D8418

PS10 (2((2,4-dihydroxyphenyl)sulfonyl)isoindoline-4,6-diol, HY-121744, MedChemExpress, Monmouth Junction, NJ, USA) was used to block PDK activity [42 (link),43 (link)]. The compound was reconstituted in DMSO (dimethyl sulfoxide, D8418, Sigma) and injected into 1–2-cell stage embryos at a concentration of 1 mM, as determined by a previous dose/toxicity experiment.

Omecamtiv mecarbil and (−)‐blebbistatin were purchased from Selleck (S2623) and Sigma (B0560), respectively. Stock solutions were prepared in DMSO (molecular biology grade, Sigma, D8418) and compound purities were estimated to be >95% by RP‐HPLC and ESI mass spectrometry.

Male Wistar rats, aged 8–10 weeks weighing 220–260 g (Japan SLC), were maintained at 22 ± 2 °C under a 12-h light/dark cycle with free access to food and water. The clinical signs of rats were observed at least twice a day. If the criteria of humane endpoints were met, rats were sacrificed. Humane endpoints were considered as severe respiratory distress, poor physical appearance, seizure, vomiting or skin problems (wounds or signs of inflammation). The animal experiments were performed under a protocol approved by the Institutional Animal Care and Use Review Committee of the Kobe University of Medicine (approval number P170705) which complied with the ARRIVE guidelines. GGA (ab146190, Abcam) was dissolved in 100% dimethyl sulfoxide (DMSO) (D8418, Sigma) and stored at − 20 °C.

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (M5655, Sigma-Aldrich, St. Louis, MO, USA) assay was used to assess cell viability. Briefly, the medium was aspirated from the culture and MTT reagent was added to a final concentration of 0.5 mg/mL in culture medium. The samples were incubated for 3 h at 37 °C in the dark. Afterwards, the solution was aspirated, and the constructs were lysed using DMSO (D8418, Sigma-Aldrich, St. Louis, MO, USA). The absorbance was read at 550 nm using a microplate reader (Biotek ELX808, Winooski, VT, USA).

STHdh cells were exposed to 125 μM Mn and incubated at 37 °C (5% CO₂) in HBSS for 3 h. This generated “vehicle” values when Mn was co-incubated with representative amounts of DMSO (Sigma-Aldrich; D8418; St. Louis, MO, USA). Other small molecules were co-incubated with the 125 μM Mn in the HBSS to assess their impact on Mn levels. After incubation, the cells were washed three times with PBS (without Ca²⁺ and Mg²⁺) to remove the extracellular Mn. Extraction buffer (PBS containing 0.1% TritonX100 (Sigma-Aldrich; T8787; St. Louis, MO, USA) and 500 nM Fura-2) was added to lyse the cells. The fluorescence of the Fura-2 in the extraction buffer was then measured at 360_Ex/535_Em so that Mn could be quantified.

Harvested parasites were cleaned of all impurities and washed several times in sterile 0.9% NaCl. In vitro culturing of A. simplex L3 larvae was carried out according to Iglesias et al. [34 (link)] in RPMI-1640 medium (R8758, Sigma Aldrich, Poznan, Poland) enriched with fetal bovine serum (Sigma Aldrich, F7524) and pepsin (Sigma Aldrich, P7125, Poznan, Poland). The medium was acidified to pH = 4 with 1M HCl. A total of 3 mL of medium was pipetted into each well of the six-well culture plate (from BD Biosciences, Warsaw, Poland). The experiment was conducted in the incubator under anaerobic conditions, at 37 °C and 5% CO₂, with the addition of ivermectin (IVM) (Sigma Aldrich, I8898, Poznan, Poland) in 0.1% DMSO solution (Sigma Aldrich, D8418, Poznan, Poland ) at a concentration of 12.5 µg/mL of culture medium according to Hu et al. [35 (link)]. Five parasites were placed in each well (45 in total). The A. simplex L3 larvae with no drug were cultured as a control (three samples; five larvae in each sample). Larvae cultured with IVM (all were alive after the treatment) were taken from the cultures after 12 h (three samples; five larvae in each sample) or 24 h (three samples; five larvae in each sample) and were preserved at −80 °C until the next step of the analysis.