A1r hd25

Live embryos were imaged using a laser scanning confocal (Leica SP8 or Nikon A1RHD25) with water Apochromat 40×1.1 NA objective. Embryos were scanned every 5 to 10 min for long-term imaging. Higher temporal resolution of around 2 minutes were used when imaging cleavage divisions. FRAP was performed at 3.5-times zoom on a 5 μm × 10 μm region of interest, photobleached using the 405 nm laser at 100%, with a pixel dwell time of 6 μs. Azido-blebbistatin was activated locally by illuminating a ROI (3 mm×5 mm) with a 405 nm laser at 20% laser power for 60 s.

To investigate the biofilm-reductive effects of peptides and ciprofloxacin, the biofilm of DRPa-4009 cells was formed on the plastic coverslips for 48 h. After treatments of peptides (64 µM) and ciprofloxacin (256 µM) for 24 h, the coverslips were washed with SP buffer and stained with FilmTracer SYPRO Ruby biofilm matrix stain (Thermo Fisher Scientific Co., Ltd., Seoul, Korea) according to the manufacturer’s protocol. Samples were analyzed by CLSM (A1R HD 25, Nikon, Japan) [30 (link),31 (link)].

Mouse bone marrow neutrophils were extracted via gradient centrifugation. Cell climbing tablets were placed in the 12-well plates, after which the extracted cells were added. After being cultured in a cell incubator at 37 °C and 5% CO₂ for 30 min, rhCD177 was added, and then Phorbol 12-myristate 13-acetate (PMA) was added 30 min later. After 4 h, the cells were fixed with 4% paraformaldehyde for 10 min and washed with PBS, and the cell membrane was broken using 0.25% TritonX-100. After another PBS wash, cells were inoculated on cell slides. The new slides were then incubated with PBS containing 5% goat serum for 1 h, and then with anti-histone H3 antibody (Abcam) overnight at 4 °C. One day later, slides were washed with PBS again and incubated at 25 °C for 2 h with Alexa Fluor 488 (green) labeled anti-rabbit IgG. Hoechst 33342 was used for nuclear staining after washing. Fluorescent immune samples were observed using a confocal microscope (Nikon A1R HD25, Tokyo, Japan) and analyzed using the NIS Elements AR 4.3 software.

Cells exposed to the indicated treatment were fixed with ice-cold 4% (w/v) paraformaldehyde (PFA, Sigma–Aldrich) for 15 min and then permeabilized with 0.1% Triton X-100 (Sigma–Aldrich) for 20 min at RT. After blocking with 3% bovine serum albumin (BSA, Sigma–Aldrich) for 30 min, specific primary antibodies, namely, anti-HTNV NP 1A8 mouse monoclonal antibody (prepared and maintained by our laboratory; 1:50 dilution) and anti-SREBP2 rabbit polyclonal antibody (Abcam, ab30682; 1:50 dilution), were added and incubated overnight at 4°C. After five washes with DPBS, the secondary antibodies, namely, FITC-conjugated goat anti-mouse IgG (Abcam, ab6785) and Cy3-conjugated goat anti-rabbit IgG (Abcam, ab6939), were used for detection (incubation at 37°C for 1 h). Cell nuclei were stained with DAPI (Thermo Fisher, D9542) for 5 min at RT. After sealing with ProLong™ Gold Antifade Mountant (Thermo Fisher, P36930), the samples were observed using a fluorescence microscope (A1R-HD25, Nikon).

HEK293 cells stably expressing GFP-mCherry-LC3 were grown on coverslips, induced by EBSS starvation for 3 h, and then fixed with 4% paraformaldehyde for 20 min. Confocal images were obtained using a 60× oil lens objective on an inverted fluorescence microscope (Nikon, A1RHD25, Japan, Tokyo). The fluorescence assay was performed as described previously [10 (link)].

AtTCP21 and melittin were labeled by adding Flamma^® 675-NHS (BioActs, Incheon, Republic of Korea), a fluorescent dye solution, to the proteins in buffer A at a molar ratio of 1:1. After 2 h of incubation, the mixtures were dialyzed against PBS for 48 h to remove any unbound fluorescent dye. The labeled protein samples were incubated with C. gloeosporioides cells at 28 °C for 4 h. The fungal cells were washed thrice with buffer A, mounted on a cover glass in 50% glycerol and 0.1% n-propyl gallate solution, and examined under a CLSM (A1R HD 25, Nikon, Japan) [19 (link)].

E. coli treated with Flamma^®675-labeled peptides were observed on a CLSM. Cells were inoculated and adjusted as the above procedure of antimicrobial assay. Flamma675-labeled peptides were added to 100 μL of the cell suspensions at each MIC, after which the cells incubated for 30 min were washed by centrifugation at 4000 rpm for 5 min in three times with ice-cold PBS buffer. Localization of Flamma675-labeled peptides was then examined using CLSM (A1R HD 25; Nikon, Tokyo, Japan).