Fig. 6.

A: VSVG transport to the plasma membrane is impaired in PC-depleted MT58 cells. MT58 and CHO-K1 transiently transfected with VSVG-GFP were cultured for 24 h at 40°C and shifted to 33°C. Cells were subsequently fixed at the indicated time. After fixation, cells were stained with PDI antibody. VSVG-GFP (direct fluorescence) is green, and PDI is red. Bar = 10 µm. B: Impaired VSVG transport in PC-depleted MT58 cells can be restored by lysoPC addition. Transiently VSVG-GFP transfected MT-58 cells and CHO-K1 cells were cultured in the absence or presence of 50 μM lysoPC during the 40°C incubation period (24 h). Cells were subsequently shifted to 33°C for 1 h and fixed and stained under nonpermeabilizing conditions with an antibody directed against the extracellular domain of VSVG (red). Bar = 10 µm. C. Gaussia luciferase secretion is impaired in PC-depleted MT58 cells. Gaussia luciferase was measured in medium and cell lysate of transiently transfected MT58 and CHO-K1 cells incubated at 33 or 40°C. For experimental details, see Materials and Methods. Gaussia luciferase secretion (RLU medium/RLU lysate) is expressed relative to the value of CHO-K1 secretion at 40°C (arbitrarily set to 1). To investigate whether the impaired secretion was dependent on PC, 50 μM lysoPC was added during the 40°C incubation period. The results represent the means ± SEM of three experiments performed in triplicate. *P < 0.05, MT58 versus CHO-K1.