Ethylene glycol tetraacetic acid (egta)

The following agents were used: ATP (Sigma Aldrich Co., St. Louis, MO; MP Biomedicals, Solon, OH, USA, Calbiochem Co., La Jolla, CA, and Roche Diagnostic Co., Indianapolis, IN), ADP (Sigma), AMP (Sigma), adenosine (Sigma), benzoylbenzoyl ATP (Sigma), oxidized ATP (Sigma), suramin (Wako, Tokyo, Japan), MIA (methyl isobutyl amiloride, Wako), DIDS (4,4′- Diisothiocyanatostilbene-2,2′-disulfonic acid, Sigma), EDTA (Dojindo, Tokyo, Japan), EGTA (Dojindo), dipyridyl (Sigma), CAS (Chrome Azurol S, Sigma), E. coli S17-1 and pK18mobSacB (kindly provided by Dr. F. Taguchi, Okayama University), Instagene matrix (Bio-Rad, Hercules, CA), KOD-Plus (Toyobo, Osaka, Japan), Wizard SV Gel and PCR cleanup system (Promeg, Madison, WI), Ligation High (Toyobo), Protein Assay Rapid Kit (Wako), [¹⁴C] isoleucine (Moravek Biochemicals, Inc, Brea, CA), [¹⁴C]uracil (Moravek Biochemicals, Inc.), α-defensin-1 (Peptide institute, Inc, Osaka, Japan.), cathepsin G (Sigma), vancomycin (Wako), clarithromycin (Taisho-Toyama Pharmaceutical Co., Tokyo), rifampin (Daiichi Sankyo Co., Tokyo), and ethambutol (Sigma), gatifloxacin (Wako), FLUOS (Sigma), LB medium (Invitrogen, San Diego, CA), M9 medium (prepared by our laboratory), Heart infusion agar (Eiken Chemical Co., Tokyo, Japan), Middlebrook 7H9 medium (Becton Dickinson, Cockeysville, MD), and Middlebrook 7H11 medium (Becton Dickinson).

Pharmacological reagents were obtained from Sigma-Aldrich, except for bilirubin, TEA (Tokyo Chemical Industry, Tokyo Japan), EGTA, and HEPES (Dojindo Molecular Technologies, Kumamoto, Japan). T16A_inh-A01 (2-[(5-ethyl-1,6-dihydro-4-methyl-6-oxo-2-pyrimidinyl)thio]-N-[4-(4-methoxyphenyl)-2-thiazolyl]-acetamide) and bilirubin were dissolved in dimethyl sulfoxide (DMSO) and 0.1 M NaOH, respectively, at a concentration of 30 mM as a stock solution.

Cryopreserved hESC-CMs were thawed, re-plated, and cultured for an additional 4 days for in vitro electrophysiological analysis. To examine the autonomic beating rate, the maximum dV/dt of depolarisation (dV/dt_max), and action potential duration at 50% and 90% repolarisation (APD₅₀, and APD₉₀, respectively), of spontaneous action potentials were recorded using a ruptured whole-cell patch-clamp technique in the current-clamp mode at 35–36 °C using a patch-clamp amplifier (Axopatch 200B, Molecular Devices) and sampled at 5 kHz after being low-pass-filtered at 2 kHz^{33 (link)}. Patch pipettes (7–8 MΩ) were fabricated from borosilicate glass capillaries (Kimax-51, Kimble Glass) and coated with Sylgard 184 (Dow Corning Toray Co.). Series resistance was always kept below 20 MΩ. Action potentials were measured using an intracellular solution containing (mmol/L): 130 potassium gluconate (Wako), 10 KCl (Wako), 5 NaCl (Wako), 1 MgCl₂ (Wako), 0.1 EGTA (Dojindo), 0.1 Mg ATP, and 10 HEPES (Dojindo) [pH 7.2 with KOH (Wako)]. The extracellular bath solution contained (mmol/L): 136.5 NaCl, 5.4 KCl, 1.8 CaCl₂ (Wako), 0.53 MgCl₂, 5.5 HEPES, and 5.5 glucose (Wako) (pH 7.4 with NaOH).

The P. falciparum 3D7 strain expressing the cytosolic yeast DHODH (3D7-yDHODH) was prepared as follows. pHHyDHODH-GFP plasmid (Kerafast, Boston, MA, USA) was transfected to the P. falciparum 3D7 strain following the established transfection method [59 (link)]. Briefly, 50 µg of plasmid dissolved in cytomix solution (120 mM KCl, 0.15 mM CaCl₂, 10 mM K₂HPO₄/KH₂PO₄ pH 7.6, 25 mM HEPES-KOH pH 7.6, 2 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, Dojindo), and 5 mM magnesium chloride (Wako)) was transfected into the red blood cells by the Gene Pulser Xcell (Bio-Rad). Percoll–sorbitol synchronized parasites were mixed with red blood cells containing plasmid and maintained under 5 nM WR99210 for 2 weeks. The growth of resistant parasites against DSM265 and atovaquone, as PfDHODH and complex III inhibitors respectively, was confirmed by PfLDH/diaphorase assay. A total of 10 µM PfDHODH inhibitors were tested against wild-type 3D7 and 3D7-yDHODH following the method described above in triplicate. The significance of difference between the inhibition of the two parasite strains was evaluated by Student’s t-test (Figure S4).

D-iPS cells were differentiated into myotubes as described in Section 2.2 by EPS culture from day 7 with the following conditions: 0.3 V/mm, 4 ms, and 1 Hz. Ethylene glycol tetra-acetic acid (EGTA) (Dojindo Laboratories, Kumamoto, Japan) was added on day 9 for 24 h and the apoptosis of myotubes was analysed as described in Section 2.7.