ACREEDORES Y DEUDORES ALIMENTISTAS

2.2.3.1 Growing and passaging cells

A subclone o f M DCK cells (Ridley et al., 1995), 293 (human embryonic kidney) cells and 911 (human embryonic retina) cells (Fallaux et al., 1996) were grown in Dulbecco's modified Eagle's m edium (DM EM ) containing 10% bovine fetal calf serum (FCS), penicillin (10,000 lU/ml) and streptomycin (10,000 pg/ml). Cells were grown at 37 °C in a humidified atmosphere containing 1 0% CO2.

Cells were passaged every 3 to 4 days in their respective media. Cells were grown in 25cm^ tissue culture flasks and were split before reaching confluency. They were washed once with 10 ml PBS to remove any remaining FCS which would inactivate the trypsin/EDTA before they were incubated with 1ml trypsin/EDTA at 37 °C for about 5 min. When the cells floated off the bottom of the flask, 10 ml of 10% FCS/DMEM was added to inactivate the trypsin. Cells were then diluted according to their growth rate at a ratio of 1:5 - 1:10.

2.2.3.2 Freezing and thawing of cells

Cell stocks were cryopreserved in liquid nitrogen for storage. To prepare for freezing, cells were pelleted by centrifugation at 200 g for 5 min and resuspended at 10^- 10^ cells/ml in freezing solution (cell culture medium containing 1 0% dimethyl sulphoxide

(DMSO) and 20% FCS). The cells were dispensed in 1 ml aliquots in cryotubes (nunc) and wrapped up in tissue to allow slow freezing overnight in a polystyrene box at -70 °C. The next day cells were transferred to liquid nitrogen for long-term storage.

To thaw cells, an aliquot was removed from liquid nitrogen and thawed rapidly in a 37°C waterbath. Cells were then transferred into a 50 ml falcon tube and 10 ml of fresh medium was then added dropwise to the cells to dilute slowly the DMSO contained in the freezing solution. After pelleting at 200 g, cells were resuspended in 10 ml fresh medium, put in a 25 cm^ tissue culture flask and incubated at 37 °C. They were checked the next day and passaged when required.

2.2.3.3 HGF/SF stim ulation

To study the effects of HGF/SF stimulation, M DCK cells were starved for 16 h prior to stimulation with 10 ng/ml HGF/SF in 0.5% FCS/DMEM. For experiments involving the inhibitors LY294002 or PD98059, the culture medium was replaced with DMEM containing 0.5% FCS for at least 1 h prior start o f the experiment. LY294002 and PD98059 were obtained from Calbiochem, Nottingham, UK. The synthetic compound PD98059 has been shown to inhibit growth factor-induced activation o f M A PK K l/2 in various cell types, and thus to inhibit p42/p44 M APK activation at 50 |LiM (IC5 0 for

M A PK K l/2; [Alessi et al., 1995]). LY294002 is a synthetic compound which blocks activation of PI3K specifically (IC50 = 1 .4 p.M) without affecting the activity of protein

serine/threonine kinases, protein tyrosine kinases and lipid kinases (PI 4-kinase) when used at concentrations of up to 50 pM (Vlahos et al., 1994).

2.2.3.4 M icroinjection

Cells for microinjection or stimulation with HGF/SF were seeded in 15 mm wells at 1 0^

cells/well on 13 mm circular glass coverslips. After 3 days, cells were transferred to DMEM containing 0.5% FCS with or without 50 jiM PD98059 or 20 |iM LY294002, and HGF/SF (10 ng/ml) was added and/or cell groups were injected at the edge and in the middle of colonies. For microinjection, proteins were diluted in 50 mM Tris-Cl pH 7.5, 100 mM NaCl, 5 mM MgCl% and m icroinjected into the cytoplasm o f M DCK cells m aintained in 0.5% FCS for 1 h before injection. To identify injected cells, tetramethylrhodamine dextran MW 10 000 (Molecular Probes, Leiden, The Netherlands) at 5 mg/ml was microinjected together with recombinant proteins. Cells microinjected with the proteins V12Ras, V12Racl, V14RhoA or NlVRacl were incubated for different timepoints according to the different experiments. In some experiments where the injected component had an inhibitory effect, cells were treated with HGF/SF (10 ng/ml) for different timepoints as indicated in the figure legends.

Cells microinjected with plasmids encoding NlTRas, AFG M A PK K l, Ala-221 M A PK K l, Glu-217/Glu-221 M A PK K l, wild-type M A PK K l, p i 10a or RafCAAX were incubated at 37 °C for 4-6 h to allow protein expression. Plasmid-injected cells were detected by using an antibody against the expressed protein. Cells were then fixed and stained for F-actin and junctional complexes as described below.

2.2.3.5 Tim e-lapse videom icroscopy

M DCK colonies chosen for each experiment contained between 15-20 cells, and in microinjection studies every cell in each colony was microinjected. For experiments studying the motility of MDCK cells, HGF/SF was added after microinjection, at a final concentration of 10 ng/ml. Cells were filmed on a Zeiss ICM 405 microscope linked to a Panasonic F15 camera and Hamamatsu image processor with a Sony U-matic time-lapse video recorder. In some experiments, after filming, cells were fixed and stained for confocal analysis.

2.2.3.6 TER measurements

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MDCK cells (passage 9-21) were plated at 6 x 10 /cm on Transwell filters (6.5 mm

diameter, 0.4 pm pore size) (Costar, Cambridge, MA) and grown in 10% FCS/DMEM. Cells were used between three and four days after plating when they reached a resistance in the range of 3000-6000 Q/cm^. Initial resistance measurements were taken with the M illicell-ER S instrum ent (M illipore C ontinental W ater System s, B edford, MA).

Resistance measurements were subsequently taken at timepoints as indicated in the figures. In all cases, appropriate solvent controls were included in the TER measurements. TER values were obtained by subtracting the contribution of the filter and the bathing solution. To normalize the results of different experiments, the transepithelial resistance for monolayers in control cells (unstimulated) was taken as 1 0 0%.