Furosemide

All S10b-follicles of a single fly (approximately 10–20 follicles) were divided into a control group and an experimental group. Inhibition was performed for 20 min in R-14 medium containing the respective inhibitor according to [16 (link)]. The following inhibitors of ion-transport mechanisms were used: Na⁺/H⁺-exchangers (NHE) and 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 without the respective inhibitor. After treatment, wild-type follicles were fixed and stained before analysis while GFP-follicles were directly analysed as described above. Each experiment was performed at least three times.

All reagents were of the highest purity commercially available. SA, 5-ALA, 4-iodophenol, TMP, furosemide, saponin, IPP, doxycycline, luminol, and DHA were purchased from Sigma (St. Louis, MO, USA). 5-[¹³C₄]-ALA was purchased from Cambridge Isotope Laboratories, Inc (Tewksbury, MA, USA).

The first injection for each animal was given at the beginning of the mice daily light cycle. Mice were randomly divided into 6 groups. All intervention groups had kanamycin (Sigma Aldrich, Oakville, ON, Canada, Cat. No. F4381-1G) (1 mg/g) injected subcutaneously followed by furosemide (Sigma Aldrich, Oakville, ON, Canada, Cat. No. F4381-1G) (0.5 mg/g) injected intraperitoneal 30 minutes later (T₀). Animals were then kept under a heat lamp and provided 100% O₂ until they returned to normal levels of activity. Those showing signs of severe dehydration or other significant illness were euthanized. All animals were monitored by trained animal care technologists supervised by a veterinarian. A total of 71 mice were used.

Alternariol (AOH) was obtained from Cfm Oskar Tropitzsch (Marktredwitz, Germany). Racemic warfarin (WAR), naproxen (NAP), human serum albumin (HSA), bovine serum albumin (BSA), porcine serum albumin (PSA), rat serum albumin (RSA), glimepiride (GLIM), furosemide (FUR), bilirubin (BIL), phenylbutazone (PBut), indomethacin (IME), racemic ibuprofen (IBU) and S-camptothecin (CPT) were purchased from Sigma-Aldrich (Budapest, Hungary). Ethinylestradiol (EE) was purchased from Serva (Budapest, Hungary). Spectroscopic grade ethanol (96%), as well as HPLC-grade acetonitrile and methanol were obtained from VWR (Budapest, Hungary). Stock solutions of AOH (5000 μM), bilirubin (500 μM), methyl orange (2000 μM), and glimepiride (2000 μM) were prepared in spectroscopic grade dimethyl sulfoxide (DMSO; Fluka, Bucharest, Romania). Stock solutions of indomethacin and ethinylestradiol (both 2000 μM), as well as ibuprofen, furosemide, phenylbutazone, and naproxen (each 2500 μM) were prepared in ethanol (96%, spectroscopic grade). The applied amounts of organic solvents did not affect significantly the fluorescence measurements (tested in each spectroscopic model). All stock solutions were stored at −20 °C, and protected from light.