FIGURE 4:

(A) HeLa cells were transfected with the indicated siRNA. After 48 h, cells were transfected with plasmids encoding GFP (pEGFP) or different version of GFP-tagged PLCG1. Cells were fixed after 24 h, and the Golgi was visualized by immunofluorescence labeling of giantin. The Golgi area was measured using ImageJ. PLCG1H335Q is a catalytically inactive mutant. (B) HeLa cells were transfected with the indicated siRNA. After 48 h, cells were transfected with plasmids encoding GFP (pEGFP) or different version of GFP-tagged PLCG1. After additional 8 h, cells were plated into ibidi migration inserts. The next day, migration was initiated by removing the ibidi insert, and cells were allowed to migrate and close the wound for 18 h. (C) HeLa cells were treated with 10 μM U73122 for 3 h, followed by fixation, giantin staining, and measurement of Golgi volume. (D) HeLa cells were transfected as indicated, and migration assay (wound healing) was performed 72 after transfection. Cells were allowed to migrate for 18 h. U73122 indicates cells transfected with control siRNA that were treated with 10 μM inhibitor for the whole duration of the migration assay. siSec16+U73122 indicates cells transfected with Sec16A siRNA and treated with the PLCG inhibitor. Asterisks indicate statistically significant differences from control (p < 0.05).