Adenosine triphosphate (atp)

To prime the NLRP3 inflammasome, 5 × 10⁵ BMDMs were pre-treated with 10 ng/mL LPS (Enzo Biochem, Farmingdale, NY, USA) for 4 h, followed by the addition of 1 mmol/L ATP (Sigma-Aldrich, St. Louis, IL, USA) for 1 h. Additionally, some cells were pre-treated with DCA to inactivate inflammasome activity for 30 min and then treated with LPS and/or ATP. Similarly, some cells were activated with 10 ng/mL LPS and then treated with DCA for 30 min before adding 1 mmol/L ATP for 1 h. In addition, some cells were treated with different concentrations (0, 5, or 10 mmol/L) of sodium succinate or with dimethyl succinate (Sigma-Aldrich, St. Louis, USA) with or without pertussis toxin (200 ng/mL, Sigma-Aldrich, St. Louis, USA) and with or without GPR91 antagonist validated at the CBMR Metabolic Receptology laboratory [cpmd0441 (IC₅₀ = 2.63E − 06M); synthesized by SIA Enamine, Latvia] for 2 h prior to experiments. IL-1β production in the supernatants of cultures was analysed by ELISA (R&D Systems, MN, USA) according to the manufacturer’s instructions.

The rabbit monoclonal antibodies against STAM1 (cat. #12434-1-AP) and Ube1 (cat # 15912-1-AP) and mouse monoclonal antibody against GAPDH (cat. #60004–1) were from Proteintech. The rabbit monoclonal antibodies against UbcH7 (cat. #8721), ubiquitin (cat. #43124), STAM1 (cat #13053), and βarrestin1 (cat. #12697) were from Cell Signaling Technology. The rabbit monoclonal antibody against AIP4 (cat. #ab108515) was from Abcam. The mouse monoclonal antibody against the His epitope (cat. #A00186) was from GenScript. The mouse monoclonal antibody against the hemagglutinin epitope was from BioLegend (cat. # MMS-101R). ATP (cat #A2383-5G) was from MilliporeSigma. UbcH7 (E2; cat# E2-640–100), lysine-less ubiquitin (0K-Ub; cat# UM-NOK) and lysine-less biotinylated ubiquitin (0K-Ub biotin; cat# UB-560) were from R&D Systems. ATP (cat# 34369–07–8) and MgCl₂ (cat# M8266) were from Sigma-Aldrich. Protease inhibitors aprotinin (cat# 97062–754), leupeptin (cat # 97063–924), pepstatin (cat # 97064–248), and pH test strips (cat# BDH35309.606) were from VWR.

ATP (Sigma) was solubilized in nuclease-free water to create a stock concentration of 25 mM. Cells were treated with 200 μM ATP for 6 h. A740003, a competitive P2X₇ receptor antagonist (MCE), was solubilized in DMSO (Sigma) at a stock concentration of 5 mM. Cells were pre-treated with 20 μM A740003 for 24 h. Citreoviridin (Cayman Chemical Company) is an impermeable ATP synthase inhibitor. We solubilized Citreoviridin in DMSO to create a stock concentration of 40 mM, before diluting it down to 2 μM for cell treatment. Inhibitors of mitochondrial fission markers Drp1 and mdivi-1 (Sigma) were solubilized in DMSO at a stock concentration of 50 mM, before being diluted to 30 μM for cell treatments lasting 24 h.

CoA pull-down was performed in the presence of purified WT NME1 or the T94D mutant. Three micrograms of WT or mutant GST-tagged or untagged NME1 was incubated overnight at 4°C (300 μl final volume) with 50 μl of pre-washed CoA-agarose resin (buffer: 20% glycerol, 3 mM MgCl₂, 50 mM Hepes, 500 mM KCl, 1 mM DTT, and one mini-tablet of EDTA-free protease inhibitor) in the presence or absence of ATP (Sigma-Aldrich). The enriched CoA-agarose was washed three times in the buffer and, after a final wash with PBS, was incubated for 1 hour at 4°C in 50 μl of 5 mM free ligand (either CoA, NAD, FAD, ADP-ribose, ATP, or dephospho-CoA; all from Sigma-Aldrich) or 50 μl of H₂O as a control. Flow-through and CoA-agarose (eluted by free CoA) fractions were analyzed by WB to detect NME1 (by an anti-GST or anti-NME2 antibody) as well as histidine phosphorylation. GST-NME1 and GST-NME2 were also pulled down using extracts from bacteria expressing these proteins, following the same protocol as the CoA pull-down from eukaryotic cell extracts.

For the in vitro experiments, the murine cardiac muscle cell line (HL-1 cells) [25 (link)] (Sigma Aldrich, St. Louis, MO, USA) and human cardiomyocytes (iPS) (Cellular Dynamics, Madison, WI, USA) were used. Murine HL-1 cells were cultured in an HL-1 expansion medium at 37°C in an atmosphere of 5% CO₂. Human cardiomyocytes (iPS) were cultured for 10 days in a maintenance medium (Cellular Dynamics, Madison, WI, USA) at 37°C in an atmosphere of 7% CO₂.
Next, HL-1 cells were treated with 20 μg/ml LPS and the human cardiomyocytes with 10 μg/ml LPS for 6 h at 37°C. For the determination of troponin I elevation of cells in the presence of LPS with either ATP or nigericin, the cells were treated for 5 h with LPS with the abovementioned concentrations and for one further hour either with 1 mM ATP (Sigma Aldrich, St. Louis, MO, USA) or with 10 μM nigericin (Sigma Aldrich, St. Louis, MO, USA). The control groups were incubated in cell culture media without any supplements.

For hypoxia/reoxygenation (H/R) studies, confluent HK2 cells were incubated under a hypoxic condition of 1% O₂, 94% N₂, and 5% CO₂ in a controlled hypoxic chamber (Thermo Scientific) for 24 h. Then, they were removed to a normoxic condition of 21% O₂, 74% N₂, and 5% CO₂ for reoxygenation. Controls were cultured under a normoxic incubator for identical times. HK2 cells were treated with ATP (Sigma-Aldrich) at indicated concentrations for 8 h. Apyrase (0.2 U/ml, 1 U/ml, or 5 U/ml) or A438079 (20 µM) was added to the culture medium for 1 h before exposing to ATP or H/R. Experiments were performed in triplicate.