Sepiapterin

RMEC (80,000 cells per well) were seeded onto 1% gelatin-coated 6-well plates and, after 24 hours, when cells were approximately 50% confluent, were incubated in growth medium containing vehicle control (1:10,000 dilution of DMSO), 0.1μM sepiapterin (dissolved in DMSO; Sigma-Aldrich), or 1 μM sepiapterin and placed under hyperoxic or normoxic conditions for 24 hours. For BH4 and BH2 measurements, cells were collected by trypsinization, and the resulting cell pellet was snap frozen on dry ice and stored at −80°C until analysis. Briefly, cell pellets were homogenized in cold extraction buffer (50 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol [DTT], 1 mM ethylenediaminetetraacetic acid [EDTA]; all Sigma-Aldrich) and centrifuged at 14,000 rpm for 15 minutes at 4°C. BH₄ levels were determined by HPLC with fluorescence detection after iodine oxidation under acidic or alkaline conditions, as described above and previously.^{15 (link)} Parallel plates were used to measure superoxide detection by dihyroethidium (DHE) or proliferation by 5-ethynyl-2′-deoxyuridine (EdU).

Glucose was measured using a FreeStyle Lite® (Abbott) glucometer with accompanying strips, and triglyceride assay reagents were procured from Sigma. Insulin (Humulin R, Lilly) was purchased at a local pharmacy. Insulin ELISA kits were from ALPCO Diagnostics. S-Nitro-N-acetyl-DL-penicillamine (SNAP) and cyclic guanosine monophosphate (cGMP) were procured from Cayman Chemical Company. Sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium bicarbonate, potassium phosphate, D-glucose, collagenase, ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), sodium pyrophosphate, sodium orthovanadate, sodium fluoride, okadaic acid, 1% protease inhibitor cocktail, dithiothreitol, magnesium chloride, K-lactobionate, taurine, potassium phosphate, HEPES, digitonin, pyruvate, malic acid, glutamic acid, adenosine diphosphate, succinic acid, oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, phenylephrine and ACh, trypsin inhibitor, and cytochrome c were procured from Sigma-Aldrich (MO, USA). Mammalian Protein Extraction Reagent (M-PER) was from Thermo Scientific, and Immobilon-P PVDF membrane was from Millipore. Sepiapterin was procured from Sigma-Aldrich.

Human liver hepatocellular cells HepG2 were cultivated in the Eagle’s Minimum Essential Medium (EMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich) in 6-well plates; each tested pFLAG-PAH variant was cultivated in duplicate. The cells were transfected at the confluency of 70%–90% by 2.5 μg of WT and mutant pFLAG-PAH using Lipofectamine^®2000 (Thermo Fisher Scientific) and incubated overnight. Subsequently, the transfecting mixture was replaced with the fresh EMEM supplemented with 10% FBS. At this time, one of the duplicates of each tested WT/mutant PAH containing transfected cells was treated with 100 μM sepiapterin (Sigma-Aldrich). The cells were collected 36 h after the transfection and lysed on ice in the NP-40 buffer containing NP-40 lysis buffer containing 150 mM sodium chloride, 1.0% Nonidet P-40, and 50 mM Tris, pH 8.0. The protein concentration was determined spectrophotometrically using Quick StartTM Bradford Protein Assay (Bio-Rad).