Fluorobrite dmem medium

U-2 OS and MRC-5 cells (1·10⁵ cells per well) were seeded on 35 mm glass bottom dishes for live-cell imaging (MatTek Corporation, USA) and left to adhere for 24 h. Then, the cells were washed with phosphate-buffered saline (PBS) and incubated with compound 5 or dansyl amide fluorophore (0.1–2.0 µM) dissolved in FluoroBrite DMEM medium (Thermo Fisher, USA) for 0.5–2.5 h. After the incubation period, the cells were washed with PBS and fresh FluoroBrite DMEM was added.

Intracellular localization of overexpressed PPO-GFP construct was determined by confocal microscopy. Briefly, a day before imaging, cells were passaged onto glass cover slips. Immediately prior to imaging, cells were successively treated at 37 °C with 50 nM Mitotracker Deep Red FM (ThermoFisher Scientific) for 25 min and then with Hoechst 33258 (100 µg/ml) for 30 min. Coverslips were immersed in a FluoroBrite DMEM medium (ThermoFisher Scientific) and cells were placed into a humidity chamber (OKOlab, Puzzuoli, Italy) and imaged by a confocal microscope (DMI8, Leica Microsystems, Wetzlar, Germany) using a 63× water immersion objective. Scans were analyzed by the ImageJ 1.53k software^{77 (link)} (National Institutes of Health, Bethesda, MD, USA) and Adobe Photoshop CS4 software (Adobe Systems, San Jose, CA, USA; https://www.adobe.com/products/photoshop.html).

For the mPTP assay, HT-29 cells were seeded at 2.0 × 10⁵ cells/well into 24-well plates overnight in complete media. Cells were infected at an MOI of 1 and incubated at 37 °C with 5% CO₂. After infection, the cells were washed with HBSS 1X, the medium was replaced with fresh complete Fluorobrite DMEM medium (Thermo Fisher Scientific, Waltham, MA, USA, A1896701), and an Image-iT LIVE Mitochondrial Transition Pore Assay Kit (Thermo Fisher Scientific, I35103) was used to acquire fluorescence images with Lionheart-FX microscopy (Agilent BioTek, Santa Clara, CA, USA) at different time points of infection. This kit utilizes 1 µM of Calcein-AM (green), which accumulates in the mitochondria of live cells, and 1 mM of Cobalt (Co²⁺) to quench the signal of Calcein when the mPTP is open for a prolonged period. As an experimental control, we use 0.5 µM of ionomycin, an ionophore that induces the opening of the mitochondrial pore. The kit was employed according to the manufacturer’s specifications, and cells were fixed with 4% formaldehyde (Thermo Fisher Scientific, 28908) and the samples were mounted using Prolong Diamond Antifade Mountant (Thermo Fisher Scientific, P36961).

For the mitochondrial fragmentation assay, HT-29 cells were seeded at 2.0 × 10⁵ cells/well into 24-well plates overnight in complete media. Cells were infected at an MOI of 1 and incubated at 37 °C with 5% CO₂. After the infection, the cells were washed with HBSS 1X, the medium was replaced with fresh complete Fluorobrite DMEM medium (Thermo Fisher Scientific, A1896701), a 0.2 µM Mito Tracker Red CMXRos (Thermo Fisher Scientific, M7512) was used for 15 min, the cells were fixed with 4% formaldehyde (Thermo Fisher Scientific, 28908), and the samples were mounted using Prolong Diamond Antifade Mountant (Thermo Fisher, P36961) to visualize HT-29 mitochondria morphology with Lionheart-FX microscopy (Agilent BioTek) at different time points of infection.

Fluorescence labeling of the proteins was performed according to the AlexaFluor568 carboxylic acid NHS ester manufacturer’s instructions (ThermoFisher Scientific (Waltham, MA, USA)). SKBR-3-Luc (JCRB1627.1), MCF-7-Luc (JCRB1372), and HCC-1954-Luc (JCRB1476) were purchased from JCRB (Tokyo, Japan). For internalization 5000 cells per well were seeded in a µ-slide 8-well chamber coverslip (ibidi (Gräfeling, Germany)) and incubated for 48 h at 37 °C, 5% CO₂ (RPMI1640, 10% (v/v) fetal bovine serum (FBS), ThermoFisher Scientific (Waltham, MA, USA)). The medium was replaced by FluoroBrite DMEM medium with 10% (v/v) FBS (ThermoFisher Scientific (Waltham, MA, USA)) and 50 nM labeled Fab or bsFab was added and incubated for 24 h. Before starting microscopy, cells were washed with FluoroBrite DMEM medium without FBS. Cells were mixed with equivalent amounts of FluoroBrite DMEM medium and life cell staining buffer (supplemented with either LysoBriteBlue (1:500), AATBioquest (Sunnyvale, CA, USA), or HOECHST33342 (1:2000), ThermoFisher Scientific (Waltham, MA, USA)) and incubated prior to microscopy for 0.5–2 h and 10 min, respectively (Eclipse TE2000-E, CFI Plan Fluor 40x oil lens, Nikon (Tokyo, Japan)). Lysosome and nucleus staining were excited at 405 nm (filter 450/35), fluorescent proteins at 568 nm (filter 650LP).