Lcms grade

Plasmid pXD70Tet(DadH) encoding the active Dadh variant Dadh(A2) under native promoter P_dadh, and plasmid pXD70Tet(DadHR) encoding Dadh(A2) under native promoter P_dadh and DadR were transformed into the ΔdadR mutant and DSM 2243 WT by electroporation. The E. lenta strains were inoculated and grown to saturation in BHIrf media with 20 μg/mL tetracycline. The saturated cultures were then inoculated 1:20 into 200 μL of BHIrf with 20 μg/mL tetracycline in quadruplicate in 96-well plates. 1 mM dopamine was then added to each culture. Plates were incubated at 37 °C anaerobically for 72 h. Next, the endpoint OD₆₀₀ of each culture was measured using a Synergy HTX Multi-Mode Microplate Reader (BioTek). The plates were then centrifuged (3220 × g, 10 min, 4 °C), and the supernatants were harvested. For each sample, 20 µL of the supernatant was diluted 1:10 with 180 µL of LC-MS grade (Honeywell) methanol, and 40 µL of the resulting mixture was then diluted 1:5 with 160 µL of Milli-Q water. The amount of dopamine and m-tyramine production in the diluted mixtures were quantified measured using UPLC-MS/MS as described above.

Candida auris strains used in this study were acquired from the National Culture Collection of Pathogenic Fungi (NCCPF), Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh (NCCPF 470156, NCCPF 470157 both isolated from blood, and NCCPF 470296, isolated from pus). CBS 10913T, the first clinical isolate of C. auris, was acquired from the Central Bureau voor Schimmel Cultures (CBS), Fungal Biodiversity Centre of the Royal Netherlands, Academy of Arts and Sciences (KNAW), the CDC & FDA Antibiotic Resistance Isolate Bank (AR 0383–0386, all isolated from blood and AR 1097 isolated from ear discharge). Strains VPCI 479/P/13 (isolated from blood), and LMDM 1219 (Li et al. 2021 (link)) were received as a kind gift from Prof. Dominique Sanglard, University of Lausanne and University Hospital Centre, Lausanne, Switzerland. All strains were archived in 25% glycerol at −80°C and revived on YPD agar at 30°C for experimental purposes.
All solvents and reagents used (unless specified) were LCMS grade, purchased from Honeywell NC, USA, and Sigma Aldrich MO, USA. Lipid standards were purchased from Avanti Polar Lipids Inc. AL, USA.

KELLY Parental (PAR), DCAF15^WT and DCAF15^KO cells were seeded in 6 well plates and allowed to attach and reach 80% confluency. Cells were then treated with fresh media with 10 µM indisulam or 0.1% DMSO (vehicle control). After 24 h incubation, media was carefully removed, and wells were washed with phosphate saline buffer. Wells were quenched with 1.5 mL of pre-chilled 100% MeOH (LC–MS grade, Honeywell), 10 µL of an internal standard mixture (5–50 µg mL⁻¹ of stable isotope-labelled modifications of 21 analytes, representative of all metabolite classes) was added to each well. All wells were scrapped, and cellular content was dried down completely under N₂ flow. Then, samples were dual-phase extracted as previously described and the polar phase was dried down completely under N₂ flow and stored at −80 °C. Upon measurement, samples were thawed on ice and resuspended with 100 µL H₂O and measured by LC–MS/MS in positive and negative modes.