Cell culture, immunofluorescence, transfection and image analysis

HeLa cells, HeLa cells stably expressing HA-Vangl2 and COS7 cells were maintained in GIBCO Dulbecco's Modified Eagle Medium containing 10% Fetal Bovine Serum (FBS), 10 mU/mL of penicillin and 0.1 mg/mL of streptomycin. Transfection of siRNA or DNA constructs into HeLa cells or COS7 cells was performed using lipofectamine 2000 as described in the manual provided by Invitrogen. For immunofluorescence, cells growing on coverslips were fixed in 4% PFA for 20 min then washed five times with 500 μl of PBS and incubated with permeabilization buffer (PBS containing 0.1% TX-100, 0.2 M Glycine, 2.5% FBS) at RT for 30 min. Then cells were incubated with primary antibody and secondary antibody in permeabilization buffer for 30 min. Each antibody incubation was following by five times wash with PBS.

Images were acquired with a Zeiss LSM 510 confocal microscope system or a Zeiss Axioobserver Z1 microscope system. Image J (http://rsb.info.nih.gov/ij/) was used for colocalization analysis (Guo et al., 2008). Briefly, the two images were adjusted to be the same average intensity of pixel value using divide function. A threshold was chosen manually to select the area stained with a Golgi marker. Subsequently, the numbers of above threshold pixels were determined for each Golgi marker (A and B). Colocalized pixels were determined using the colocalization function with a fixed ratio of 0.75 (C). Finally the value of colocalization was determined by the average value of C/A and C/B.