35 mm glass bottom dishes

Fluorescent labeling of acidic organelles was performed using LysoSensor Green DND-189 probe (Life Technologies). The LysoSensor reagent exhibits a pH-dependent increase in fluorescence intensity upon acidification. Vero cells were seeded on glass bottom 35-mm dishes (Ibidi), washed with PBS, and treated (4 h) with 25 mM NH₄Cl, or 10 μM of either D or EGCG in EMEM supplemented with 25 mM HEPES pH 7.4. Then, the culture medium was replaced by fresh medium containing 1 μM of LysoSensor probe, and cells were incubated during 5 min at 37°C, washed three times with the same media without LysoSensor probe, and observed under a fluorescence microscope. Images were acquired and processed using the same microscope settings. Control cells were treated in parallel with the same amount of drug solvent (H₂O for NH₄Cl and DMSO for polyphenols).

Cells were grown on no. 1.5 glass coverslips and fixed in 4% paraformaldehyde for 10 min at room temperature. Cells were washed three times with PBS containing 1.5 mg/mL glycine, permeabilized in 0.25% Triton X‐100 for 5 min, and washed three times with PBS. Cells were blocked with 5% donkey serum for 30 min followed by a 1‐ to 2‐h incubation with primary antibody at room temperature. Cells were washed three times with PBS and incubated with Alexa Fluor conjugated secondary antibodies for 30 min at room temperature. Cells were washed three times with PBS and mounted on slides using ProLong Diamond antifade mountant (Thermo Fisher, P36970).
For live‐cell imaging, cells were seeded on μ‐Slide 8 well or glass bottom 35 mm dishes (Ibidi) and incubated for 24 h. Imaging was performed in DMEM without phenol red (Sigma‐Aldrich) and supplemented with 20 mM HEPES.
Imaging was performed on a Leica SP8 FALCON inverted confocal system (Leica Microsystems) equipped with a HC PL APO 63×/1.40 oil immersion lens and a temperature‐controlled hood maintained at 37°C and 5% CO₂, for live‐cell imaging. The microscope was controlled by Leica Application Suite X (LASX). Hoechst 33342/TagBFP were excited using a 405 nm Diode laser, and EGFP/Alexa488, mCherry/Alexa568, and Alexa647 fluorescence were excited using a tuned white light laser. Scanning was performed in line‐by‐line sequential mode.

HeLa and HEK293 cells were maintained in Dulbecco's minimal essential medium (DMEM; Gibco, Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% of a penicillin/streptomycin commercial antibiotic mixture (Gibco; Invitrogen), under controlled conditions of temperature and CO₂ (37°C, 5% CO₂). Cell culture dishes were purchased from Techno Plastic Cultures (AG) unless otherwise indicated. For all experiments, cells were seeded at a density of 10 000 cells/cm² regardless dish size. For flow cytometry assays, cells were grown on 6‐well plates (35 mm diameter). For cell viability and adenosine triphosphate (ATP) assays, cells were grown on 96‐well and 24‐well dishes, respectively. For microscopy, cells were seeded on glass‐bottom 35 mm dishes (10 mm glass surface diameter; IBIDI) and fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) right before imaging. For protein extraction (PAGE and filter trap assays) cells were seeded on 60 or 100 mm dishes.

The Chinese hamster ovary CHO-K1 cells were cultured in DMEM/F12 (Gibco, Carlsbad, CA, USA) medium supplemented with 10% FBS (Gibco, Carlsbad, CA, USA), 2 mM glutamine (Gibco, Carlsbad, CA, USA), and penicillin/streptomycin at a concentration of 100 U/mL (Thermo Fisher Scientific, Waltham, MA, USA), maintained in a humidified incubator at 37 °C with 5% CO₂, and passaged routinely using trypsin-EDTA (Thermo Fisher Scientific, Waltham, MA, USA). For living-cell confocal microscopy, the cells (1 × 10⁵ CHO-K1 cells in 1.5 mL growing media) were seeded in glass-bottom 35 mm dishes (Ibidi GmbH, Gräfelfing, Germany) and incubated for 48 h until reaching a confluence of ~70%. Complexes 1–4 were dissolved in DMSO at a concentration of 2 mM, diluted with supplemented growing media reaching the concentration of 0.5 mM, and added to the cells in a final concentration of 5–25 µM. After incubation with the probe for 24 h, cells were washed with fresh media with all supplements.