Cells were treated with MET antibody (Santa Cruz) on 24-well plates, cultured for additional 3 days with different concentrations of drugs, fixed, and washed 3 times. Crystal violet staining was performed after fixing of cells, followed by 5 times washing with water. For cell cycle assay, cells were transfected by plasmid containing vehicle or nMET (Addgene) as described above and subjected to fixation by 70% ethanol followed by protocol provided by the manufacture using Muse® Cell Cycle Assay Kit (Cat# MCH100106, Merck) with Muse cell analyzer and analysis (Merck).
For growing colonies in soft agar [29 (link)] in 6 well plates, cells were resuspended in 0.4% agarose top layer and seeded on 0.6% agarose base layer. The operation was under sterile conditions by mixing medium containing 20% FBS in 6-well plates and cells were grown for 3–4 weeks (37 °C, 5% CO2) with further feeding medium with or without membrane MET recognized antibody (Abcam, EP1454Y) for inhibiting mMET treatment. Finally, formed colonies were stained with Crystal Violet, or directly observed and counted under light microscope.
For growing colonies in soft agar [29 (link)] in 6 well plates, cells were resuspended in 0.4% agarose top layer and seeded on 0.6% agarose base layer. The operation was under sterile conditions by mixing medium containing 20% FBS in 6-well plates and cells were grown for 3–4 weeks (37 °C, 5% CO2) with further feeding medium with or without membrane MET recognized antibody (Abcam, EP1454Y) for inhibiting mMET treatment. Finally, formed colonies were stained with Crystal Violet, or directly observed and counted under light microscope.