Evaluation of Cell Signaling Proteins

The cells were washed with phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Sigma-Aldrich, St. Lois, MO, USA). Western blot analyses were conducted as previously described [15 (link)]. Antibodies to the following proteins were used: ZEB1 and ZEB2 (Novusbio, Littleton, CO, USA), cyclin-D1 (Merck, Darmstadt, Germany), N-Cadherin (Merck), E-cadherin (Merck), vimentin (Merck), CD200 (R&D systems, Minneapolis, MN, USA), CD200R1 (R&D systems), c-Myc (Cell Signaling Technology, Danvers, MA, USA), β-catenin (Merck), Fibronectin (Merck), and β-actin (Santa Cruz, Dallas, TX, USA). For flow cytometry analysis, all cells (1 × 10⁷) or dissociated tumors were incubated for 15 min in the dark with anti-mouse CD16/CD32 antibody (BD Biosciences, San Diego, CA, USA). Cells (1 × 10⁷) were incubated for 30 min with anti-human CD200 PE-Cy7-conjugated antibody (BD Biosciences) or anti-mouse CD200 PE-conjugated antibody (BioLegend, San Diego, CA, USA). After washing again with FACS buffer, the MEER cell lines were resuspended in FACS buffer and analyzed. Densitometry readings/intensity ratio of each band was performed by using ImageJ software (ImageJ, NIH, Bethesda, MD, USA). Details information of western blot could be found in Supplementary Figure S7.