L kynurenine

IDO-1 enzymatic activity was analyzed by determining L-kynurenine concentrations in conditioned media from hPDL-MSCs. Briefly, the conditioned medium was diluted 1:3 (v/v) with 30% trichloroacetic acid (Sigma-Aldrich, St. Louis, USA), which was followed by incubating samples for 30 minutes at 65° Celsius. After centrifugation, 125µl Ehrlich’s Reagent, consisting of 0.8% P-dimethylbenzaldehyde in glacial acetic acid (Sigma-Aldrich, St. Louis, USA), was added 1:1 to the supernatant. After 10 minutes of incubation at room temperature, absorbance was measured in duplicates at 492nm (OD₄₉₂) using a microplate reader (Synergy HTX multiplate reader, BioTek, Winooski, USA). L-kynurenine concentrations were calculated by plotting measured OD₄₉₂ against a linear regression curve with L-kynurenine (Sigma-Aldrich, St. Louis, USA) concentrations ranging from 7.8µM to 1000µM. The calculated L-kynurenine concentrations were normalized to the total protein amount and the unstimulated control (= 0). The Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA) was used according to the manufacturer’s instructions to determine the total protein amount in mg. In brief, quantified absorbance at 562nm (OD₅₆₂) was plotted against a linear regression curve composed of known BSA (Capricorn Scientific, Ebsdorfergrund, Germany) concentrations which ranged between 31.25µg/ml and 2000µg/ml.

System L amino acid transport was measured using the fluorescent properties of kynurenine.²² After staining to distinguish macrophages, each sample was split into four tubes: HBSS alone (FMO/background), l‐kynurenine (Sigma; 200 μm final), l‐kynurenine + leucine (Sigma, 5 mm final) and kynurenine + 2‐amino‐2‐norbornanecarboxylic acid (BCH)—a LAT1 inhibitor (Sigma; 10 mm final). Samples were placed at 37°C for 5 min, before addition of Cytofix Fixation Buffer (BD Biosciences), pulse vortex and incubation at 4°C for 20 min. After washing, cells were analysed by flow cytometry. kynurenine was detected on the e450/BV421 channel (BV450/50 filter on LSR Fortessa).

Cu treatment was performed by culturing the gemmae on BCDAT agar medium supplemented with Cu in the form of CuSO₄, Cu(NO₃)₂, or CuCl₂. Co-treatment with ethylenediaminetetraacetic acid (EDTA) and Cu was performed by culturing the gemmae on BCDAT agar medium containing 400 µM EDTA and 400 µM CuSO₄. According to the chemical speciation program Geochem-EZ (Shaff et al., 2009 ), 99% of Cu ions are chelated with EDTA under these conditions. Other heavy metals were supplemented in the form of 400 µM MnSO₄, CoSO₄, NiSO₄, or ZnSO₄. Treatment with auxins, the auxin antagonist α-(phenylethyl-2-one)-indole-3-acetic acid [PEO-IAA; stock solutions made in dimethyl sulfoxide (DMSO) to 20 mM] (Hayashi, 2012 (link); Hayashi et al., 2012 (link)), or l-kynurenine (stock solutions prepared in 50% DMSO solution diluted in sterile distilled water to 40mM; Sigma-Aldrich) were performed by culturing the gemmae on BCDAT agar medium supplemented with auxin in the form of 0.5 µM indole acetic acid (IAA) or naphthalene acetic acid (NAA), 20 µM PEO-IAA, or with 20 and 40 µM l-kynurenine. Cytokinin treatment was performed by culturing the gemmae on BCDAT agar medium supplemented with 0.5 or 2 µM 6-benzylaminopurine (BAP).