Jasplakinolide

Amyloid-β 1-42 (GenicBio, A-42-T-1), Rapamycin (Sigma Aldrich, R0395), Bafilomycin A1 (Sigma Aldrich, B1793), Latrunculin B (Sigma Aldrich, L5288), Jasplakinolide (AdipoGen Life Sciences, 102396-24-7), p62 siRNA (Cell Signaling Technology, 6399S), SSH1 siRNA (Dharmacon GE Healthcare, 5′-GAG GAG CUG UCC CGA UGA C-3′), Cofilin siRNA (Dharmacon GE Healthcare, 5′-GGA GGA CCU GGU GUU CAU C-3′).

NIH3T3 cells stably expressing nAC-mCherry were seeded on each glass-bottom dish (IWAKI, No. 3911–035). The concentration of NIH3T3 cells seeded on a glass-bottom dish was 1.0 × 10⁵ cells/dish. After 24 hours of seeding, Hoechst 33342 (Hoechst, Dojindo) was applied to the culture medium (500 ng/ml) to stain nuclei for an hour. After staining with Hoechst, jasplakinolide (Adipogen Life Sciences) diluted in dimethyl sulfoxide (DMSO) was added, and time-lapse imaging microscopy started immediately (IX83 wide-field fluorescence microscopy, Olympus). Time-lapse images were taken every 15 minutes. Three fields were taken during each experiment. The observation was continued for 15–18 hours. To capture images, a 60x lens (Olympus, UPlanSApo, numerical aperture of 1.35) and ORCA Flash 4.0 LT PLUS Digital CMOS camera (Hamamatsu, Model C11440-42U30) were used.