FIGURE 5.

COH1 is important for Golgi reassembly and tubulation activities. A and B, HeLa cells transfected with scramble or COH1 siRNA were treated with nocodazole for 75 min at 37 °C. Subsequently, nocodazole was washed out, and cells were incubated at 37 °C for 5 min and 60 min to allow Golgi reassembly. Cells were fixed and stained for giantin (blue), TGN46 (green), and β-tubulin (red) by immunofluorescence and analyzed by confocal microscopy. HeLa cells transfected with scrambled siRNA display a discernible reassembled compact Golgi structure after 60 min of nocodazole washout (A) whereas in COH1-deficients cells the Golgi remained fragmented (B). C–E, siRNA-transfected HeLa cells were treated with 5 μg/ml BFA for 5 min and processed for immunofluorescence analysis using an antibody against the small GTPase RAB6. D and E, quantifications of mean RAB6 tubule length (D) and RAB6 tubule number/cell (E) were performed using AxioVision. At least 30 cells were analyzed per condition and experiment (n = 3). F, representative confocal images showed reduced tubulation activity in COH1-deficient cells upon 5 μg/ml BFA treatment for 5 min not only for RAB6 (green) but also for TGN46 (red). Nuclei were stained with DAPI (blue). Scale bars, 10 μm. Error bars show S.D. Statistical significance was calculated by t test.