Each experiment was done 3 times in tripli cates and measurements represent the average. For wounding experiments, cells were plated in 24 well plates and allowed to grow to a confluency selleck inhibitor of 100%. Experimental wounds were made by dragging a Gilson plastic yellow pipette tip across the cell culture. Cultures were then rinsed with PBS and replaced with fresh culture media. Cell motility was then monitored at selected time points under the inverted light microscope. L 005057 00 0005, L 003536 Inhibitors,Modulators,Libraries 00 0005 and L 004610 00 0005 respectively or the siCONTROL RISC free siRNA using Invitrogen Lipofectamine accord ing to the manufacturers instructions. The day before transfection cells were plated into 6 well plates, so that they reached about 70% confluency the day of transfec tion.
The amount of siRNA used was 160 pmol for Cdc42 and Rac1, 80 pmol for RhoA and 32 pmol for each ROCK1 and ROCK2 were applied in combination. Treatments with siRNA were replaced every 24 hours and western blot analysis verified the desired specific gene silencing 48 hours Inhibitors,Modulators,Libraries after Inhibitors,Modulators,Libraries transfection. 3D culture For 3D culture experiments, cells were grown on cover slips in 24 well plates in medium with 5 mg ml Matri gel. Briefly, 1 104 cells were mixed with the Inhibitors,Modulators,Libraries Matrigel containing medium and a total volume of 300 ul was added in each well in order to form a gel of 1 mm thickness. Plates were placed in a cell incubator at 37 C for 1hour, so that gel was formed and 500 ul of com plete medium was added on the top of it. Medium was changed every 2 days and cells left to grow for 12 days.
Photographs of the 3D cultures were taken under light and confocal microscopes after the appropriate staining. Statistical analysis Data are represented throughout the text with Stan dard deviation error bars. Statistical significance was tested with the unpaired Student t test. Results BRAFV600E induces distinct morphological Inhibitors,Modulators,Libraries changes in colon adenocarcinoma cells as compared to KRASG12V and loss of their epithelial architecture in 3D culture Previously established Caco BR cells have adopted a substantially different morphology when compared to the parental Caco 2 cells. The elongated morphol ogy acquired by Caco BR cells was characterized by long membrane protrusions. We present evidence that the morphology of Caco BR13 cells show properties of both Caco 2 epithe lial nature and of the mesenchymal phenotype of Caco H2 cells.
On the other hand, Caco K15 cells, which overexpress KRASG12V, have retained the overall paren tal morphology of Caco 2 cells. For comparison, estab lished adenocarcinoma cell lines HT29 and DLD 1, bearing mutant BRAFV600E and KRASG13D respectively, have also been analyzed www.selleckchem.com/products/Imatinib(STI571).html in the present study. It is of interest that the phenotype of Caco BR cells resembles that of DLD 1 cells, especially since both of these cell types share high levels of p BRAF.