The GFP–COL transport is COPII dependent. (A and B) Still images from confocal live-cell imaging of RPE-1 stably expressing GFP–COL (green; GFP–COL–RPE) cotransfected with the inner-layer COPII marker mSc-Sec23A (magenta) derived from Video 9. Acquisition at one frame every 2.79 s. Time points indicate time in presence of asc/biotin (500 µg·ml⁻¹ and 400 µM, respectively) and 20 h after transfection. Scale bars, 10 µm; in enlargements, 0.25; n = 9. (A) Large panels show the entire cell with enlargements displayed in the top right corner. (B) The zoomed in area of interest from A (also indicated by the square) with the separate channels in gray scale, followed by the overlay image. Arrowheads indicate a GFP–COL structure that colocalizes with an mSc-Sec23A structure and at ∼1 min 38 s before the GFP–COL structure becomes part of the Golgi network. Scale bar, 0.25 µm. (C) Example image of GFP–COL–RPE cells cotransfected with ManII-mSc and the GTP-locked form of Sar1 (Sar1-H79G), which results in a COPII block. Panels show the separate channels GFP–COL (green) and MannII-mSc (red), as well as antibody labeling with a cis-Golgi marker giantin, in gray scale followed by the overlay image including nuclear labeling for DAPI (imaged as a separate channel in pseudocolor magenta). The time point indicates the incubation in presence of asc/biotin before fixation with PFA and 7.5 h after transfection. Scale bar, 10 µm. Cells expressing Sar1H79G show a scattered a disrupted Golgi apparatus, marked by ER labeling, compared with visible Golgi stacks in cells not affected by the COPII block (indicated by i). No transport of GFP–COL can be observed in COPII blocked cells (indicated by ii and iii).