Cell culture
The MDA-MB-231 cell line, a human breast cancer cell line, was cultured in Dulbecco’s modified Eagle’s media (DMEM) without phenol red and supplemented with 10% fetal bovine serum (FBS) and 1% glutamic acid.
Plasmid and invasion assay
For generating the vector pSV-CapG-eGFP, we amplified the coding sequence of the human CapG gene by polymerase chain reaction (PCR) using the following primers (Invitrogen): 5’-TCG AGC TCA AGC TTC GAA TTC GGC- 3’ and 5’-TAA TAA CCG CGG TTT CCA GTC CTT GAA AAA TT-3’. The amplified fragment was inserted into the EcoRI and SacII site of the pSV-eGFP vector (BD Biosciences Clontech, Heidelberg, Germany). The construct was sequenced.
MDA-MB-231 cells (ATCC) were transfected with the pSV-CapG-eGFP construct using Transfectin (Biorad, Hercules, CA). Stable cell lines expressing CapG-eGFP were established using neomycin/ G418 for selection.
Invasion assays were performed in 24-well format cell culture inserts (Corning, Biocoat Matrigel Invasion Chamber) with 8-mm pores and Matrigel matrix, a basement membrane preparation, according to the manufacturer’s instructions. In brief, 24-well inserts were rehydrated for 2 hours. 25,000 cells were suspended in Dulbecco’s modified Eagle’s cell culture media and seeded into the 24-well insert which was placed into the companion plate. The companion plate contained cell culture media with 10% fetal bovine serum and 200 ng/ml epidermal growth factor (Sigma) acting as chemotactic agents. The invasion assay was incubated at 37˚C, 5% CO2 atmosphere. Imaging experiments were performed at 37˚C in Hepes-buffered medium (pH = 7.4) 6-8 and 16 hrs after seeding, respectively.
Epifluorescence and confocal microscopy
MDA-MB-231 cells expressing CapG-eGFP were examined live using an Olympus CK40 epifluorescence microscope. Images were captured using an Optronis VX45 camera.
A laser-scanning confocal microscope (Nikon C1Si) with a 25-mW Argon ion laser was used to perform the imaging experiments with a 25 x 0.75 N.A. water objective. To move the porous membrane into the working distance of the objective, the inserts were placed on small sterilized plastic rings within a Lab-Tek chamber from Nunc. The confocal microscope was used to perform z-stack imaging. With the software Fiji the z-stacks were converted into movies and 3-D projections.