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8 protocols using ch223191

1

AHR Regulation of Intestinal Barrier

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Human colorectal adenocarcinomas (Caco-2) cells obtained from the American Type Culture Collection (Invitrogen, Manassas, VA, United States) were cultured in Eagle’s Minimum Essential Medium supplemented with 10% heat-inactivated fetal bovine serum (Gemini Bioproducts, Calabasas, CA, United States) and 1% non-essential amino acids. Caco-2 cells were seeded on six-well plates at a density of 1 × 106 cells/well. Once the monolayers reached 70–80% confluence, they were cultured with serum-free MEM basic media overnight and then co-cultured with 1 × 105 CFU/mL concentration of C. albicans for 24 h, with or without 10 μM KynA (Selleck, United States), 100 nM FICZ (CAS No:172922-91-7, MeChemExpress, United States), and 10 μM CH223191 (CAS No:301326-22-7, MedChemExpress, United States), to examine the expression of AHR, CYP1A1, MLCK-pMLC, MK2-p-MK2, ZO-1, and occludin proteins.
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2

Regulation of Cellular Signaling Pathways

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All chemicals, including IS and ketoconazole, were from Sigma-Aldrich (St. Louis, MI, USA) unless otherwise stated. IS was used at concentrations of 2 μM and 4 μM, which correspond to the normal human serum concentration of IS [53 (link),95 (link),96 (link)]. The aryl hydrocarbon receptor (AHR) inhibitor, CH223191, was obtained from MedChemExpress (MCE, Monmouth Junction, NJ, USA) and was applied at a concentration of 10 μM. Pregnane X receptor (PXR) downstream signaling was inhibited using ketoconazole at a final concentration of 25 μM [75 (link),76 (link)].
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3

Metabolite-Mediated SCFA Protection

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For the SCFA protection study, WT and Gpr43/ mice were given 200 mM propionate in drinking water for 3 weeks and then administered 2.5% DSS. To test the effect of Dub-derived metabolites, mice were gavaged with L-lactic acid (Lac; 0.24 mg/kg), ketoleucine (4-MOV; 2.67 mg/kg), N-acetyl-L-Asp (NAA; 250 mg/kg), L-Lys (20 mg/kg), L-α-aminobutyric acid (Abu; 30 mg/kg) daily for 5 days before DSS treatment. For the Kyn protection study, WT or Foxp3-DTR mice were i.p. injected with 10 mg/kg Kyn every other day, 7 total doses from the 6th day previous to DSS to the 6th day post-DSS administration. For the Lys protection study, WT mice, Foxp3-DTR, or Ido1/ mice were given L-Lys (20 mg/kg) via oral gavage once per day for 5 days before DSS administration. For AhR antagonist experiments, WT mice were treated i.p. with CH-223191 (10 mg/kg, MedChemExpress) daily for 10 consecutive days (from day −7 to day 2). During this period, mice were treated with 20 mg/kg Lys via oral gavage for 5 days (from day −5 to day 0) and then administered DSS.
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4

Inflammatory Pathway Modulation Protocol

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Lipopolysaccharide and caerulein Meilinbio (Dalian, China). CH223191, dexamethasone (DEX), and naringenin were purchased from MedChemExpress (New Jersey, United States). NLRP3, AhR, ZO-1, and occludin antibodies were purchased from Abcam (Cambridge, United Kingdom). Antibodies against β-actin and histone H3 were purchased from BOSTER (Wuhan, China). Goat anti-rabbit IgG was purchased from Absin (Wuhan, China).
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5

Investigating Endocrine Disruption Pathways

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BPA, glucose and kynurenine were purchased from Sigma-Aldrich (MO, USA). DHEA, corn oil, CH223191 were purchased from MedChemExpress (NJ, USA). Insulin and 2-NBDG were purchased from Thermo Fisher (MA, USA). siRNAs targeting human AhR and negative control siRNAs (NC) were purchased from RiboBio and transfected into KGN cells using Lipofectamine 2000 Transfection Reagent (Invitrogen).
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6

Investigating Aryl Hydrocarbon and Pregnane X Receptor Modulation

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Chemicals, among them, indolepropionic acid (IPA), glutathione (GSH), N-acetyl-cysteine (NAC), Mito-TEMPO, and ketoconazole were from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. IPA was used, 0.4 μM and 0.8 μM, which corresponds to the normal human serum concentration of IPA [40 (link),49 (link),50 (link)]. GSH and NAC antioxidants were used at a final concentration of 5 mM. The mitochondria-targeted antioxidant Mito-TEMPO was used at a concentration of 5 μM. The aryl hydrocarbon receptor (AHR) inhibitor, CH223191, was obtained from MedChemExpress (MCE, Monmouth Junction, NJ, USA) and was applied at a concentration of 10 μM. Pregnane X receptor (PXR) downstream signaling was inhibited using ketoconazole at a final concentration of 25 μM [51 (link),52 (link)]. The Silencer Select siRNAs targeting AHR (AHR—siRNA ID: s1198) and PXR (NR1I2—siRNA ID: s16910) and the negative control siRNA #1 (cat.no. 4390843) were obtained from Thermo Fisher Scientific (Waltham, MA, USA) and each was used at a final concentration of 30 nM.
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7

Indole-3-acetic acid and LPS Inflammation

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Indole-3-acetic acid and LPS (lipopolysaccharides from Escherichia coli O55:B5) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Tin protoporphyrin IX (SnPP) was obtained from Cayman Chemical (Ann Arbor, MI, USA). CH-223191 was purchased from MedChem Express (Monmouth Junction, NJ, USA). Dulbecco’s modified Eagle’s medium (DMEM) was from Biological Industries (Kibbutz Beit Haemek, Israel). Fetal bovine serum (FBS) was purchased from Hyclone (South Logan, UT, USA). Cell counting kit (CCK-8), TRIzol RNA extraction reagent, nitric oxide assay kit, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and Hoechst 33,342 were obtained from Applygen Technologies Inc. (Beijing, China). Hieff® qPCR SYBR® Green Master Mix and Hifair® II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) were procured from Yeasen biotech Co. (Shanghai, China). Antibody against β-actin was from Santa Cruz Bio. (Santa Cruz, CA, USA). Anti-HO-1 was purchased from ABclonal Inc. (College Park, MD, USA). Anti-NF-κB p65 was from Beyotime (Shanghai, China). ECL Western blotting detection kit, horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG), and anti-mouse IgG were purchased from Huaxingbio. (Beijing, China). ELISA kits for IL-1β, IL-6, TNF-α, and MCP-1 were bought from ExCell Biotech (Taicang, Jiangsu, China).
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8

Analyzing Basal IDO1 Levels in CLL

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To analyze basal IDO1 levels, CD19+ CLL cells were serum starved for 1 h in RPMI-1640 at 37°C prior to RNA extraction. In all other experiments, CD19+ CLL cells were resuspended in RPMI-1640 medium + 10% FBS. To mimic microenvironmental stimulation, CLL cells were treated with one of the following soluble factors: IFN-γ 500 U/ml (PeproTech Cat# 300-02, Rocky Hill, NJ, USA); LPS 5 μg/ml (Sigma-Aldrich Cat# L5293, St. Louis, MO, USA); goat F(AB′)2 fragment to human IgM (5FCµ) 10 μg/ml (Thermo Fisher Scientific Cat# ICN55055, Waltham, MA, USA); Type B CpG oligonucleotides 1 μg/ml (ODN 2006) (InvivoGen Cat# tlrl-2006, San Diego, CA, USA); and CD40L 200 ng/ml + interleukin (IL)-4 20 ng/ml (both from PeproTech Cat# 310-02 and 200-04) or tumor necrosis factor (TNF)-α 5 ng/ml (PeproTech Cat# 300-01A). Control cells were cultured in parallel without stimulation. L-Kynurenine (Sigma-Aldrich Cat# K8625) was used at 100 μM. INCB018424 (ruxolitinib) (SelleckChem Cat# S1378, Houston, TX, USA) was used at 0.1 and 1 μM as previously described (30 (link)). CH-223191 (MedChemExpress Cat# HY-12684, Princeton, NJ, USA) was used at 10 μM. ABT-199 (venetoclax) (SelleckChem Cat# S8048) was used at 1 nM. AMG-176 (MedChemExpress Cat# HY-101565) was used at 100 or 300 nM.
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