Ketamine-induced 7-day-old neurotoxicity rats were produced according to a previously reported method [31 (link), 32 (link)]. All rats were randomized into six groups:
Vx 765
Manufactured by MedChemExpress
Sourced in United States, China
VX-765 is a laboratory product manufactured by MedChemExpress. It is a small molecule compound used for research purposes. The core function of VX-765 is to inhibit the activity of the enzyme caspase-1, which plays a role in the inflammatory response.
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Lab products found in correlation
Hy 13205, by MedChemExpress (3 mentions)
Mcc950, by MedChemExpress (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Z vad fmk, by MedChemExpress (2 mentions)
Fluperlapine, by MedChemExpress (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Hy 17589a, by MedChemExpress (1 mentions)
Hy 19312, by MedChemExpress (1 mentions)
Hy b0215, by MedChemExpress (1 mentions)
G9422, by Merck Group (1 mentions)
Hy 10219, by MedChemExpress (1 mentions)
Irisin, by Phoenix Pharmaceuticals (1 mentions)
Movas 1, by American Type Culture Collection (1 mentions)
Hy 12815a, by MedChemExpress (1 mentions)
Necrosulfonamide, by Selleck Chemicals (1 mentions)
Nec 1, by Selleck Chemicals (1 mentions)
3 methyladenine, by MedChemExpress (1 mentions)
Vitamin e, by MedChemExpress (1 mentions)
23 protocols using vx 765
1
Ketamine-Induced Neurotoxicity Rat Model
2
Modulating Vascular Smooth Muscle Cell Calcification
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The mouse vascular smooth muscle cell line (MOVAS-1) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in DMEM (Hyclone, Logan, UT, USA) containing 10% FBS (Gibco, Waltham, MA, USA) and 1% (v/v) penicillin/streptomycin at 37 °C in a humidified incubator with 5% CO2. Cells between passages 6 and 8 were used for all experiments. VSMC calcification was induced according to previous protocols [20 (link), 21 (link)]. Briefly, VSMCs were cultured with growing medium in the presence of β-glycerophosphate (β-GP) (10 mM; G9422; Sigma, St. Louis, MO, USA) for 7 days. To investigate the role of Irisin in VSMC calcification, Irisin (067-29; Phoenix Pharmaceuticals Inc, Burlingame, CA, USA) at different concentrations (50 or 100 ng/ml dissolved in PBS) was used to treat VSMCs in the presence of β-GP medium for 7 days with medium changes every 2–3 days. For pharmacological treatment, the CASP1 inhibitor VX-765 (10 μM; HY-13205), NLRP3 inhibitor MCC950 (100 μM; HY-12815A), ROS scavenger N-acetyl-L-cysteine (NAC, 20 μM; HY-B0215), autophagy inducer rapamycin (200 nM; HY-10219), autophagy inhibitor 3-methyladenine (3-MA, 5 mM; HY-19312), and chloroquine (CQ, 25 μM; HY-17589A) were purchased from MedChem Express (NJ, USA) and used to treat VSMCs according to the experimental design.
3
Caco-2 Cell Viability Assay
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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.
4
Traumatic Brain Injury Mouse Model
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In this study, we purchased adult male C57BL/6 mice (8-10 weeks old, 20-25 g) from the Shanghai Slaccas Experimental Animal Limited Liability Company (Shanghai, China). Under 12 h of light and dark circulation, the mice were placed in animal care facilities and were free to access food and water. The experiment was carried out under the supervision of Wenzhou Medical University. Mice were isolated and reared for 1 week and then randomized into four groups: sham surgery, controlled cortical impact (CCI), CCI+vehicle (DMSO), and CCI+VX765. VX765 (catalog number: HY-13205; MedChemExpress, Monmouth Junction, NJ, USA) dissolved in DMSO was injected intraperitoneally (100 mg/kg) 1 h after CCI and every 2 days after CCI for two weeks [33 (link), 35 (link)]. At the same time point, the CCI+vehicle group was given an equal volume of DMSO. In this study, we followed the methods of Xu et al. [36 (link)].
5
Flow Cytometry Strategy for B Cell Subsets
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A flow cytometry gating strategy for B cell subsets was performed as previously described (21 (link)). All antihuman fluorochromes were obtained from BD Biosciences. Harvested PBMCs were stained for 30 min at 4°C using CD19 (APC-H7; HIB19), CD10 (PerCP-Cy5.5; HI10a), CD21 (PE-Cy7; B-ly4), CD27 (BV510; L128), CD95 (PE; DX2), and the corresponding isotype controls with or without caspase-3 (Z-DEVD-FMK [Sigma-Aldrich, St. Louis, MO]) and caspase-1 (VX-765 [MedChem Express, Monmouth Junction, NJ]) inhibitors. When conducting in vitro inhibitor experiments, PBMCs were transferred to preheated complete medium (Roswell Park Memorial Institute 1640/10% fetal calf serum) and treated with Z-DEVD-FMK (50 μM) or VX-765 (2 μM), then cultured at 37°C for 4 h before harvesting cells.
6
Pyroptosis Inhibitor Modulates AZU1 Sensitivity in TNBC
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MDA-MB-231 (TNBC), MCF-7 (luminal A) and BT-549 (TNBC) cell lines were cultured in high-sugar DMEM or RPMI-1640 containing 10% fetal bovine serum (FBS; all from Zhejiang Ginocell Biological Technology Co., Ltd.; www.genomcell.com ) plus 1% streptomycin/penicillin at 37°C in an environment of 5% CO2. Subsequently, the cells were pre-treated with 50 µM pyroptosis inhibitor (VX765; MedChemExpress) which was prepared in DMSO and was further diluted in cell culture medium for 2 h, followed by the exposure to various concentrations (0, 250, 500 and 1,000 ng/ml) of AZU1 (Beijing Solarbio Science & Technology Co., Ltd.) for 24 h at 37°C in an environment of 5% CO2. Then, 10 µl of Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology) reagent was added to each well and incubated at 37°C for 2 h to detect the viability of BC cell lines.
7
Priming and Activation of ASCs
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ASCs were primed with 0.5 μg/ml LPS for 4 h and then transfected with 1 μg/ml LPS, 0.25 μg/ml ultrapure flagellin, 2 μg/ml dsDNA, or 10 μM nigericin, plus 0.025% v/v FuGENE HD. In addition, LPS-primed ASCs were also incubated with the specific supernatants collected from macrophages or 3T3 cells for 2 h. The caspase-1 inhibitor VX765 (HY-13205; MedChemExpress) or caspase pan-inhibitor z-VAD-FMK (HY-16658; MedChemExpress) pretreated ASCs before priming of LPS. In the IFNβ (B276240; BioLegend) treatment assay, ASCs were primed with LPS (0.5 μg/ml) and IFNβ (0.4 ng/ml) for 4 h followed by related stimulation.
8
Hemin-Induced Differentiation of K562 Cells
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K562 cells (CRL‐3343; American Type Culture Collection) were maintained in RPMI culture medium supplemented with 10% fetal calf serum (FCS), 2 mM Glutamine, and 1% penicillin–streptomycin (Life Technologies). They were tested for mycoplasma contamination and authenticated by STR profiling. Cells were maintained and subcultured before confluence every 72 h. For differentiation, cells were treated with 50 μM hemin (#16009‐13‐5, Sigma‐Aldrich), prepared as previously described (Smith et al, 2000 (link)), in the presence of 0.1% DMSO alone or with the caspase‐1 inhibitor VX‐765 (100 μM), the tyrosine kinase inhibitor nilotinib (0.1 μM), the RNA polymerase I inhibitor CX‐5461 (200 nM, 1 μM and 10 μM) (all from MedChemExpress). Cells were collected at different time points (0, 24, 48 h post‐hemin addition), centrifuged, washed twice with PBS, and stored at −80°C for further analysis.
9
Murine Macrophage Culture and Stimulation
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The mouse peritoneal macrophages (MPMs) extracted from C57BL/6 female mice and MH-S cells and were cultured routinely in Roswell Park Memorial Institute (RPMI) medium (HyClone) supplemented with 10% fetal bovine serum (FBS), l -glutamine, and penicillin. Immortalized bone marrow-derived macrophage (iBMDM) cells and Raw264.7 cells were cultured routinely in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone) supplemented with 10% FBS, l -glutamine, and penicillin. All cells were cultured in antibiotic-free medium before PA stimulation or inhibitor pretreatment. The cells were pretreated with VX-765 (HY-13205, Med Chem Express), Nig (HY-13205, Med Chem Express), IL-1β (HY-P7073, Med Chem Express), IL-1β receptor inhibitor (TLR-1, HY-W011400, Med Chem Express), AKT inhibitor VIII (HY-10355, Med Chem Express), or IFN-β (CAT 300-02BC, PeproTech) for the indicated times before stimulation with PA, as indicated; multiplicity of infection (MOI) values are given for the different times.
10
Therapeutic Effects of Caspase-1 Inhibitor in Rat MCAO Model
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All animal studies were conducted according to the guidelines of the Animal Care and Use Committee of Jinan University. Sprague–Dawley rats aged 12 months and weighing between 350 and 500 g were supplied by the Guangdong Medical Laboratory Animal Center. The rats were anesthetized using 10% chloral hydrate (3 ml/kg, BBI, China) (i.p.) solution, and a distal middle cerebral artery occlusion (dMCAO) model was established as previously described (Bederson et al. 1986 (link)). Sham-operated rats underwent the same surgical procedures except dMCAO electrocoagulation. At 1 h after dMCAO, the caspase-1 inhibitor VX-765 (50 mg/kg, MedChem Express, USA) was intraperitoneally injected once daily for 28 consecutive days after dMCAO as previously described (Flores et al. 2018 (link)). SD rats were randomly assigned to 3 groups: (1) the sham group, (2) the MCAO + vehicle group, and (3) the MCAO + VX-765 therapy group. Cognitive and learning function was determined 24 d after dMCAO in the Morris water maze test.
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