Oleic acid

To obtain crystals of a BSP30b_F61M_F115M-oleic acid complex, purified protein was concentrated to ~2 mg.mL^-1 and incubated with 20 μL of oleic acid (≥99%, Sigma-Aldrich) at 4 °C overnight. This mixture was purified using SEC to remove unbound oleic acid and then concentrated to 8 mg.mL^-1 for crystallization. Crystals were obtained after two weeks under the same conditions as for the protein alone. A complete diffraction dataset was collected at the Australian Synchrotron MX2 beamline using the new ACRF Eiger 16M detector [24 (link)]. The structure was solved by molecular replacement using Phaser [25 (link)] with the solved BSP30b structure as the reference model. The structure was built and refined as above. A polder OMIT map was calculated using phenix.polder in the PHENIX software package [26 (link)].

After silencing total p63 and TAp63α in THLE2 cells, cell culture medium was supplemented with 1 mM of oleic acid (Sigma Aldrich, USA) bound to fatty-acid-free bovine serum albumin (BSA) (Capricorn) (2:1 molar ratio). Controls were supplemented with BSA alone. THLE2 cells were incubated with oleic acid and BSA during 12 h. After treatments, the coverlips were placed in a 24-well plate with 500 μl of Minimum Essential Medium Eagle (Sigma-Aldrich, USA) complete medium and incubated (37 °C) during 40 min with BODIPY (green) 493/503 (1:200 dilution) (Thero Fisher, USA). After incubation, coverlips were washed with phosphate-buffered saline (PBS) and fixed with 4% formaldehyde during 10 min. The coverslips were mounted in aqueous medium (FluoroGel #17985-10) with DAPI (D-9,542, Sigma-Aldrich, USA) (blue) (1:1,000) for preserving cell fluorescence. Confocal images were collected using a Leica confocal microscope (Leica A0B5-SPSX) equipped with a high grade colour corrected plan apochromat lens for confocal scanning × 63/1.32 objective. Leica Confocal Software was used for acquisition and analysis. Images are combinations of optical sections taken in the z axis at 0.5-μm intervals.

NRK52E cells (2 × 10⁴ cells/well) were incubated in a 24-well plate for 16 h, and then treated with the nifedipine (Sigma, St. Louis, MO, USA) at different concentrations (0, 7.5, 15, or 30 μM) or with 150 μM oleic acid (Sigma, St. Louis, MO, USA) as a positive control for 48 h. The nifedipine dose was selected to correspond to internal levels in humans, and the positive control oleic acid dose was selected according to a preliminary 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) test. The cells were washed with phosphate-buffered saline (PBS) and incubated with 0.5 mg/mL MTT solution for 4 h at 37 °C prior to removing the culture medium. Dimethyl sulfoxide was then added and mixed for 5 min at 26 °C. Cell viability was determined by measuring the absorbance at 560 nm. Cell viability for each experimental group was calculated as a percentage of that of the control group.

Cybrids derived from the 143B.TK^-Rho-0 cell line were established as described previously [27 (link)]. Haplogroup genotyping was used to verify that the mtDNA of the cybrids line was the same as in the donor platelets. The mtDNA haplogroups were assessed using established methods [26 (link)].
Transmitochondrial cybrids were kept in Dulbecco´s modified Eagle’s medium with 5.5 mM (low) glucose (Gibco, Grand Island, NY, USA) (from here on described as DMEM-glu). For experiments, cybrids were cultured in DMEM-glu, DMEM no glucose (Gibco) supplemented with 100 µM oleic acid (Sigma–Aldrich, Merck KGaA, Darmstadt, Germany) (from here on described as DMEM-ole) or DMEM low glucose supplemented with 100 µM oleic acid (consequently described as DMEM-glu/ole). All media were further supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) (Gibco). The cells were incubated in a humidified 5% CO₂ atmosphere at 37 °C. At least two clones were obtained of each cybrid for each experiment.

Apoptosis was assessed using PE Annexin V Apoptosis Detection Kit I (BD Biosciences) and analyzed by flow cytometry. Briefly, cells were plated into six-well culture dishes (1 × 10⁶ cells/well) for 24 h. prior to the addition of oleic acid (Sigma Cat#75090) at a concentration of 150 µM. Following 24 hrs incubation with oleic acid the percentage of apoptotic cells was determined by the annexin V-PE/7-AAD assay following manufacturer’s instructions. Fluorescence of the cells was immediately determined by a Sony SA3800 spectral analyzer.

PCLSs were cultured in Williams medium E with GlutaMAX™ (Cat. 32551020, Invitrogen, Bleiswijk, The Netherlands), supplemented with gentamycin (50 µg/mL; Cat. 15750-037, Invitrogen) and glucose (25 mM; Cat. 1.08342.1000, Merck, Darmstadt, Germany), hereinafter referred to as “WEGG”. To induce steatosis, additional glucose (36 mM), fructose (5 mM, Cat. F3510-100G, Sigma-Aldrich, St. Louis, MO, USA), human insulin (1 nM, Cat. I9278, Sigma-Aldrich), palmitic acid (240 µM, Cat. P0500-10G, Sigma-Aldrich), and oleic acid (480 µM, Cat. 75096-1L, Sigma-Aldrich) were added, a combination denoted as “GFIPO”. palmitic acid and oleic acid were solubilized in 0.04% bovine serum albumin (BSA; Cat. A2153-100G, Sigma-Aldrich). The fatty acids were initially dissolved in 0.1 M sodium hydroxide (Cat. 1.06469.5000, Merck) at 70 °C, followed by combination with pre-warmed BSA in water at 55 °C. It was observed that the final concentrations of BSA and sodium hydroxide did not influence the pH of the medium nor affect the PCLSs’ viability. The medium preparation and final concentrations were based on previous experiments [10 (link)]. PCLSs were incubated at 37 °C in an atmosphere containing 20% O₂ and 5% CO₂ for periods ranging from 24 to 96 h. The medium was renewed at 24 h intervals, and samples were collected at the same frequency.