Rhodamine 123

Mitochondria were isolated from cells grown in YPGlyA medium at 28 °C by enzymatic method according to the protocol described previously^{47 (link)}. For all assays, they were diluted to 75 µg/mL in respiration buffer (10 mM Tris-maleate pH 6.8, 0.65 M mannitol, 0.35 mM EGTA, and 5 mM Tris–phosphate). Oxygen consumption rates were measured using a Clarke electrode adding consecutively 4 mM NADH (state 4 respiration), 150 µM ADP (state 3) or 4 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP) (uncoupled respiration), as described previously^{48 (link)}. The rates of ATP synthesis were determined under the state 3 conditions with 750 µM ADP; every 15 s, 100 µl aliquots were withdrawn from the oxygraph cuvette and added to 50 µl of the 3.5% (w/v) perchloric acid and 12.5 mM EDTA solution already prepared in the tubes to stop the reaction. The samples were then neutralized to pH 6.5 by the addition of KOH and 0.3 M MOPS. The synthetized ATP was quantified using a luciferin/luciferase assay (Kinase-Glo Max Luminescence Kinase Assay, Promega) in a Beckman Coulter Paradigm plate reader. The participation of F₁F_O-ATP synthase in ATP production was assessed by measuring the sensitivity of ATP synthesis to oligomycin (3 μg/mL). The specific ATPase activity at pH 8.4 of non-osmotically protected mitochondria was measured using the procedure previously described^{49 (link)}. The oxygen consumption was quantified in nmol O₂ min⁻¹ mg⁻¹, the ATP synthesis in nmol of ATP min⁻¹ mg⁻¹ and ATPase activities in µmol Pi min⁻¹ mg⁻¹. Variations in transmembrane potential (ΔΨ) were evaluated by monitoring the fluorescence quenching of Rhodamine 123 (0.5 μg/mL; λ_exc of 485 nm and λ_em of 533 nm) from mitochondrial samples (0.150 mg/mL) in the respiration buffer under constant stirring at 28 °C using a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) as described previously^{50 (link)}.

P-glycoprotein (P-gp/ABCB1) activity was determined by measuring the cellular accumulation of the efflux pump ligand rhodamine 123 (R123). BECs were seeded in 24-well plates and incubated in 10 µM R123 in Ringer-HEPES for 1 h at 37 °C after cytokine treatments. Cyclosporine A (1.6 µM), which blocks P-gp and breast cancer-resistant protein/ABCG2 was used as a reference inhibitor molecule. Following cytokine treatments and incubation with R123, BEC monolayers were washed 3 times with ice-cold PBS, then solubilized in 0.1 M NaOH. Fluorescence intensity indicating intracellular R123 concentration was measured in a 96-well plate (Fluostar Optima; excitation: 485 nm, emission: 520 nm).

Assays were performed in DPBS-H (10 mM HEPES, 25 mM glucose, in Dulbecco's phosphate-buffered saline with calcium chloride and magnesium chloride) in 24-well transwell plates on a rocking shaker at 20 rpm, 37 °C, 95% humidity, and 5% CO₂. All substrates were dissolved at specific concentrations in DPBS-H and added to the luminal (A) or abluminal (B) side. The incubation times for the substrate were 15, 30, 45, and 60 min. The concentrations of the substrates were measured by a fluorescence microplate reader (Fluoroskan Ascent FL, Thermo Fisher Scientific). Digoxin, dantrolene, and salazosulfapyridine samples were pretreated with acetonitrile precipitation of proteins and measured using an LC-MS/MS system (ExionLC-QTRAP6500+, SCIEX, Framingham, Massachusetts, USA). The compound concentrations were as follows: rhodamine 123 (10 μM), Hoechst 33,342 (200 μM), 2-NBDG (100 μg/ml), digoxin (5 μM), dantrolene (5 μM), and salazosulfapyridine (sulfasalazine, SASP) (5 μM).
In this study, we employed P_e as the permeability coefficient because researchers can eliminate the influence of the insert membranes. The permeability coefficient (P_e) was calculated according to Nakagawa et al. [16 (link)] by dividing the amount of substrate in the luminal compartment (A) by the substrate concentration in the abluminal compartment (B). The volume was obtained at multiple timepoints according to the following formula: where [C]r is the amount of the compound in the receiving compartment, [C]d represents the amount of the compound in the donor compartment, and [R] is the volume of the receiving compartment. When the volume is plotted over time, the slope equals the permeability × surface area product (PS) of the membrane. The PS of the membrane with cells is called the total PS (PS_total), and the PS of the membrane without cells is called the membrane PS (PS_mem). The P_e can be computed from the PS_total and PS_mem:
1/PS_e = 1/PS_total-1/PS_mem where the units of PS and surface area are μL/mL and cm², respectively.
To calculate P_e (cm/min), the PS_e value was divided by the surface area (S) of the membrane: