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Fig. 6. VAMP8 but not VAMP3 is mislocalized in Caco2 cells after transient RNAi of TI-VAMP. Caco2 cells were electroporated with 60 pmol of siRNA targeting human TI-VAMP or against human β-globin and grown on filters for 4 days. (A) Confocal Z and X-Y sections (top and bottom) of control (siRNA beta glob) and TI-VAMP transient-knockdown (siRNA TI-VAMP) Caco2 cells. Cells were labeled with antibodies against human VAMP3 (left panel) and VAMP8 (right panel) and secondary antibodies coupled to FITC. Note that the lateral localization of VAMP3 is unaffected by the transient knockdown of TI-VAMP. Compared with its vesicular localization in control Caco2 cells, VAMP8 seems to be less concentrated in vesicular structures and slightly mislocalized to the lateral membrane in Caco2 TI-VAMP transient-knockdown cells. (B) Digitized images of confocal X-Y sections of control (siRNA beta glob) and TI-VAMP (siRNA TI-VAMP) Caco2 cells. Cells were labeled with antibody against human VAMP8 and secondary antibody coupled to FITC. Fluorescent images were converted and analyzed quantitatively using ImageJ® software (see Materials and Methods). A statistical analysis of the area of each fluorescent object was performed (data not shown). We found fewer objects in the case of TI-VAMP knockdown compared with the control (130 versus 164 in the control). We also found that there were more objects of small area (less than five pixels) and fewer objects of large area (greater than ten pixels) in the case of the TI-VAMP knockdown. Bar, 10 µm.
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