were treated with various inhibitors in CD4+ T-cell stimulation assay. Z-VAD (cat #HY-16658B), Z-IETD (cat #HY-101297), Ferrostatin-1 (cat # HY-100579), GSK872 (cat #HY-101872), Nec1 (cat # HY-15760), AZD7762 (cat # HY-10992), and Rabusertib (cat # HY-14720) were purchased from MedChemExpress; Nec-1s (cat #S8641) was purchased from Selleck.
Experiment to restore the phenotype of Ddb1 deficient activated CD4+ T cells and CD4+ T cells were treated with various inhibitors in CD4+ T-cell stimulation assay. Z-VAD (cat #HY-16658B), Z-IETD (cat #HY-101297), Ferrostatin-1 (cat # HY-100579), GSK872 (cat #HY-101872), Nec1 (cat # HY-15760), AZD7762 (cat # HY-10992), and Rabusertib (cat # HY-14720) were purchased from MedChemExpress; Nec-1s (cat #S8641) was purchased from Selleck.
7
Necroptosis Inhibition Assay
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GSK’872, BAY 11–7082, and MITO-TEMPO were purchased from Medchem Express (USA). Necrostatin-1 (NEC-1) and RBC lysing buffer were from Sigma-Aldrich (USA). Antibodies against RIPK3, RIPK1, p-P65, and actin were from Cell Signaling Technology and Abcam. RIPK3, RIPK1 and p-P65 were diluted at 1:1000 and actin were at 1:2500. Goat anti-Mouse and Goat anti-Rabbit secondary antibodies (diluted at 1:2500) were from Erath. Recombinant mouse M-CSF were from R&D Systems.
8
Emodin Modulates Cell Death Pathways
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Dulbecco’s modified Eagle’s medium/nutrient mixture F12 (DMEM/F12), fetal bovine serum (FBS), and dimethyl sulfoxide (DMSO) were purchased from GIBCO (Grand Island, NY, USA). Emodin (CAS No. 518–82-1, HPLC>98%) was obtained from Dalian Meilun Biotechnology Co. Ltd. (Dalian, China). RNAiso plus, SYBR Premix Ex Taq II and ROX reference dye were purchased from TAKARA (Dalian, China). All primers were designed and synthesized by TSINGKE (Wuhan, China). The following antibodies for western blot analysis were purchased from ABclonal Biotechnology Co., Ltd. (Wuhan, China): GAPDH; caspase-3; caspase-8; TNF-α; RIP1; RIP3; MLKL; and HRP-labeled goat anti-rabbit IgG. Emodin was dissolved in DMSO as a stock solution and stored in a dark bottle at 4 °C. Nec-1 (CAS No.: 4311-88-0) and GSK872 (CAS No.:1346546–69-7) were purchased from MedChemExpress (Shanghai, China). The FITC Annexin V Apoptosis Detection Kit I (556547) was purchased from BD (USA). CCK-8 was purchased from Dojindo (Japan). The Apoptosis and Necrosis Assay Kit, Cell Cycle and Apoptosis Kit, and LDH Assay Kit were all purchased from Beyotime (China).
9
Spinal Cord Injury Rat Model
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This experimental object was the male SD rats (6–7 weeks, 210–260 g) provided by the Shandong University Laboratory. All experimental rats were fed with a 12-h light/dark cycle at 25°C and had free access to rodent water and food. The Animal Care and Use Committee at Shandong University approved our experimental designs and operation procedures.
All rats were randomly divided into four groups (n = 4–7 per group for various analyses): sham group, SCI group, vehicle group, GSK872 group. In the sham group, only the vertebral lamina was removed without SCI; In the vehicle group, rats with SCI received an intrathecal injection of 10% dimethyl sulfoxide (DMSO) (Cell Signaling Technology, USA); In the GSK872 group, rats with SCI received an intrathecal injection of GSK872 (Med Chem Express, China). GSK872 was dissolved in DMSO.
All rats were randomly divided into four groups (n = 4–7 per group for various analyses): sham group, SCI group, vehicle group, GSK872 group. In the sham group, only the vertebral lamina was removed without SCI; In the vehicle group, rats with SCI received an intrathecal injection of 10% dimethyl sulfoxide (DMSO) (Cell Signaling Technology, USA); In the GSK872 group, rats with SCI received an intrathecal injection of GSK872 (Med Chem Express, China). GSK872 was dissolved in DMSO.
10
TREM-1 Activation in Macrophages
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To activate TREM-1 in vitro, we coated 24-well plates with the agonist anti-TREM-1 mAbs (10 μg/mL, Mab1187, R&D Systems, USA) at 37 ℃ overnight and washed twice with PBS. Then purified macrophages (0.5 × 106 cells/well in RPMI 1640) or macrophages pre-treated with the RIPK3 inhibitor (GSK872, 1 μM, Medchem Express, USA), the DRP1 inhibitor (Mdivi-1, 100 nM, Medchem Express) or the mTOR inhibitor (Rapamycin, 100 nM, Beyotime, Jiangsu, China) were added. Cells were cultured for 12 h or 24 h and harvested for gene or protein detection.
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