Cls 2

The F_oF₁ ATP-synthase (ATP Synthase) activity was evaluated incubating 2 × 10⁵ cells at 25 °C for 10 min in a medium containing: 50 mM Tris-HCl (pH 7.4), 50 mM KCl, 1 mM EGTA, 2 mM MgCl₂, 0.6 mM ouabain, 0.25 mM di(adenosine)-5-Penta-phosphate (an adenylate kinase inhibitor), and 25 μg/mL ampicillin (0.1 mL final volume); then, 10 mM pyruvate plus 5 mM malate or 20 mM succinate were employed to stimulate complexes I, III, and IV or complexes II, III, and IV pathways, respectively [17 (link)]. As for OCR evaluation, 3 µM BPTES, 4 µM Etomoxir, and 2 µM UK5099 were used for the cellular energy substrate affinity evaluation. In this case, cells were suspended in the growth medium diluted 1:1 with the solution described above. In each case, ATP synthesis was induced by adding 0.1 mM ADP. The reaction was monitored every 30 s for 2 min with a luminometer (GloMax^® 20/20 Luminometer, Promega Italia, Milano, Italy), using the luciferin/luciferase chemiluminescent method (luciferin/luciferase ATP bioluminescence assay kit CLS II, Roche, Basel, Switzerland). ATP standard solutions in a concentration range between 10⁻⁸ and 10⁻⁵ M were used for calibration. Data were expressed as nmol ATP/min/10⁶ cells [24 (link)].

Mechanical stretching experiments and photon imaging of ATP release were performed as previously described15 (link)51 (link). In brief, an elastic silicone stretch chamber (STB-CH-04, STREX, Osaka, Japan) and an extension device (STB150, STREX) were set on a photon imaging system, and the chamber medium was replaced with extracellular solution containing a luciferase reagent (ATP bioluminescence assay kit CLS II, Roche Diagnostics, Basel, Switzerland). ATP bioluminescence during stretch stimulation was detected and visualized with a VIM camera (C2400-35, Hamamatsu Photonics, Hamamatsu, Japan). The standard calibration curve yielded a correlation coefficient for bioluminescence vs. ATP concentration of 0.981 over a concentration range of 0 nM to 2 μM (Fig. S2). To measure ATP release measurement in vivo, 25 μl or 200 μl of saline containing ARL 67165 (100 μM) was slowly injected into the ureters ligated-bladder of WT or VNUT-KO mice using a PE-10 tube (Clay-Adams, Parsippany, NJ). The saline was collected and the ATP concentration measured by using the luciferase reagent. Although the previous report by Yu52 (link) showed that extracellular ATP concentration and its distribution in the urothelium was affected by ectonucleotidases, the activity of these enzymes was unaffected by the deletion of VNUT (data not shown).

ATP content was measured using the ATP bioluminescence assay kit CLS II (Roche, Heidelberg, Germany) as previously described (Shu et al., 2021 (link)). Protein content was measured using the Pierce BCA Assay kit. 20 μg of protein was run in duplicate in a white, flat-bottom 96-well microplate. Determination of free ATP was performed against an ATP standard curve. Luminescence was determined at 60 ms integration using a Synergy H1 Plate Reader (BioTek, Winooski, VT, United States).

After the reaction, platelets were removed by centrifugation in the presence of 5 mM of ice cold EDTA and 10 µg/ml indomethacin. The amounts of adenosine triphosphate (ATP) and serotonin in platelet-free supernatant fraction was measured using an ATP bioluminescence assay kit CLS II (Roche Applied Science, Mannheim, Germany) and EIA Serotonin kit (IMMUNOTECH SAS, Marseille, France), respectively, according to the manufacturer’s protocol.

ATP in the swim-up-prepared fresh sperm and the vitrified sperm of each treatment group was measured using the ATP bioluminescence assay kit CLS II (#11699695001, Roche) following the manufacturer’s instructions. Briefly, 50 μl washed sperm suspension (∼5 million sperm/ml) was mixed with 450 μl of boiling cell lysis reagent (100 mM Tris, 4 mM EDTA, pH 7.75) and incubated for another 2 min at 100°C. The lysed sperm suspension was centrifuged at 1,000 × g for 60 s and the supernatant collected on ice. The ATP standard was serially diluted with water in a range from 10^–5 to 10^–10 M of ATP. In each well, 50 μl of luciferase reagent and 50 μl of the sample/standard were added; following a 1-s delay, light emission was measured over a 10-s integration period using a luminometer (MPL1, Berthold Detection System, Pforzheim, Germany). A standard curve was constructed using solutions containing known concentrations of ATP, and the sample’s ATP was expressed as micromolar ATP/million sperm.

F_o-F₁ ATP synthase activity was evaluated in mNVCM and mNVFib. For each experiment, 50,000 cells were incubated for 10 min at 37 °C in a medium containing: 10 mM Tris-HCl pH 7.4, 100 mM KCl, 5 mM KH₂PO₄, 1 mM EGTA, 2.5 mM EDTA, and 5 mM MgCl₂, 0.6 mM ouabain, 0.3 mM P1,P5-Di(adenosine-5′) pentaphosphate, and 25 mg/mL ampicillin. Afterward, ATP synthesis was induced by the addition of 10 mM pyruvate plus 5 mM malate or 20 mM succinate, to stimulate the pathways composed of Complexes I, III, and IV or Complexes II, III, and IV, respectively. The reaction was monitored every 30 s for up to two minutes in a luminometer (GloMax^® 20/20n Luminometer, Promega Italia, Milan, Italy), by the luciferin/luciferase chemiluminescent method, with ATP standard solutions between 10⁻⁸ and 10⁻⁵ M (luciferin/luciferase ATP bioluminescence assay kit CLSII, Roche, Milan, Italy). Data are expressed as nmol ATP produced/min/10⁶ cells [27 (link)].

F₁F_o-ATP synthase (ATP synthase) activity was detected by measuring the ATP production by the highly sensitive luciferin/luciferase method. The assays were conducted at 37 °C, for 2 min, and data were collected every 30 s. Cells (1 × 10⁵) were added to the incubation medium (0.1 ml final volume), which contained 50 mM KCl, 1 mM EGTA, 2 mM EDTA, 5 mM KH₂PO₄, 2 mM MgCl₂, 0.6 mM ouabain, 1 mM P¹,P⁵-Di(adenosine-5′) pentaphosphate, 0.040 mg/ml ampicillin, 0.2 mM adenosine diphosphate (ADP), 10 mM Tris-HCl pH 7.4, and the metabolic substrates (5 mM pyruvate + 2.5 mM malate or 20 mM succinate). Cells were equilibrated for 10 min at 37 °C, and then ATP synthesis was induced by the addition of 0.2 mM ADP. ATP synthesis was measured using the luciferin/luciferase ATP bioluminescence assay kit CLSII (11699695001, Roche) and a Luminometer (GloMax® 20/20 Luminometer, Promega). ATP standard solutions in the concentration range 10⁻¹⁰–10⁻⁷ M were used for calibration [27 (link)].