Oil immersion lens

The MDA-MB-231, MCF7, and T47D cells with endogenous production of HtrA4 were seeded on coverslips (16 × 16 mm, Marienfeld, Germany) and allowed to grow overnight. The cells were fixed in PBS, containing 4% formaldehyde, for 10 min at room temperature. Next, the coverslips were placed on ice, and the cells were permeabilized by adding 0.2% Triton-X100 in PBS for 5 min before blocking with a PBSB solution (i.e., PBS containing 4% BSA) for 60 min. After blocking, the cells were incubated with the anti-HtrA4 rabbit IgG (1:87) in PBSB for 1 h. Next, the cells were washed three times with PBS and incubated for 1 h with the secondary anti-rabbit antibodies (1:2500) conjugated with Alexa Fluor 488.
The A549 cells expressing HtrA4/ΔN-HtrA4-GFP were observed alive using fluorescent properties of GFP. Mitochondria were labeled for 30 min by adding 150 nM Mito RED. Specimens were imaged using a confocal laser scanning microscope (Leica SP8X) with a 63× oil immersion lens (Leica, Wetzlar, Germany). The analyses of the HtrA4 and partner protein co-localizations were performed using the LAS AF 3.3.0 software. The pixel intensities were quantified and are presented as Pearson’s correlation and overlap coefficient.

Starch granules were visualized in leaf chloroplasts as described in Chen et al. (2022) (link). Briefly, 1.5 mm × 1.5 mm square leaf samples were harvested at the base of the lowest leaf from 2-wk-old seedlings in fixative (glutaraldehyde (2.5%), sodium cacodylate, pH 7.4 [0.05 M]). Samples were then postfixed with osmium tetroxide (1% w/v) in sodium cacodylate, pH 7.4 (0.05 m). After dehydration in an ascending series of ethanol, samples were embedded in LR white resin using EM TP embedding machine (Leica). Semithin sections (0.5 µm thick) were produced from the embedded leaves using a glass knife and were dried onto polytetrafluoroethylene-coated slides. Starch was stained using reagents from the Periodic Acid-Schiff staining kit (Abcam): using a 30-min incubation with periodic acid solution, followed by 5 min with Schiff's solution. Chloroplasts and cell walls were stained using toluidine blue stain (0.5% toluidine blue “O”, 0.5% sodium borate) for 1 min. The sections were mounted with Histomount (National Diagnostics) and imaged on a DM6000 microscope with 63× oil immersion lens (Leica).

Cells were divided into four groups as described above. They were grown on polymer coverslips (µ-slide, Ibidi, Gräfelfing, Germany), fixed with 4% paraformaldehyde in PBS for 10 min (1 h after irradiation), and permeabilized with 0.2% Triton X-100 in PBS for 5 min. Next, they were washed in PBS, incubated with the rabbit anti-gamma H2A.X (phospho S139) antibody (1:4000, Abcam, Cambridge, UK), followed by anti-rabbit AlexaFluor 546-conjugated IgG (1:1000, Thermofisher, Waltham, MA, USA), and analyzed using a Leica TCS SP8X confocal laser scanning microscope with a 63× oil immersion lens (Leica Microsystems, Wetzlar, Germany). Nuclear staining was performed using 4′,6-diamidino-2-phenylindole (DAPI). γH2A.X foci were quantified as foci per nucleus. γH2A.X foci were scored using ImageJ software in at least 80–100 cells.

Cells were washed with PBS, fixed and permeabilized in 3% paraformaldehyde containing 0.1% Triton X-100 for 30 min at 4°C. The cells were blocked and incubated with primary antibodies and then TRITC-conjugated secondary antibodies. The cells were washed twice with PBS and stained with DAPI for 10 min before being imaged with a TCS-SP laser-scanning confocal microscope with a 63× oil immersion lens (Leica Microsystems, Mannheim, Germany). Uptake of Texas Red-EGF was performed by first starving cells in serum-free medium for 16 h at 37°C. The cells were then incubated with cold serum-free medium containing 1000 ng/ml Texas Red-EGF for 30 min at 4°C. Endocytosis was initiated by replacing the ligand-binding medium with pre-warmed medium. At the end of each time point, the cells were rapidly chilled, washed, fixed, and then imaged as described above.