Ch223191

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).

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).

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.

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).

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.