Protein Expression Analysis of MSC-MG63 Co-culture

After different proportions of MSC and MG63 were co-cultured to a predetermined time, the supernatant of the upper and lower chambers of the 6-well cell culture plate was aspirated, and cells were cleaned twice with PBS. A protein extraction kit (SD-001, Invent Biotechnologies, Inc, China) was used for protein extraction. Lysis buffer was added and spilled on the ice for 5 min. Then, the protein lysate was filtered through a centrifugal column and placed into a centrifuge for 10 minutes under the condition of 10000 rpm and 4°C, and the clear fluid in the collection tube is the protein. NanoDrop 2000 was used to measure protein concentration. After that, the protein was prepared with a 5x loading buffer in proportion, and the sample was performed using publishing protocols [16 (link), 17 (link)] with antibodies directed against α-smooth muscle actin (α-SMA, ab5694, Abcam, Cambridge, UK), E-cadherin (ab212059, Abcam), N-cadherin (AF4039, Affinity), Slug (ab51772, Abcam), Snail (ab216347, Abcam), and Vimentin (ab92547, Abcam); the dilution of these antibodies was 1 : 1000, and β-actin (ab8227, Abcam) and GAPDH (ab9485, Abcam) ratio of these two antibodies was 1 : 100000.