ahr inhibitor gnf 351  (MedChemExpress)

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: eLife

Article Title: Uremic toxin indoxyl sulfate induces trained immunity via the AhR-dependent arachidonic acid pathway in end-stage renal disease (ESRD)

doi: 10.7554/eLife.87316

Figure Lengend Snippet: Monocytes were pretreated with or without GNF351 (AhR antagonist; 10 μM) followed by IS (1000 μM)-training for 6 days. ( A ) On day 6, nuclear and cytosol fraction were prepared and immunoblotted for AhR protein. Band intensity in immunoblots was quantified by densitometry. β-ACTIN was used as a normalization control. ( B–D ) On day 6, IS-trained cells with or without GNF351 were restimulated with lipopolysaccharide (LPS) (10 ng/ml), for 24 hr. TNF-α and IL-6 in supernatants were quantified by enzyme-linked immunosorbent assay (ELISA) ( B ). Expression of TNF-α and IL-6 ( C ) and IL-1β, MCP-1, and IL-10 mRNA ( D ) was analyzed by RT-qPCR. ( E ) Monocytes were transfected with siRNA targeting AhR (siAhR) or negative control (siNC) for 1 day, followed by stimulation with IS for 24 hr. After a resting period of 5 days, cells were re-stimulated with LPS for 24 hr. mRNA expression levels of TNF-α and IL-6 were assessed using RT-qPCR. ( F ) Enrichment of H3K4me3 on promoters of TNFA and IL6 loci was assessed on day 6 after IS-training. 1% input was used as a normalization control. n=5 ~ 8. Bar graphs show the mean ± SEM. *=p < 0.05, **=p < 0.01, and ***=p < 0.001 by two-tailed paired t -test. Figure 4—source data 1. Raw data for . Figure 4—source data 2. PDF file containing and the relevant western blot analysis with highlighted bands and sample labels. Figure 4—source data 3. Original image files for all western blot bands analyzed in .

Article Snippet: Chemical compound, drug , GNF351 , Sigma-Aldrich , 182707 , .

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Transfection, Negative Control, Two Tailed Test

Journal: eLife

Article Title: Uremic toxin indoxyl sulfate induces trained immunity via the AhR-dependent arachidonic acid pathway in end-stage renal disease (ESRD)

doi: 10.7554/eLife.87316

Figure Lengend Snippet: ( A ) Monocytes were pretreated with FICZ (100 nM), an AhR agonist, followed by training for 5 days. Cells were restimulated with lipopolysaccharide (LPS) (10 ng/ml) for 24 hr. TNF-α and IL-6 in supernatants were quantified by enzyme-linked immunosorbent assay (ELISA) (n=7 ~ 9). ( B, C ). On day 6, IS-trained cells with or without GNF351 (10 μM) were restimulated with LPS for 24 hr. Glycolysis and mitochondrial stress test were conducted with IS-trained macrophages (n=4 ~ 5) using Seahorse XF-analyzer. Extracellular acidification rate (ECAR) levels were measured after sequential treatment with glucose, oligomycin, and 2-DG. Cellular glycolysis and glycolytic capacity were analyzed ( B ). Oxygen consumption rate (OCR) levels were measured after sequential treatment with oligomycin, FCCP, and Rotenone/antimycin A (Ro/AA). Basal respiration, maximal respiration, and ATP production were analyzed ( C ). ( D ) Monocytes were pretreated with or without GNF351 followed by IS-stimulation for 24 hr. Cell lysates were prepared and immunoblotted for phosphorylated S6K protein. Band intensity in immunoblots was quantified by densitometry. β-ACTIN was used as a normalization control. Bar graphs show the mean ± SEM. *=p < 0.05, **=p < 0.01, and ***=p < 0.001 by two-tailed paired t -test. Figure 4—figure supplement 1—source data 1. Raw data for . Figure 4—figure supplement 1—source data 2. PDF file containing and the relevant western blot analysis with highlighted bands and sample labels. Figure 4—figure supplement 1—source data 3. Original image files for all western blot bands analyzed in .

Article Snippet: Chemical compound, drug , GNF351 , Sigma-Aldrich , 182707 , .

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control, Two Tailed Test

Journal: eLife

Article Title: Uremic toxin indoxyl sulfate induces trained immunity via the AhR-dependent arachidonic acid pathway in end-stage renal disease (ESRD)

doi: 10.7554/eLife.87316

Figure Lengend Snippet: ( A ) RNA-sequencing (RNA-Seq) analysis was performed on IS (1000 μM)-trained monocytes. Volcano plots show differentially expressed genes between IS-trained and non-trained macrophages. ( B ) Functional annotation of upregulated or downregulated genes (FC >±2, p<0.05) in IS-trained macrophages analyzed by Gene Ontology (GO) analysis with the Reactome Gene Set. ( C, D ) Gene Set Enrichment Analysis (GSEA) ( C ) and heatmap ( D ) of genes related to the AA metabolism in IS-trained macrophages compared to non-trained cells or compared to IS-trained macrophages with GNF351 (10 μM) treatment were analyzed. ( E, F ) On day 6 after IS-training with or without GNF351, expression of CYP1B1 , arachidonate 5-lipoxygenase ( ALOX5) , ALOX5 activating protein ( ALOX5AP) , and LTB4R1 mRNAs were quantitated using RT-qPCR ( E ) and cell lysates were prepared and immunoblotted for ALOX5 and ALOX5AP proteins ( F ). Band intensity in immunoblots was quantified by densitometry. β-ACTIN was used as a normalization control. ( G ) Monocytes were transfected with siRNA targeting AhR (siAhR) or negative control (siNC) for 1 day, followed by stimulation with IS for 24 hr. After a resting period of 5 days, mRNA expression level of each gene was assessed using RT-qPCR. ( H ) Monocytes were pretreated with zileuton (ALOX5 inhibitor, 100 μM) and trained with IS for 6 days followed by restimulation with lipopolysaccharide (LPS) (10 ng/ml) for 24 hr. TNF-α and IL-6 in supernatants were quantified by enzyme-linked immunosorbent assay (ELISA). ( I ) A chromatin immunoprecipitation (ChIP) assay was performed in IS-trained macrophages pre-treated with zileuton. 2% input was used as a normalization control. ( J ) The pooled normal serum (NS) from healthly controls (HCs) or uremic serum (US) from patients with end-stage renal disease (ESRD) were used to treat monocytes isolated from HCs for 24 hr at 30% (v/v) followed by resting for 5 days. Expression of ALOX5 , ALOX5AP , and LTB4R1 mRNAs were quantitated using RT-qPCR in trained macrophages with NS or US for 6 days. n=5 ~ 6. Bar graphs show the mean ± SEM. *=p < 0.05, **=p < 0.01, ***=p < 0.001 by two-tailed paired t -test. Figure 5—source data 1. Raw data for . Figure 5—source data 2. PDF file containing and the relevant western blot analysis with highlighted bands and sample labels. Figure 5—source data 3. Original image files for all western blot bands analyzed in .

Article Snippet: Chemical compound, drug , GNF351 , Sigma-Aldrich , 182707 , .

Techniques: RNA Sequencing Assay, Functional Assay, Expressing, Quantitative RT-PCR, Western Blot, Control, Transfection, Negative Control, Enzyme-linked Immunosorbent Assay, Chromatin Immunoprecipitation, Isolation, Two Tailed Test

Journal: eLife

Article Title: Uremic toxin indoxyl sulfate induces trained immunity via the AhR-dependent arachidonic acid pathway in end-stage renal disease (ESRD)

doi: 10.7554/eLife.87316

Figure Lengend Snippet: ( A ) Heatmaps of RNA-sequencing (RNA-seq) analysis between IS (1000 μM)-trained and non-trained macrophages. ( B ) Gene Set Enrichment Analysis (GSEA) of genes related to the leukotriene metabolic process and cyclooxygenase pathway were compared between IS-trained macrophages [IS(T)] and non-trained cells (Control). ( C ) Purified monocytes were pretreated with IS (1 mM), FICZ (100 nM), or KA (0.5 mM) for 1 day, followed by 5 day resting period. mRNA expression of arachidonate 5-lipoxygenase ( ALOX5) and ALOX5 activating protein ( ALOX5AP) was analyzed via RT-qPCR. ( D ) Heatmaps show changes in expression of ALOX5 , ALOX5AP , LTB4R1 , and CYP1B1 of monocytes under the indicated conditions (1t lane: IS-trained macrophages, 2nd lane: peripheral monocytes isolated from end-stage renal disease [ESRD] patients). Comparison of the fold changes of RNA-seq data in the present study and microarray data reported previously (GSE155326). ( E ) Schematic diagram of the AA metabolism and target molecules of inhibitors such as zileuton and U75302. ( F ) Monocytes were pretreated with U75302 (BLT1 inhibitor, 5 μM) and trained with IS for 6 days, followed by restimulation with lipopolysaccharide (LPS) (10 ng/ml) for 24 hr. TNF-α and IL-6 in supernatants were quantified by enzyme-linked immunosorbent assay (ELISA). ( G ) RNA-Seq analysis was performed on IS-trained macrophages pretreated with or without GNF351. Heatmaps of 71 upregulated DEGs including AA metabolism-related genes in IS-trained macrophages [IS(T)] compared to non-trained macrophages (Con) , illustrates their expression changes following GNF351 (10 μM) pre-treatment (IS +G). ( H ) Monocytes were transfected with siRNA targeting ALOX5 (siALOX5) or negative control (siNC) for 1 day, followed by stimulation with IS for 24 hr. After a resting period of 5 days, cells were re-stimulated with LPS for 24 hr. mRNA expression levels of TNF-α and IL-6 were assessed using RT-qPCR. ( I ) Monocytes were pretreated with zileuton (ALOX5 inhibitor, 100 μM) and stimulated with IS for 1 day. Cell lysates were analyzed by immunoblotting. ( J ) The pooled normal serum (NS) from healthly controls (HCs) or uremic serum (US) from patients with ESRD were used to treat monocytes isolated from HCs for 24 hr at 30% (v/v) followed by resting for 5 days. After stimulation with LPS for 24 hr, expression of ALOX5 , ALOX5AP , and LTB4R1 mRNAs were quantitated using RT-qPCR. n=5 ~ 8. Bar graphs show the mean ± SEM. *=p < 0.05 and **=p < 0.01, by two-tailed paired t -test. Figure 5—figure supplement 1—source data 1. Raw data for . Figure 5—figure supplement 1—source data 2. PDF file containing and the relevant western blot analysis with highlighted bands and sample labels. Figure 5—figure supplement 1—source data 3. Original image files for all western blot bands analyzed in .

Article Snippet: Chemical compound, drug , GNF351 , Sigma-Aldrich , 182707 , .

Techniques: RNA Sequencing Assay, Control, Purification, Expressing, Quantitative RT-PCR, Isolation, Comparison, Microarray, Enzyme-linked Immunosorbent Assay, Transfection, Negative Control, Western Blot, Two Tailed Test

Journal: eLife

Article Title: Uremic toxin indoxyl sulfate induces trained immunity via the AhR-dependent arachidonic acid pathway in end-stage renal disease (ESRD)

doi: 10.7554/eLife.87316

Figure Lengend Snippet: IS-induced trained immunity in human monocytes is mediated by epigenetic reprogramming and metabolic rewiring via histone modification H3K4m3 and increased glycolysis and mitochondrial respiration, respectively. Direct interaction of uremic toxin IS with the aryl hydrocarbon receptor (AhR) in human monocytes activates AhR signaling pathways that are involved in enhanced expression of the arachidonic acid metabolism-related genes arachidonate 5-lipoxygenase (ALOX5), ALOX5 activating protein (ALOX5AP), and LTB4R1 and augmented production of TNF-α and IL-6 upon stimulation with lipopolysaccharide (LPS) as secondary stimulus via epigenetic regulation. A pivotal role of each pathway or molecule was confirmed by in vitro assay with inhibitors including GNF351 (an AhR antagonist), zileuton (an ALOX5 inhibitor), U75302 (a BLT1 receptor inhibitor), 2-deoxy-d-glucose (2-DG) (a glycolysis inhibitor), and 5’-methylthioadenosine (MTA) (a non-selective methyltransferase inhibitor). Meanwhile, the AhR-independent mechanism contributes to metabolic rewiring, such as increased glycolysis in IS-trained macrophages, which leads to enhanced proinflammatory responses upon secondary stimulation.

Article Snippet: Chemical compound, drug , GNF351 , Sigma-Aldrich , 182707 , .

Techniques: Modification, Expressing, In Vitro

Journal: eLife

Article Title: Uremic toxin indoxyl sulfate induces trained immunity via the AhR-dependent arachidonic acid pathway in end-stage renal disease (ESRD)

doi: 10.7554/eLife.87316

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , GNF351 , Sigma-Aldrich , 182707 , .

Techniques: Transfection, Construct, Isolation, Recombinant, Sequencing, Enzyme-linked Immunosorbent Assay, In Vitro, Software