Fig 2: Carvones influence the cellular functions of AhR.Cells were incubated for 90 min with carvones (1000 µM) in combination with vehicle (0.1% DMSO) or AhR agonists TCDD (20 nM), BaP (7 µM), or FICZ (8 nM). (A) The nuclear translocation of AhR is not influenced by carvones. Microscopic specimens from LS180 cells were prepared using Alexa Fluor 488-labeled primary antibody against AhR and DAPI. AhR was visualized and evaluated using a fluorescence microscope. Experiments were performed in two consecutive cell passages (n = 2) with all tested compounds in duplicate. Representative images are shown. Scale bar = 50 µM. (B) Carvones inhibit the formation of AhR-ARNT heterodimers. Protein coimmunoprecipitation – formation of AhR-ARNT heterodimers in LS180 cells. Representative immunoblots of immunoprecipitated protein eluates and total cell lysates are shown. Experiments were performed in three consecutive cell passages (n = 3). (C) Carvones inhibit the binding of AhR to the CYP1A1 promoter. Chromatin immunoprecipitation ChIP – binding of AhR to the CYP1A1 promoter in HepG2 cells. The bar graph (top) shows enrichment of the CYP1A1 promoter with AhR compared to vehicle-treated cells. Representative DNA fragments amplified by PCR analyzed on a 2% agarose gel are from the 2nd experiment (bottom). Experiments were performed in three consecutive cell passages (n = 3). Data are shown as mean ± SD. (D) Schematic depiction of the cellular effects of carvones binding to AhR. Abbreviations: BaP, benzo[a]pyrene; FICZ, 6-formylindolo[3,2-b]carbazole; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Source data are provided as a Source Data file.

Fig 3: Evaluation of off-target effects of carvones.A PKC inhibition assay: The catalytic activity of PKC was measured in lysates from HepG2 cells incubated with vehicle (DMSO, 0.1% V/V), staurosporine (1 µM), and carvones (10 µM; 100 µM; 1000 µM). Data are the mean ± SD from three independent experiments (n = 3). The significance was calculated using 2-way ANOVA multiple comparison test, and p-values are indicated in the graph. Incubations and measurements were performed in uniplicate (technical replicates). B KINOMEscanTM profiling: The interaction between 100 µM S-carvone and 468 human protein kinases, employing KINOMEscanTM (scanMAX assay), a proprietary active site-directed competition binding assay. A low-resolution interaction map is shown. C Hypoxia-mimic VEGF induction: HaCaT cells were incubated for 24 h with carvones (10 µM; 100 µM; 1000 µM) in combination with vehicle (0.1% DMSO) or deferoxamine (DFX; 200 µM). The expression of VEGF and CYP1A1 mRNAs was measured using RT-PCR. Incubations and measurements were performed in duplicate (technical replicates). Data are the mean ± SD from three independent cell passages (n = 3) and are expressed as fold induction over the vehicle-treated cells. The results were normalized using GAPDH as a housekeeping gene. D Counterscreen radioligand binding assay with human AR, ERa, ERß, PR, GR, RXRa, and PPAR? receptors. The bar graph shows the percentage of displacement of the radioligand by S-carvone (100 µM; 1000 µM). The data are mean from two independent incubations (n = 2). A value >50% is considered to indicate interaction. E Protein phosphatase inhibition assay: The catalytic activity of PTPN11/SHP2 and PTPN6/SHP1 was measured with recombinant enzymes incubated with vehicle (DMSO, 0.1% V/V), PTP1B inhibitor (33.3 µM), and S-carvone (100 µM; 1000 µM). Data are the mean from two independent experiments (n = 2). Incubations and measurements were performed in duplicate (technical replicates). F Competitive fluorescence binding assay for HSP90: The bar graph shows the percentage displacement of fluorescently labeled geldanamycin by S-carvone (100 µM) and nonlabeled geldanamycin (0.12 µM). Data are the mean from two independent experiments (n = 2). Incubations and measurements were performed in duplicate (technical replicates). AR androgen receptor, ARNT AhR nuclear translocator, DFX deferoxamine, ERa/ß estrogen receptor alpha/beta, GR glucocorticoid receptor, HSP90 heat shock protein 90 kDa, PKC protein kinase C, PPAR? peroxisome proliferator-activated receptor gamma, PR progesterone receptor, PTPN6(11)/SHP1(2) tyrosine-protein phosphatases non-receptor type 6(11), RXRa retinoid X receptor alpha, VEGF vascular endothelial growth factor. Source data are provided as a Source Data file.