Carbonyl cyanide 3 chlorophenylhydrazone

The selected membrane-active peptides gramicidin D (formyl-VGALAVVVWLWLWLWG-NHCH₂CH₂OH), cecropin A (KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK-NH₂), magainin 2 (GIGKFLHSAKKFGKAFVGEIMNS-OH), and melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH₂) were purchased from Sigma-Aldrich^® (St. Louis, MO, USA). cecropin A and gramicidin D were dissolved in DMSO and magainin 2 and melittin in pyrogenic water. The stock solutions were kept at −20 °C. The uncouplers carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), the membrane potential-sensitive fluorescent distributional probes bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC₄(3)) and 3,3′-dipropylthiadicarbocyanine iodide (diSC₃(5)), and the membrane-impermeable fluorescent dye propidium iodide (PI) were purchased from Sigma-Aldrich. Stock solutions were prepared as follows: 400 µM diSC₃(5) in 100% DMSO, 50 µM DiBAC₄(3) in 100% DMSO, and 1 mg/mL PI in ddH₂O (PI would precipitate in a more concentrated solution). All stocks, were protected from light by aluminum foil and were stable at −20 °C for at least 6 months.

In the experiments described herein, the culture media used were the following: cation-adjusted Mueller–Hinton broth (MHB II; Sigma-Aldrich, St. Louis, MO, USA and Biokar Diagnostics, Allone, Beauvais, France), Luria–Bertani broth (LB-B; Sigma, St. Louis, MO, USA), Tryptic Soy broth (TSB; Scharlau Chemie S. A., Barcelona, Spain), and Trypto-Casein Soy agar (TSA; Biokar Diagnostics, Allone, Beauvais, France). For the QS inhibition assays, the agar medium used was Luria–Bertani with a few modifications (LB*-A) and was prepared in house. The composition was as follows: 1.0 g yeast extract (Merck, Darmstadt, Germany), 10.0 g tryptone (Biolab, Budapest, Hungary), 10.0 g NaCl (Molar Chemicals, Halásztelek, Hungary), 1.0 g K₂HPO₄ (Biolab, Budapest, Hungary), 0.3 g MgSO₄·7 H₂O (Reanal, Budapest, Hungary), 5 mL Fe-EDTA stock solution and 20.0 g of bacteriological agar (Molar Chemicals, Halásztelek, Hungary) per liter of media.
The chemicals used—DMSO, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), phosphate-buffered saline (PBS; pH 7.4), ethidium bromide (EB), reserpine, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), PMZ, PAβN, and CV—were bought from Sigma-Aldrich Chemie GmbH (Steinheim, Germany), and doxorubicin (2 mg/mL) was purchased from Teva Pharmaceuticals, Budapest, Hungary.

Trypan Blue, Dulbecco’s Modified Eagle Medium (DMEM), penicillin (10,000 units), streptomycin (10 mg/mL), trypsin from porcine pancreas, 2-bromopalmitate (2-BP), palmitate, dimethyl sulfoxide (DMSO), potassium chloride, propidium iodide, RNase A, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), acridine orange, Giemsa stain, formaldehyde, paraformaldehyde, glutaraldehyde, Hoechst staining solution and poly-L-lysine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Transferrin-AlexaFluor 633, Albumin-AlexaFluor 488, rhodamine-123, goat anti-mouse IgG AlexaFluor 488 conjugate, goat anti-mouse IgG AlexaFluor 594 conjugate and Prolong Gold were purchased from Molecular Probes/Life Technologies (Eugene, OR, USA). Potassium ferrocyanide, Permount, sodium cacodylate, osmium tetroxide and PolyBed-812 resin were purchased from Electron Microscopy Sciences (Hatfield, PA, USA). Sodium dodecyl sulfate (SDS) was purchased from Ludwig Biotecnologia (Alvorada, RS, Brazil). Fetal bovine serum (FBS) was purchased from Gibco/Invitrogen/Life Technologies (Eugene, OR, USA). Nonidet 40 (NP-40) was purchased from Anresco Laboratories (San Francisco, CA, USA).

Human embryonic kidney cells (HEK293T, ATCC^®) and human adenocarcinoma cells (HeLa, ATCC^®) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher) supplemented with 10% fetal bovine serum (Thermo Fisher) at 37°C and 5% CO₂. To assess PINK1 protein levels, HeLa cells were treated with 4 µM MG132 (Sigma-Aldrich) and with 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma-Aldrich) for 24 h and 4 h, respectively, at 37°C before cell lysis. Human neuroblastoma SH-SY5Y cells (IST, Genova, Italy) were cultured in a 1:1 mixture of Ham's F12 and Dulbecco Modified Eagle Medium (Life Technologies) supplemented with 10% fetal bovine serum, in a 5% CO₂ humidified incubator at 37°C. Previously described SOD1 and SOD2 stably overexpressing SH-SY5Y cells were used (17 (link)). When required, SH-SY5Y cells were treated with 10 µM SOD mimetic drug M40403 for 24 h at 37°C.

Clorgyline (Sigma Aldrich, St. Louis, MO) was dissolved in distilled water and stored at 4°C. Fluconazole (Sigma Aldrich) was dissolved in dimethyl sulfoxide (DMSO), and stored at 4°C. Amphotericin B (Sigma Aldrich), carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Sigma Aldrich) and milbemycin A4 oxime (Novartis Animal Health, Basel, Switzerland) was dissolved in DMSO and stored at -20°C. Micafungin (Astellas, Tokyo, Japan) was dissolved in distilled water and stored at -20°C.