Fv3000 confocal laser microscope system

FRAP experiments were performed using the FV3000 confocal laser microscope system (Olympus) at 37°C with 5% CO₂. Photobleaching of mGFP-FTH1 was achieved using a 488-nm laser with a bleaching time of 55.461 ms. Images were captured at 10-s intervals for 20 min (120 time points) or 4-s intervals for 128 s (32 time points).

Cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with digitonin (50 μg/ml; Sigma-Aldrich, D141) in PBS for 5 min, and blocked with 3% bovine serum albumin in PBS for 30 min. After incubation with the indicated antibodies, the specimens were mounted in SlowFade with 4′,6-diamidino-2-phenylindole (Invitrogen, S36939) and observed using a confocal FV3000 confocal laser microscope system (Olympus). For the final output, images were processed using Adobe Photoshop 2021 v22.5.9 software (Adobe). The number of LC3 puncta was counted using Fiji. Briefly, the images were applied with a Gaussian filter for noise suppression and processed with a Top-Hat filter. After setting a threshold and watershed segmentation, the number of LC3 puncta was counted by the Analyze Particles command. Cells were counted by using the Cell Counter plug-in. Microscopy and image analysis were done blindly.

Cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilised with 50 μg/ml digitonin (D141; Sigma-Aldrich) in PBS for 5 min, blocked with 3% BSA in PBS for 30 min, and then incubated with anti-p62 antibody for 1 hr. After washing five times with PBS, cells were incubated with Alexa Fluor 568 conjugated goat anti-rabbit IgG secondary antibody for 1 hr. These specimens were observed using a confocal FV3000 confocal laser microscope system (Olympus). For the final output, images were processed using Adobe Photoshop 2021 v22.3.1 software (Adobe). Average of p62 were measured using the open-source software Fiji.