Fig. 6.

Atg9/Bif-1 trafficking from the Golgi is impaired in CAPNS1-depleted cells. (A) Control and shCAPNS1 U2OS cells were transiently co-transfected with AmCyan-Bif-1 and HcRed-LC3. 16 h later, the cells were fixed and analysed by a confocal microscope. Arrows indicate areas of colocalization of AmCyan-Bif-1 and HcRed-LC3. Scale bars: 20 μm. (B) 100 cells for each sample were considered and the number of cells where Bif-1 forms aggregates or single dots was counted. The graph represents the means and s.d. of three independent experiments. (C) U2OS cells stably expressing Flag-Atg9 were transiently silenced with control or CAPNS1-specific siRNA and then transfected with AmCyan-Bif-1. 24 h later the cells were treated for 1 h with or without 100 nM thapsigargin, and then fixed and analysed by immunofluorescence to visualize endogenous GM130, Flag-Atg9 and AmCyan-Bif1. Representative confocal merged images are shown. Scale bars: 20 μm. (D) 50 cells for each sample were considered and the number of cells where Atg9/Bif-1 positive vesicles colocalize with endogenous GM130 was counted. Mean and s.d. of three independent experiments.