Citreoviridin

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.

The levels of extracellular ATP secreted by A549, SK-N-BE(2)C, and T47D cells were assayed with a bioluminescence assay kit (Sigma) according to the manual. Cultures of 1 × 10⁴ cells of each type were incubated for 24 h. The cells were refreshed with medium containing 2 μM citreoviridin (Cayman Chemical Company, Ann Arbor, MI, USA) or dimethyl sulfoxide (DMSO, sigma) for 24 h at 37 °C. Next, 200 μM ADP (Sigma) was added for 10 min. The concentration of extracellular ATP was determined using the bioluminescence assay kit. In Brief, we added 100 μl ATP assay mix solution to the assay tube and stand for 3 min at room temperature for hydrolyzing endogenous ATP. ATP standard solution were diluted with DMEM from 10⁻³ to 10⁻⁷ moles/liter. In all, 100 μl of each sample and standard were added and the plate was read immediately using a luminometer. For the starvation experiments, 1 × 10⁴ cells of each type were seeded in 12-well plates and incubated for 24 h. The cells were refreshed with medium containing 10% or 0.1% FBS for a further 18 h incubation.