Amiloride

deg‐1 cRNA and MDEG cRNA were separately injected into oocytes and incubated at 18°C for 3–6 days before electrophysiological recordings were performed. We held the membrane potential at −80 mV and recorded the macroscopic current using the two‐electrode voltage‐clamp technique with a bath clamp amplifier (OC‐725C; Warner Instruments, USA) and pClamp software (Molecular Devices, USA) in bath solution containing 100 mM NaCl, 2 mM MgCl₂, and 10 mM HEPES (pH 7.3). Temperature stimulation was regulated using a lab‐made temperature controller with a range of 10–35°C and was monitored by both a thermistor probe adjacent to the oocytes and a thermometer (Digital Thermometer PTC‐401; Unique Medical, Japan). An Arrhenius plot was created, indicating the current amplitude induced by temperature changes on the y‐axis (log scale) versus the inverse of temperature on the x‐axis (1,000/K). Temperature thresholds were determined by the intersection of the two linear regions (magenta lines), and all thresholds were then averaged. As a negative control experiment, we used amiloride, an inhibitor of DEG/ENaC ion channel, and oocytes incubated at 18°C in bath solution with 500 μM amiloride (Sigma‐Aldrich) for 48 h.

Ribavirin (Sigma-Aldrich, St. Louis, MO, USA), 5-fluorouracil (Sigma-Aldrich), 5-azacytidine (Sigma-Aldrich), and amiloride (Sigma-Aldrich) were used in this study. All of these mutagens were dissolved in RPMI-1640 medium (Sigma-Aldrich) at stock concentrations of 15 mM (Ribavirin) and 20 mM (5-fluorouracil, 5-azacytidine, and amiloride), sterile-filtered using a 0.22-μm syringe filter, aliquoted, and stored at −20°C until use.

To compensate for the variability observed between different flies, all S10b-follicles of a single fly (approximately 10–20 follicles) were divided into a control group and an experimental group. Inhibition and staining of living follicles, using either CFDA or DiBAC, were done simultaneously for 20 or 60 min, depending on the type of experiment, in R-14 medium containing the respective inhibitor and the fluorescent probe.
The following inhibitors of ion-transport mechanisms were used: Na⁺/H⁺-exchangers (NHE) and amiloride-sensitive Na⁺-channels were blocked with amiloride (Sigma-Aldrich, Germany; 10 μM; dissolved in DMSO), V-ATPases with bafilomycin A1 (Sigma-Aldrich; 160 nM; dissolved in DMSO), ATP-sensitive K⁺-channels with glibenclamide (Biomol, Germany; 100 μM; dissolved in DMSO), voltage-dependent L-type Ca²⁺-channels with verapamil-HCl (Sigma-Aldrich; 50 μM; dissolved in ethanol), Cl⁻-channels with 9-anthroic acid (Sigma-Aldrich; 100 μM; dissolved in ethanol) and Na⁺/K⁺/2Cl⁻-cotransporters with furosemide (Sigma-Aldrich; 1 mM; dissolved in DMSO). Control experiments were performed in R-14 medium containing 0.1–1% v/v ethanol or DMSO, respectively, without the inhibitor.

Whole cell recording was performed using the protocol by Hayashi et al. (1992) with slight modification. A coverslip on which cells were grown was mounted on a recording chamber affixed on the stage of a Nikon Eclipse T1 inverted microscope, equipped with a MP‐225 motorized micromanipulator. Cells were perfused with solution containing (in mmol/L) 116 NaCl, 6 KCl, 2.4 CaCl₂, 6 glucose, 10 HEPES, 10 tetraethylamonium (TEA), and 1 BaCl₂, pH 7.4. The pipette had 116 KCl, 1.2 MgCl₂, 6 glucose, 10 HEPES, 10 TEA, 10 BAPTA, and 0.01 BaCl₂ (pH 7.2). Recording was made using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale). Currents were recorded with 20 mmol/L NH₄Cl, at a holding voltage of ‐70 mV. For amiloride experiments, currents induced by NH₄Cl were recorded in the absence and then presence of 1 μmol/L amiloride (Sigma‐Aldrich; Cat#: A7410). I–V relationship was determined by a staircase voltage command between −80 to +15 mV (170 msec duration). Currents were recorded using pClamp 8.0 (Molecular Devices) and signals were low‐pass filtered (3 db at 2 kHz, 8‐pole Bessel filter). Experiments were performed at room temperature.

Ussing chamber measurements of FDLE cells were performed 4 days after cell isolation, as previously reported (5 (link)). Only monolayers with a transepithelial resistance (R_te) exceeding 300 Ω·cm² were included in the analyses. Electrophysiological solutions consisted of: 145 mM Na⁺, 5 mM K⁺, 1.2 mM Ca²⁺, 1.2 mM Mg²⁺, 125 mM Cl⁻, 25 mM ${\text{HCO}}_3^{-}$ , 3.3 mM H₂ ${\text{PO}}_4^{-}$ , and 0.8 mM ${\text{HPO}}_4^{2-}$ (pH 7.4). For the basolateral solution, 10 mM glucose was used, while 10 mM mannitol was used in the apical solution. During measurements, the solutions were continuously bubbled with carbogen (5% CO₂ and 95% O₂). Equivalent short-circuit currents (I_SC) were determined every 20 s by measuring transepithelial voltage (V_te) and R_te with a transepithelial current clamp (Physiologic instruments, San Diego, CA) and calculating the quotient I_SC = V_te/R_te. After the I_SC reached a stable plateau (I_base), amiloride (10 μM, # A7410, Sigma-Aldrich) was applied to the apical chamber to assess the amiloride-sensitive ΔI_SC (ΔI_amil). The current reduction induced by amiloride (ΔI_amil) was used as a measure of ENaC activity. amiloride was dissolved in water.

HBEC were mounted in Ussing chambers connected to a VCC MC6 or MC8 voltage clamp unit (Physiological Instruments, San Diego, CA, USA). CFTR activity was measured as the change in short-circuit current (I_SC) caused by CFTR inhibition with 10 µM CFTR_inh-172 (#C2992; MilliporeSigma, Burlington, MA, USA) after CFTR stimulation with 10 µM forskolin (#F3197; MilliporeSigma) in the presence of 10 µM amiloride (#A7410; MilliporeSigma) under a basolateral-to-apical Cl⁻ gradient. ENaC activity was measured as the change in I_SC after amiloride. For measurements of BK channel function, the basolateral membrane of HBEC mounted in Ussing chambers was permeabilized with 20 µM Amphotericin B (#A2411; MilliporeSigma), 10 µM Nigericin (#4312; Bio-Techne Corporation, Minneapolis, MN, USA), and 10 µM Valinomycin (#3373; Bio-Techne Corporation). BK channel activity was then measured as the change in I_SC following stimulation with 10 µM ATP (#A9187; MilliporeSigma) in the presence of 10 µM amiloride under a basolateral-to-apical K⁺ gradient and an apical-to-basolateral Na⁺ gradient as previously described^{24 (link)}.

Cells were seeded in 6-well culture plates at a density of 500000 cells/well, after 24 h incubated with different endocytosis inhibitors [2.5 mM amiloride (Sigma-Aldrich), 2.5 μg/mL chlorpromazine (Sigma-Aldrich) and 100 μM indomethacin (Sigma-Aldrich)] and then maintained for 1 h at 37°C with 5% of CO₂-air. The concentrations and treatment times of each chemical inhibitors were optimized in a preliminary experiment to select the maximum non-toxic doses and treatment times. After 1 h incubation, the inhibitors were removed, and the cells exposed to 2 μg/mL AuNPs in complete medium for 24 h incubation. Then ICP-MS protocol was followed as described in the previous section.