Figure 4.
Figure 4. Newly identified factors are essential for secretory pathway organization. (A and B) HeLa cells were transfected with indicated specific individual siRNAs and after 3 d, analyzed by immunofluorescence microscopy using antibodies against GM130 (A) and TGN46 (B), respectively. Nuclei were stained with DAPI. Scale bars, 20 µm. (C) Quantification of the percentage of cells with fragmented Golgi complex after transfection with the indicated specific individual siRNAs. For unbiased analysis of Golgi membrane morphology, the classifier of the CellProfiler Analyst was used (see Materials and methods). At least 200 cells were analyzed for each independent experiment (mean of n = 3 ± SEM; two-tailed Student’s t test for comparison to control condition siCtr; *, P < 0.05). (D and E) Left: HeLa cells were transfected with indicated specific individual siRNAs and after 3 d, analyzed by immunofluorescence microscopy using an anti-GM130 antibody. Nuclei were stained with DAPI. Scale bars, 50 µm. Right panel in D shows the quantification of Golgi surface area. For unbiased analysis of Golgi membrane surface area, cells (465 control cells and 388 TTC17-depleted cells) were analyzed with the CellProfiler Analyst (See Materials and methods). Mann–Whitney U test for comparison to control condition siCtr; ***, P < 0.001. Right panel in E shows the quantification of the percentage of cells with Golgi membranes with a ring-shape structure using the classifier of the CellProfiler Analyst (See Materials and methods). At least 200 cells were analyzed for each independent experiment (mean of n = 3 ± SEM; two-tailed Student’s t test for comparison to control condition siCtr; *, P < 0.05). (F) Transmission electron microscopy of Golgi membranes in control cells and TTC17- and CCDC157-depleted cells. Scale bars, 500 nm. In a given cell with altered Golgi membranes, all individual Golgi stacks had altered ultrastructure characterized by enlarged and swollen Golgi cisternae (blue arrowheads) in TTC17-depleted cells, and most likely accumulated and coalesced transport carriers in close proximity to Golgi membranes (red arrowheads) in CCDC157-depleted cells, respectively.

Newly identified factors are essential for secretory pathway organization. (A and B) HeLa cells were transfected with indicated specific individual siRNAs and after 3 d, analyzed by immunofluorescence microscopy using antibodies against GM130 (A) and TGN46 (B), respectively. Nuclei were stained with DAPI. Scale bars, 20 µm. (C) Quantification of the percentage of cells with fragmented Golgi complex after transfection with the indicated specific individual siRNAs. For unbiased analysis of Golgi membrane morphology, the classifier of the CellProfiler Analyst was used (see Materials and methods). At least 200 cells were analyzed for each independent experiment (mean of n = 3 ± SEM; two-tailed Student’s t test for comparison to control condition siCtr; *, P < 0.05). (D and E) Left: HeLa cells were transfected with indicated specific individual siRNAs and after 3 d, analyzed by immunofluorescence microscopy using an anti-GM130 antibody. Nuclei were stained with DAPI. Scale bars, 50 µm. Right panel in D shows the quantification of Golgi surface area. For unbiased analysis of Golgi membrane surface area, cells (465 control cells and 388 TTC17-depleted cells) were analyzed with the CellProfiler Analyst (See Materials and methods). Mann–Whitney U test for comparison to control condition siCtr; ***, P < 0.001. Right panel in E shows the quantification of the percentage of cells with Golgi membranes with a ring-shape structure using the classifier of the CellProfiler Analyst (See Materials and methods). At least 200 cells were analyzed for each independent experiment (mean of n = 3 ± SEM; two-tailed Student’s t test for comparison to control condition siCtr; *, P < 0.05). (F) Transmission electron microscopy of Golgi membranes in control cells and TTC17- and CCDC157-depleted cells. Scale bars, 500 nm. In a given cell with altered Golgi membranes, all individual Golgi stacks had altered ultrastructure characterized by enlarged and swollen Golgi cisternae (blue arrowheads) in TTC17-depleted cells, and most likely accumulated and coalesced transport carriers in close proximity to Golgi membranes (red arrowheads) in CCDC157-depleted cells, respectively.

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