Valinomycin

All cell lines used in this study were obtained from ATCC (Manassas, VA, USA). MCF7, A549, and SH-SY5Y cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM), and SKBR3 cells were cultured in McCoy’s5A supplemented with 10% FBS and penicillin/streptomycin in a humidified 5% CO₂ incubator at 37 °C. MDA-MB-231 and MDA-MB-468 cells were cultured in Leibovitz’s L-15 medium supplemented with 10% FBS and penicillin/streptomycin in a humidified incubator at 37 °C. For mitochondrial inhibition, carbonyl cyanide m-chlorophenyl hydrazine (CCCP) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) treatment was performed at 2.5, 5, or 10 μM for an optimal hour. MG132 (Peptide Institute, Osaka, Japan) and N-acetyl cysteine (NAC) (Nacalai Tesque, Kyoto, Japan) treatments were performed at 10 μM and 10 mM, respectively, 30 min before CCCP treatment. For alternative mitochondrial inhibition, cells were treated with antimycin A (Santa Cruz Biotechnology, Dallas, TX, USA), oligomycin A (Cayman Chemicals, Ann Arbor, MI, USA), deferiprone (DFP) (Tokyo Chemical Industry, Tokyo, Japan), rotenone (Cayman), or valinomycin (Cayman) for 24 h. Cells were cultured up to 1 month after thawing. Mycoplasma contamination was routinely tested using the e-Myco Mycoplasma PCR Detection kit (iNtRON Biotechnology, Inc., Korea).

HEK293 cells [American Type Culture Collection (ATCC); CRL-1573] were grown in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Euroclone, Pero, Italy), 200 U/ml penicillin, 200 μg/ml streptomycin and 2 mM glutamine at 37°C, 5% CO₂. Calu-6 cells (ATCC; HTB-56) were grown in Eagle's minimum essential medium (LGC Standards, London, UK; 30-2003) supplemented with 10% FBS, 200 U/ml penicillin and 200 μg/ml streptomycin at 37°C, 5% CO₂. Cells were transiently transfected using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer's protocol. All cell lines were routinely tested by PCR for the absence of mycoplasma contamination. When indicated cells were treated with 100 nM valinomycin (Cayman Chemical, Ann Arbor, MI, USA; 10009152) for 20 h.

The mitochondrial membrane potential assay can be used to evaluate cell function and health; thus, cationic carbocyanine dye (JC-1) staining cells were used [35 (link)]. Thus, ConA-stimulated T lymphocytes exposed to IC₅₀ doses of P. alata extract and polyphenols were washed with PBS 0.1 M (pH 7.4). After centrifuging at 300× g for 5 min at room temperature, the cells were labelled with 1 µg of JC-1 dye (Santa Cruz Biotechnology, Dallas, TX, USA), diluted in 100 µL of PBS and incubated at 37 °C for 30 min. The cells were then washed with PBS, centrifuged for 5 min at 300× g and analyzed immediately by flow cytometry. T cells were submitted to valinomycin (Cayman Chemical, Ann Arbor, MI, USA) as a positive assay control at 100 µM. The results are expressed as the percentage of depolarized (green fluorescent) and polarized (orange–red fluorescent) T lymphocyte mitochondrial membrane potential.

H1299, A549, and MRC-9 cells (5 × 10⁴ cells/ well) seeded in chamber slides were treated with DMSO and CMLD-2 (30 µM). Cells treated with valinomycin (30 µM; Cayman, Ann Arbor, MI) served as positive control^{40 (link)}. After 24 h and 48 h of treatment, cells were stained using the cationic dye JC-1 (Sigma Aldrich) as described previously^{27 , 34 (link)}. Briefly, the cells were incubated with JC-1 staining solution for 20 min at 37 °C. At the end of the incubation period, the staining solution was aspirated and cells were washed twice with culture medium. Cells were subsequently overlaid with fresh culture medium and observed under an inverted Leica SP2 MP confocal microscope (Leica Microsystems, Buffalo Grove, IL). Based on the changes in the membrane potential, the presence of JC-1 aggregates and JC-1 monomers was determined by excitation/emission at 525 nm/590 nm and 490 nm/530 nm, respectively. The quantitative difference in membrane potential was measured by determining the ratio of green over red fluorescence intensity and analyzed for statistical significance.

Carbonyl cyanide m-chlorophenyl hydrazone (CCCP; Sigma Aldrich, C2759) was used at a concentration of 10 µM for all experiments with HEK293 and HeLa and 20 µM for all ReN cell experiments. Valinomycin (Cayman Chemical, 10009152) was used for experiments with fibroblasts at 1 µM. HeLa cells were plated 20–24 h before transfection with siRNA/DNA using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019). The following amounts were used for transient siRNA and DNA transfection per 6-well: 3 µl of 20 nM siRNA, 500 ng of PINK1 cDNA, and 4.5 µl of Lipofectamine per 250 µl of Opti-MEM media (Thermo Fisher Scientific, 51985034). For HCI, media was replaced after 4 h and cells were cultured for an additional 20 h. For immunoblot analysis, media was replaced after one day. Control siRNA (all stars negative control, Qiagen, 1027281) or PINK1-specific siRNA (5’-GACGCTGTTCCTCGTTATGAA-3’, Qiagen) were used along with siRNA-resistant PINK1 cDNA. PINK1 constructs with C-terminal V5 or mCherry tag have been described previously [27 (link)]. PINK1 G411 substitutions were introduced by site-directed mutagenesis of WT or KD (kinase dead: K219A+D362A+D384A) PINK1 cDNA constructs and confirmed by sequencing.

Venetoclax (Cat. N° HY-15531), BIRB796 (doramapimod; HY-10320), SB203580 (HY-10256), M3258 (HY-111790), ML604440 (HY-114170), marizomib (HY-10985), dithiothreitol (HY-15917; DTT), ONX-0914 (HY-13207), quinoline-Val-Asp-difluorophenoxymethylketone (QVD-OPh; HY-12305), and adenosine 5’-triphosphate (HY-B2176; ATP) were from MedChemExpress (Monmouth Junction, NJ, USA). 4′,6-Diamidino-2-phenylindole (DAPI; 14285) and valinomycin were from Cayman Chemical (Ann Arbor, MI, USA). IFNγ (Cat. N° IF002), phorbol 12-myristate 13-acetate (PMA; P8139), and ionomycin calcium salt (I0634s) were from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human sCD40 ligand (sCD40L; 310-02) was from PeproTech (London, UK).