Fig 2: Transcriptome analysis of constitutively activated hAHRΔe8-9 mutant. (a) Western blot results for transgenic mouse cell lines, expressing Cas9 (negative control), hAHR WT, hAHRΔe8-9, or a DNA-binding deficient version of hAHRΔe8-9 (hAHRΔe8-9 DBD). (b) Rlog values of the 172 most differentially expressed genes between the hAHRWT and hAHRΔe8-9 transgenic mouse bladder organoids in untreated and TCDD treated conditions. (c) Boxplot of normalized counts of RNA reads of AHR target genes for the untreated mouse bladder organoids expressing hAHR mutants (n = 4). (d) Images of Dapi stained mouse bladder organoids constitutively expressing hAHR mutants. The lower panel shows the magnification of the boxed area. Scale bar 250 µm. (e) GO enrichment analysis of hAHRΔe8-9 mutant for the cellular component domain. (f) Rlog values of basal and luminal cell layer makers for hAHRΔe8-9 and hAHRWT transgenic mouse bladder organoids.

Fig 3: GTP cyclohydrolase 1 (GCH1)/L-5-hydroxytryptophan (5-HTP) metabolic axis activates aryl hydrocarbon receptor (AHR) to upregulate indoleamine 2,3-dioxygenase 1 (IDO1). (A) Schematic diagram depicting the two main pathways of Trp metabolism. (B) Relative N-acetyl-5-hydroxytryptamine (NAS) and 5-HTP levels in BT549 cells expressing vector or GCH1 detected by liquid chromatography with tandem mass spectrometry. (C) Relative IDO1 transcription levels detected by quantitative reverse transcription PCR (qRT-PCR) analysis and IDO1 levels detected by western blot in BT549 cells treated with dimethyl sulfoxide (DMSO), tetrahydrobiopterin (BH4, 10 µM), 5-HTP (500 µM), serotonin (10 µM), NAS (100 µM), and melatonin (1 µM) for 24 hours. (D and E) Relative IDO1 transcription levels detected by qRT-PCR analysis and IDO1 levels detected by western blot in BT549 cells treated with 5-HTP at different concentrations for 24 hours (D) and with 5-HTP (500 µM) for different durations (E). (F) IDO1 levels detected by western blot in MDA-MB-453 cells expressing control or GCH1 short hairpin RNAs treated with 5-HTP (500 µM) for 24 hours. (G and H) Representative immunofluorescence images of AHR (G) and the cytoplasmic and nuclear protein fractions of the AHR protein (H) in BT549 cells treated with phosphate buffer saline (PBS) or 5-HTP (500 µM) for 24 hours. Scale bar=10 µm. (I) Relative IDO1 transcription levels detected by qRT-PCR analysis and IDO1 levels detected by western blot in BT549 cells treated with small interfering RNA targeting AHR (siAHR) for 48 hours followed by 5-HTP (500 µM) supplementation for 24 hours. (J) Chromatin immunoprecipitation followed by quantitative PCR analysis of BT549 cells treated with 5-HTP (1 mM) for 48 hours. (K) Analysis of luciferase activity in human embryonic kidney (HEK) 293 T cells. HEK-293 T cells concomitantly expressing an IDO1 promoter–luciferase reporter and AHR were treated with 5-HTP (500 µM) or PBS for 24 hours. (L) Analysis of luciferase activity in HEK-293 T cells. HEK-293 T cells concomitantly expressing an IDO1 promoter–luciferase reporter (ABS–WT) or IDO1 promoter mutation–luciferase reporter (ABS1-Del and ABS2-Del) and AHR were treated with 5-HTP (500 µM) for 24 hours. The data are presented as the mean±SD (B, C, D, E, I, J, K, and L): two-tailed unpaired Student’s t-test for B, C, D, E, I, J, K, and L; n=3 independent experiments (B, C, D, E, I, J, K, and L). *P<0.05; **P<0.01; ***P<0.001; n.s., not significant. 5-HTP, L-5-hydroxytryptophan; AADC, dopa decarboxylase; AANAT, aralkylamine N-acetyltransferase; ABS, AHR binding site; ASMT, acetylserotonin O-methyltransferase; BH4, tetrahydrobiopterin; Del, deletion; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC1, histonedeacetylase 1; Kyn, kynurenine; NAS, N-acetyl-5-hydroxytryptamine; PBS, phosphate buffer saline; shGCH1, short hairpin GCH1; siAHR, small interfering RNA targeting AHR; TPH, tryptophan hydroxylase; WT, wild type.

Fig 4: Kyn activated NRF2 through AhR signaling. (A) Macrophages were pretreated with CH-223191 (CH, 20 μM) for 2 h before the addition of Kyn to the culture for 24 h. The mRNA levels of AhR, CYP1A1, CYP1B1, and NRF2 were measured by RT-qPCR. (B) The total protein levels of NRF2 and AhR were detected by western blotting. (C) The nuclear and cytoplasmic protein levels of AhR in macrophages were detected by western blotting. (D) The nuclear and cytoplasmic protein levels of NRF2 in macrophages were detected by western blotting. (E) (a) The location of the gene sequence of NFE2L2 (also known as NRF2) on chromosome 2. (b) The locations of the binding sites were upstream of the TSS. (F) ChIP-qPCR analysis of the interaction between AhR and the NRF2 promoter. (G) A schematic overview of IDO-mediated ASC regulation on macrophages in periodontitis. TSS, transcription start site; IDO, indoleamine 2,3-dioxygenase; ASC, adipose-derived stromal/stem cells.

Fig 5: Carbidopa inhibits proliferation of LNCaP cells via AHR.a Colony formation assay: LNCaP cells were treated with or without Carbidopa (100 μM). Where indicated, AHR blocker CH223191 was present (10 μM). b TUNEL assay: LNCaP cells were treated with or without Carbidopa (100 μM). Where indicated, AHR blocker CH223191 was present (10 μM). Scale bars = 100 μm. c Quantitative analysis of TUNEL+ cells in at least three separate fields; data are given as means ± SEM. **p < 0.01 versus control. #p < 0.05 versus Carbidopa treatment. d Migration assay: LNCaP cells were treated with or without Carbidopa (100 μM). Where indicated, AHR blocker CH223191 was present (10 μM). Scale bars = 100 μm. e Quantification of the migration assay. Data are given as means ± SEM of three independent experiments. **p < 0.01 versus control. #p < 0.05 versus Carbidopa treatment.