Zen 2.3 imaging software

Cells on coverslips were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and blocked with 1% BSA (Sigma-Aldrich) plus 0.1% Triton X‐100 (Sigma-Aldrich) in PBS. Mouse monoclonal anti-MDM4 (clone 8C6, Merck-Millipore) was applied at 1:200 dilution in PBS-Tween overnight at 4 °C. Next, the coverslips were washed in PBS and incubated for 1 h with Alexa 488-conjugated anti-mouse secondary antibody (Thermo Fisher Scientific) diluted 1:500 in PBS-Tween. Cell nuclei were counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich), and fluorescence microscopy was performed using the LSM700 confocal microscope (Carl Zeiss). To analyze the MDM4 foci, the number of foci formed inside the nuclei, as well as the total number of nuclei in the picture, was counted manually in 10 representative snapshots of each sample. The foci/nuclei ratio was calculated as total foci inside nuclei / total nuclei. The number of foci-containing nuclei per snapshot was also assessed. The nuclear area was analyzed on maximum intensity projections (MIP) of confocal z-stacks using the Zen 2.3. imaging software (Zeiss).

Immunofluorescence (for LC3B-II, Tom20, and LAMP1 detection) and fluorescence intensity of mtKeima and mitoTimer was observed under a fluorescence microscope to assess mitophagy. For immunofluorescence staining, samples were pre-treated as described above, fixed in 4% paraformaldehyde, and permeabilized with PBS-Triton (0.4%). Subsequently, the samples were incubated overnight with primary antibodies against LC3B‐II, Tom20, and LAMP1. Next, the samples were rinsed and incubated with a secondary antibody in a humidified container for 1 h at 37 °C. Finally, the samples were stained with DAPI. Random fields of view were selected, and each group was arranged for several biological duplications. The primary and secondary antibodies used in this study are listed in Supplemental material. 2.
mtKeima was detected at an excitation wavelength of 458 nm at neutral pH (autophagosome) and an excitation wavelength of 561 nm at an acidic pH (autolysosome) using confocal microscopy- Zeiss LSM 800^{50 (link)}. The red intensity area and total intensity area (red and green intensity area) of mtKeima proteins were evaluated using Zeiss ZEN 2.3 imaging software. MitoTimer shifts its color as the protein matures, and thus, its fluorescence shifts over time from green (excitation wavelength = 483 nm) to red (excitation wavelength = 558 nm)^{51 (link)}.

Exosome uptake assay was carried out as previously described [56 (link)]. Exosomes derived from EMP2-transfected H2170 cells were labeled with 2 µM PKH67 dye (Sigma-Aldrich, Munich, Germany) for 5 min. The staining reaction was stopped by adding an equal volume of 1% ED-FBS in PBS. The cells were washed twice with PBS by centrifugation at 100,000× g for 70 min. H2170 cells were seeded into 6-well plates and incubated with PKH67-labeled exosomes (500 ng/mL) for 24 h in 2 mL RPMI 1640 medium containing ED-FBS. For confocal analysis, 7500 cells were seeded in Ibidi Chambered Coverslips (Ibidi GmbH, Gräfelfing, Germany) and treated with PKH67-stained exosomes or an equivalent volume of PBS/PKH67 as a control. After 24 h, cells were washed with ice-cold PBS and fixed with 2% formaldehyde for 10 min at RT. The slide was incubated with primary and secondary antibodies (Table S4) for 2 h and 60 min, respectively. DAPI (1 µg/mL; Sigma Aldrich) was added to visualize the nuclei. Cellular uptake of exosome was observed using a Zeiss LSM 710 confocal laser microscope (Carl Zeiss Microscopy GmbH) with 405 nm (DAPI) and 488 nm (PKH67) lasers. Confocal images and the mean fluorescence intensity (MFI) were analyzed with ZEN 2.3 imaging software (Carl Zeiss Microscopy GmbH).

For the metaphase assay and immunofluorescence, we performed the imaging on a microscope (Carl Zeiss, imager.M2) fitted with a 63× oil-immersion objective lens and 10× ocular lens. We processed and analysed the images with ZEN 2.3 imaging software (Carl Zeiss Microscopy GmbH, Jena, Germany). For the Boyden chamber and wound-healing assays, we imaged the pictures using a microscope (Carl Zeiss, Vert.A1) fitted with a 10× or 5× objective lens and 10× ocular lens. We analysed the images using ZEN 2.3 imaging software and ImageJ software (ImageJ 1.51f, Wayne Rasband, National Institutes of Health, USA). For the gelatine matrix degradation assays, we performed the imaging on a microscope (Carl Zeiss, imager.M2) fitted with a 40× objective lens and 10× ocular lens. We processed and analysed the images using ZEN 2.3 imaging software and ImageJ software.