Sodium dl lactate

The hESC lines (NKX2.5^eGFP/w HES3, H1 and H9-GCaMP6f) with passages 55-65 were routinely maintained and differentiated as described previously 22 (link), 23 (link). In brief, the hESCs were cultured on feeder-free Matrigel (356231, Corning, USA) in E8 medium (A1517001, Thermo Fisher, USA). Before cardiomyocyte differentiation, the hESCs were passaged every 4 days at 80% confluence using 0.5 mM EDTA and re-plated at a density of 2 × 10⁴ cells/cm². To initiate cardiomyocyte differentiation, the medium was changed with CDM3 (chemically defined medium, three components) containing 5 μM CHIR99021 (C-6556, LC Laboratories, USA) for 48 hours (day 0-2). After a 2-day culture in CDM3, the medium was changed with CDM3 containing 5 μM IWR-1 (I0161, Sigma, USA) for 48 hours (day 4-6). The subsequent culture was in CDM3. The medium was changed every day. Spontaneous beating was commonly observed on day 10. For cardiomyocyte purification, the cells were re-plated on gelatin-coated dishes in RPMI 1640 lacking of glucose and containing 5 mM sodium DL-lactate (L4263, Sigma, USA) from day 11 to day 15 of differentiation. RA (R2625, Sigma, USA) was dissolved in DMSO and added to the CDM3 at the indicated times for cardiomyocyte differentiation and maturation. The final concentration of RA was 1 μM, and the final concentration of DMSO was 0.05% in both groups.

ESCs were split and cultured as described above. When cells reached ~ 90% confluence, cardiomyocyte differentiation was initiated by changing the culture medium to differentiation medium CDM3 [20 (link)]. During cardiomyocyte differentiation, cells were treated with 5 μM of the glycogen synthase kinase 3-β inhibitor CHIR99021 (Sigma, USA) on days 0–2 and 2 μM of the Wnt pathway inhibitor Wnt-C59 (Selleck Chemicals, USA) on days 4–6. The medium was changed daily, and spontaneous beating was noted from day 9. For cardiomyocyte purification, the cells were replated and cultured in CDM3L medium, which consisted of glucose-free RPMI 1640 (Thermo Fisher, USA) and 5 mM sodium dl-lactate (Sigma, USA). Cardiomyocytes were then split at 1:4 with 0.25% trypsin (Sigma, USA) containing 0.1 mM EDTA and seeded on 0.1% gelatin-coated dishes in CDM3.

CDM3 hiPSC-CM differentiation was performed with minor modifications as detailed below^{64 (link),65 (link)}. All hiPSC lines were differentiated with CDM3 media consisting of RPMI 1640 (11875119, Gibco), 500 µg/mL recombinant human albumin (Sigma-Aldrich, A0237 or Sciencell, OsrHSA), and 213 µg/mL l-ascorbic acid 2-phosphate sesquimagnesium (Sigma-Aldrich, A8960-5G) until day 10. ERRγ KO and its control hiPSC-CMs were maintained with glucose-deficient CDM3 with sodium DL-lactate (Sigma-Aldrich, L4263-100ML) supplementation for 5 days. WTC11 hiPSC-CMs were enriched with glucose-deficient media for 2 days. ERRα/γ KO and control hiPSC-CMs were maintained until day 22 with CDM3 media. We confirmed the cardiac differentiation efficiency in both lines was similar, which was more that 90%^{10 (link)}. The purified cardiomyocytes were replated on matrigel-coated plates in 10% FBS supplemented with RPMI 1640 media (Thermo Fisher Scientific, 11875119) and 5 μM Y27632 (Selleck Chemical LLC, S104910MG). Two days after replating, the ERRγ ΚΟ and its control cardiomyocytes were maintained with the glucose-deficient media for an additional 5 days. siRNA transfection was conducted with WTC11 hiPSC-CMs 2 days after replating as described in siRNA transfection section. Adenoviral infection was performed after the 2nd purification with CDM3 media supplemented with 5% FBS.

A chemically defined molecular-based method was used for CM differentiation (Burridge et al., 2014 (link)). Spontaneous contracting could be observed 7–8 days after differentiation. Then, the CMs were purified by using a metabolic-selection method using the purification medium composed of RPMI 1640 without glucose, 213 μg/mL of l-ascorbic acid 2-phosphate, 500 μg/mL of Oryza sativa-derived recombinant human albumin, and 5 mM of sodium dl-lactate (Sigma, USA).

Cy3B-STORM was done following the procedure described on Joshua et al (Vaughan et al., 2012 (link)). We labeled neurons with Cy3B-SA (2 μM) following the procedure of sQD labeling. We then fixed the cells with a solution of 4% PFA and 0.1% Glutaraldehyde in HBSS for 10 min. Wash with three tines PBS. Treat the sample with 10 mM of NaBH4 in PBS freshly prepared for 10 min. Wash with PBS. Before imaging, we add imaging buffer consisting of 5 μL of PCD, 20 μL of PCA, 4 μL of Trolox in 471 μL of T-100 (100 mM Tris at pH 8.0). Images were acquired at 10 Hz using 405 nm laser at low power to do activation and 561 nm laser to excite fluorescence.
In order to image native AMPARs, we labeled AMPARs using Anti-GluA2-Alexa647 after fixation. For STORM imaging, we added imaging buffer consisting of 5 mM MEA (Sigma: 30070, St. Louis, MO) solution (~pH 8.0) and additionally added 40 mM Sodium D/L-lactate (Sigma: 71720, St. Louis, MO) and EC-Oxyrase (Sigma: SAE0010, St. Louis, MO) in PBS in order to improve the photo-stability.

To evaluate growth in the presence of different carbon sources SC5314 and the eed1Δ/Δ mutant were diluted to OD₆₀₀ of 0.1 in YPD medium with 2 or 0.1% glucose or in SD minimal medium (0.67% yeast nitrogen base; BD Biosciences) in the presence of 2% glucose, 0.1% glucose, 2% N-acetyl-glucosamine (GlcNAc; Sigma–Aldrich), 2% sodium-DL-lactate (Sigma Aldrich), 2% potassium acetate (Merck), 2% citric acid monohydrate (Roth) or 2% casamino acids (BD Biosciences). For testing of proteolytic activity, C. albicans strains were grown in SD medium overnight, washed twice with PBS and OD₆₀₀ was set to 0.1 in YCB-BSA (1.17% Yeast Carbon Base (BD Biosciences), 1% glucose, 0.5% BSA (Serva)), pH 4.0. For growth in kidney homogenates, kidneys of uninfected mice were removed aseptically, homogenized and diluted to 25 mg/ml in DPBS and filtered through 70 µm and 40 µm cell strainers before filter sterilization. Growth was recorded by measuring OD₆₀₀ in a microplate reader at 37 °C. Measurements were performed in 30 min intervals over a course of 60 h. Blank values were subtracted from all measurements.