Live-cell imaging was performed on H1299 cells stably expressing RAB6A-EGFP. Cells were plated on 35-mm glass-bottom plates (MatTek), transfected with siRNAs, and imaged 48 to 72 hours later using a Nikon A1+ confocal microscope equipped with 488 nm laser line, GaAsP detectors, and ×100 1.45 NA objective. Live-cell imaging conditions (5% CO2, 37°C, and 90% humidity) were maintained on stage using an Okolab incubation chamber. For optimal spatial and temporal resolution, Nyquist sampling was applied in bidirectional image acquisition mode without averaging. Images were acquired at 1 fps for 2.5 to 3 minutes. After acquisition, images were bleach-corrected (Huygens Professional). RAB6A vesicles were identified and tracked using the spots module in Imaris (Bitplane). A spot diameter of 500 nm was applied to segment the vesicles before tracking them using an autoregressive motion algorithm. To avoid unnecessary splitting of tracks due to uneven illumination or out-of-plane movement, a gap-closing algorithm was used. As described previously (28), unfissioned tubular structures were defined as RAB6A-expressing tubular structures that were attached to the Golgi. For each cell, a mask that isolated the Golgi and connected tubular structures was generated, and unfissioned tubules were counted and measured.

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