Journal: Frontiers in Oncology

Article Title: Long-term exposure to BAY2416964 reduces proliferation, migration and recapitulates transcriptional changes induced by AHR loss in PyMT-induced mammary tumor cells

doi: 10.3389/fonc.2024.1466658

Figure Lengend Snippet: BAY2416964 inhibits kynurenine-induced AHR activity in PyMT mouse mammary cancer cells. Kynurenine increased Cyp1a1 (A) , Cyp1b1 (B) and Parp7 (C) mRNA levels in a dose-response manner in PyMT cells as measured by RT-qPCR. Relative mRNA levels of Cyp1a1 (D) and Cyp1b1 (E) after dose-response treatment with BAY2416964. Cyp1a1 (F) and Cyp1b1 (G) mRNA levels after treatment with increasing doses of GNF351. Relative Cyp1a1 and Cyp1b1 mRNA levels after treatment with increasing amounts of BAY2416964 (H, I) or GNF351 (J, K) in the presence of 100 µM kynurenine. RT-qPCR results are generated by samples treated 6 h with test compounds and presented as mean ± S.E.M n=3. (L) Western blot of PyMT cells treated for 6 h with kynurenine, BAY2416964 or GNF351 at the concentrations indicated. Representative image n=3. (M) Quantification of western blot. (N) AHR protein in cytoplasmic and nuclear fractions after 6 h treatment with BAY2416964 or GNF351. Representative image n=3. (O) Protein quantification of cytoplasmic AHR relative to loading control (Tubulin). (P) Protein quantification of nuclear AHR relative to loading control (Lamin A/C). *p<0.05 compared with control (DMSO).

Article Snippet: BAY2416964 and GNF351 were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Activity Assay, Quantitative RT-PCR, Generated, Western Blot, Control

Journal: Frontiers in Oncology

Article Title: Long-term exposure to BAY2416964 reduces proliferation, migration and recapitulates transcriptional changes induced by AHR loss in PyMT-induced mammary tumor cells

doi: 10.3389/fonc.2024.1466658

Figure Lengend Snippet: Loss of AHR prevents increases in kynurenine-induced AHR target genes and reduces proliferation of PyMT cells. (A) Ahr mRNA levels in PyMT Ahr KO cells compared with WT cells as determined by RT-qPCR. Significance was determined with Student’s t-test, n=3 (B) Western blot of WT and Ahr KO cells detected no AHR protein in the PyMT Ahr KO cell line. Representative image of n=3. AHR activity was determined in the PyMT WT and Ahr KO cell lines by treatment with 100 µM kynurenine, 10 µM BAY2416964 or combination for 6 hours. Kynurenine and high concentration of BAY2416964 failed to increase Cyp1a1 (C) , Cyp1b1 (D) and Parp7 (E) expression levels in Ahr KO cells. RT-qPCR results are presented as mean ± S.E.M. of n=3. Significance was determined using Two-Way ANOVA, p<0.05. * Significant from PyMT WT control (DMSO). (F) Ahr KO cells proliferate more slowly than WT cells. (G) BAY2416964 did not affect proliferation of PyMT WT cells. (H) Proliferation of PyMT WT cells treated with increasing doses of GNF351. Treatment with 10 µM GNF351 decreased proliferation of PyMT WT cells. Proliferation of Ahr KO cells treated with increasing concentrations of BAY2416964 (I) or GNF351 (J) . Only 10 µM GNF351 affected proliferation of Ahr KO cells compared to DMSO control. WT was added for comparison. Data are presented as mean ± S.E.M of n=12, measured by IncuCyte. Significance was determined using Area under curve with p<0.05.

Article Snippet: BAY2416964 and GNF351 were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Quantitative RT-PCR, Western Blot, Activity Assay, Concentration Assay, Expressing, Control, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: Inducible CD147 up-regulation boosts extended SARS-CoV-2 infection triggering severe COVID-19 independent of ACE2

doi: 10.1038/s41392-025-02551-x

Figure Lengend Snippet: SARS-CoV-2-induced CD147 expression is regulated by transcription factor AHR. a The expressions of CD147 and ACE2 in BEAS-2B cells were determined by western blot. b The expressions of CD147, AHR, AHRR, and CYP1A1 in BEAS-2B cells were determined by western blot with SARS-CoV-2 pseudovirus infection or RBD incubation. c The expression of CD147 was determined in BEAS-2B cells by western blot with the addition of AHR agonist 2 or AHR antagonist 5. d The relative luciferase signals in pseudovirus-infected BEAS-2B cells were determined by dual-luciferase reporter assays with the addition of AHR agonist 2 or AHR antagonist 5, *** p < 0.001. e The expression of CD147 in BEAS-2B-shCD147 cells and its control was determined by western blot. f The transfection efficiency of plasmid AHR was evaluated by western blot in BEAS-2B-shCD147 cells. g The relative luciferase signals were determined by dual-luciferase reporter assays in pseudovirus-infected BEAS-2B cells and BEAS-2B-shCD147 cells with or without the transfection of plasmid AHR, ns, not significant, *** p < 0.001. h The expression of CD147 was determined in AHR knockdown BEAS-2B cells by western blot. i The expression of CD147 was determined in pseudovirus-infected BEAS-2B cells by western blot with the addition of AHR antagonist 5. j Dual-luciferase reporter assays were performed after co-transfecting of the pGL3-Basic-CD147 promoter (−2000– − 1), pRL-TK, and plasmid AHR into HEK293T cells. The relative luciferase signal was detected using a Dual-Luciferase® Reporter Assay System, *** p < 0.001. k Nuclear and cytoplasmic protein extraction assays were performed to determine the nuclear expression and cytoplasmic expression of AHR in BEAS-2B cells with pseudovirus infection or RBD incubation. Lamin B1 and GAPDH were selected as controls for nuclear protein and cytoplasmic protein, respectively

Article Snippet: AHR agonist 2 (HY-144339, MCE) and AHR antagonist 5 (HY-141609, MCE) are potent agonist and inhibitor of AHR, respectively, which can regulate the transcription of AHR’s downstream genes by affecting its nuclear localization.

Techniques: Expressing, Western Blot, Infection, Incubation, Luciferase, Control, Transfection, Plasmid Preparation, Knockdown, Reporter Assay, Protein Extraction

Journal: Toxicology Reports

Article Title: Characterization of marine-derived halogenated indoles as ligands of the aryl hydrocarbon receptor

doi: 10.1016/j.toxrep.2022.05.016

Figure Lengend Snippet: Antagonism of CYP1A1 catalytic activity induced by halogenated indoles. HepG2 cells were treated with 10 µM of the halogenated indoles for 24 h both alone and in combination with the AhR antagonist, GNF-351 (0.25 µM). CYP1A1 activity was determined via the EROD assay and is expressed as a percentage of the vehicle control (0.5% DMSO). Bars represent the mean ± SEM of three independent experiments performed in duplicate. Data were analyzed using a one-tailed t-test. *significantly decreased compared to the respective indole-only response, p < 0.05.

Article Snippet: 2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD, 50 μg/mL in DMSO, >99.9%) was purchased from Cambridge Isotope Laboratories (Tewksbury, MA) and GNF-351 was purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Activity Assay, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: The Aryl Hydrocarbon Receptor in Energy Balance: The Road from Dioxin-Induced Wasting Syndrome to Combating Obesity with Ahr Ligands

doi: 10.3390/ijms22010049

Figure Lengend Snippet: Current areas of research regarding the aryl hydrocarbon receptor (AHR) and energy metabolism. Current research interests include: ( A ) the role of the AHR in regulating FGF21 and adiposity; ( B ) how the AHR influences liver metabolism; ( C ) interactions between the AHR and circadian proteins, and their effect on glucose metabolism and energy balance; ( D ) the use of AHR antagonists to treat obesity and other metabolic disorders; ( E ) the relationship between the AHR, gut microflora, and the compounds excreted by these microorganisms; and ( F ) the role of the AHR in regulating adipocyte differentiation and adipogenesis.

Article Snippet: AHR antagonists have since exhibited such success that a few have found themselves in the drug pipelines of pharmaceutical companies, including Bayer, Magenta Therapeutics, Celgene, and JAGUAHR Therapeutics.

Techniques:

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Role of the Aryl Hydrocarbon Receptor in Sugen 5416–induced Experimental Pulmonary Hypertension

doi: 10.1165/rcmb.2017-0260OC

Figure Lengend Snippet: Effect of the AhR antagonist CH223191 on Su/Hx pulmonary hypertension in female rats. (A) Right ventricular systolic pressure (RVSP; n = 5–6 per group), (B) right ventricular hypertrophy (RV/[LV+S]; n = 8 per group), and (C) the percentage of remodeled arteries in lungs without treatment (control) or with CH223191 alone, Su/Hx treatment with vehicle, or CH223191; n = 5–6. (D) Representative images showing elastic laminae stained with elastin/Picrosirius red. Scale bar: 20 μm. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 as indicated, determined by one-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: Stimulation with Sugen increased proliferation in BOECs ( A ); however, both the AhR antagonist CH223191 (1 μM) and Sugen 5416 (1 μM) reduced cell viability in BOECs by Trypan blue exclusion ( B ), n = 3, repeated three times.

Techniques: Control, Staining

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Role of the Aryl Hydrocarbon Receptor in Sugen 5416–induced Experimental Pulmonary Hypertension

doi: 10.1165/rcmb.2017-0260OC

Figure Lengend Snippet: Effect of the AhR antagonist CH223191 on the protein expression of AhR, CYP1A1, and AhR nuclear translocator (ARNT) in female rat lungs. (A) AhR expression (n = 4), (B) CYP1A1 expression, and (C) ARNT expression, with (D) representative immunoblots (n = 4–6). (E) Representative CYP1A1 immunostaining in pulmonary arteries from rats. Scale bar: 50 μm. (F) Representative AhR immunostaining in pulmonary arteries from rats. Scale bar: 50 μm. Data are shown as mean ± SEM. *P < 0.05 as indicated, determined by one-way ANOVA followed by Bonferroni’s post hoc test. HIF = hypoxia-inducible factor.

Article Snippet: Stimulation with Sugen increased proliferation in BOECs ( A ); however, both the AhR antagonist CH223191 (1 μM) and Sugen 5416 (1 μM) reduced cell viability in BOECs by Trypan blue exclusion ( B ), n = 3, repeated three times.

Techniques: Expressing, Western Blot, Immunostaining

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Role of the Aryl Hydrocarbon Receptor in Sugen 5416–induced Experimental Pulmonary Hypertension

doi: 10.1165/rcmb.2017-0260OC

Figure Lengend Snippet: Sugen stimulates the proliferation of blood outgrowth endothelial cells (BOECs) from female patients with PAH. Stimulation with Sugen increased proliferation in BOECs (A); however, both the AhR antagonist CH223191 (1 μM) and Sugen 5416 (1 μM) reduced cell viability in BOECs by Trypan blue exclusion (B), n = 3, repeated three times. Data are displayed as mean ± SEM. *P < 0.05, **P < 0.01 as indicated, determined by one-way ANOVA followed by Bonferroni’s post hoc test. (C) Sugen caused nuclear translocation of AhR after 60 minutes. AhR protein expression was normalized to α-tubulin and nucleoporin as markers for cytosolic and nuclear enrichment, respectively. The data are displayed as mean ± SEM. *P < 0.05 as indicated, determined by area under the curve. (D) Our data suggest that Su may activate AhR nuclear translocation and subsequent activation of CYP1A1, apoptosis, and aromatase expression. The resulting increase in E2 synthesis and metabolism may contribute to experimental PH. We also demonstrate directly that Su and hypoxia synergize, perhaps via ARNT, to cause hPASMC proliferation, suggesting that inhibition of AhR is a potential approach to the treatment of PAH.

Article Snippet: Stimulation with Sugen increased proliferation in BOECs ( A ); however, both the AhR antagonist CH223191 (1 μM) and Sugen 5416 (1 μM) reduced cell viability in BOECs by Trypan blue exclusion ( B ), n = 3, repeated three times.

Techniques: Translocation Assay, Expressing, Activation Assay, Inhibition