Figure 6.
Figure 6. CCDC157 is required for fusion events with Golgi membranes. (A) Control HeLa cells and cells depleted of TTC17 or CCDC157 were incubated with 5 µg/ml BFA for the indicated times, after which cells were analyzed by immunofluorescence microscopy using an anti-GM130 antibody. For BFA washout, cells were incubated with BFA for 30 min, washed extensively, and incubated in BFA-free medium before immunofluorescence microscopy analysis. Nuclei were stained with DAPI. Scale bars, 20 µm. (B) Quantification of cells with dispersed Golgi membranes using an anti-GM130 antibody as described in A. 200 cells were counted per condition for each individual experiment (mean of n = 3 ± SEM). (C) Control HeLa cells and cells depleted of CCDC157 or TTC17 were analyzed by immunofluorescence microscopy using anti-ERGIC and anti-GM130 antibodies. Nuclei were stained with DAPI. Scale bars, 10 µm. (D) Pearson’s correlation coefficient of colocalization between ERGIC53 and GM130 (n = 24; Two-tailed Student’s t test for comparison to control condition siCtr; ***, P < 0.001). (E) Control HeLa cells and cells depleted of CCDC157 or TTC17 were analyzed by immunofluorescence microscopy using anti-TfR and anti-TGN46 antibodies. Nuclei were stained with DAPI. Scale bars, 10 µm. Arrowheads point at enlarged and coalesced TfR-positive vesicles, surrounded by Golgi membranes. (F) Pearson’s correlation coefficient of colocalization between TfR and TGN46 (n = 27; two-tailed Student’s t test for comparison to control condition siCtr; **, P < 0.01; ***, P < 0.001). (G) Control HeLa cells and cells depleted of CCDC157 were analyzed by immunofluorescence microscopy using anti-EEA1 and anti-TfR antibodies. Nuclei were stained with DAPI. Scale bars, 10 µm. (H) Pearson’s correlation coefficient of colocalization between EEA1 and TfR (n = 27; two-tailed Student’s t test for comparison to control condition siCtr; ***, P < 0.001).

CCDC157 is required for fusion events with Golgi membranes. (A) Control HeLa cells and cells depleted of TTC17 or CCDC157 were incubated with 5 µg/ml BFA for the indicated times, after which cells were analyzed by immunofluorescence microscopy using an anti-GM130 antibody. For BFA washout, cells were incubated with BFA for 30 min, washed extensively, and incubated in BFA-free medium before immunofluorescence microscopy analysis. Nuclei were stained with DAPI. Scale bars, 20 µm. (B) Quantification of cells with dispersed Golgi membranes using an anti-GM130 antibody as described in A. 200 cells were counted per condition for each individual experiment (mean of n = 3 ± SEM). (C) Control HeLa cells and cells depleted of CCDC157 or TTC17 were analyzed by immunofluorescence microscopy using anti-ERGIC and anti-GM130 antibodies. Nuclei were stained with DAPI. Scale bars, 10 µm. (D) Pearson’s correlation coefficient of colocalization between ERGIC53 and GM130 (n = 24; Two-tailed Student’s t test for comparison to control condition siCtr; ***, P < 0.001). (E) Control HeLa cells and cells depleted of CCDC157 or TTC17 were analyzed by immunofluorescence microscopy using anti-TfR and anti-TGN46 antibodies. Nuclei were stained with DAPI. Scale bars, 10 µm. Arrowheads point at enlarged and coalesced TfR-positive vesicles, surrounded by Golgi membranes. (F) Pearson’s correlation coefficient of colocalization between TfR and TGN46 (n = 27; two-tailed Student’s t test for comparison to control condition siCtr; **, P < 0.01; ***, P < 0.001). (G) Control HeLa cells and cells depleted of CCDC157 were analyzed by immunofluorescence microscopy using anti-EEA1 and anti-TfR antibodies. Nuclei were stained with DAPI. Scale bars, 10 µm. (H) Pearson’s correlation coefficient of colocalization between EEA1 and TfR (n = 27; two-tailed Student’s t test for comparison to control condition siCtr; ***, P < 0.001).

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