Csu x m2 n

Rat pituitary GH3 cells were cultured at 37 °C in a humidified atmosphere containing 95 % air and 5 % CO₂. The culture medium was DMEM supplemented with 10 % fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin. GH3 cells were transfected with small hairpin RNA (shRNA) constructs (Sigma-Aldrich) or the VSVG-mEmerald plasmid (modified from Addgene plasmid #31947) with Lipofectamine 2000 (Invitrogen). For growth hormone secretion assay, cells were transferred to serum free medium 48 h after transfection and incubated for 2 h before ELISA. Live cell confocal images were acquired 48 h after transfection, using spinning disk confocal scan head (CSU-X/M2 N, Yokogawa) attached to an inverted microscope (IX-81, Olympus) and an EMCCD camera (DU897BV, Andor) controlled by Micro-Manager software. Images (512 × 512 pixels, voxel size 0.0946 μm/pixel) were taken every 0.5 s for 400 frames. Live images were analyzed in NIH ImageJ with the MTrackJ plugin. VSVG containing vesicles (10/cell) were randomly selected in 11 Otg1 knockdown cells and 11 scramble shRNAs treated cells. Directionality of each vesicle was defined as its real transport distance divided by linear distance between the start and end positions.