Glycan-deficient CHO cells Transfected with Human Podoplanin

All methods were carried out in accordance with the approved guidelines. Chinese hamster ovary (CHO) cells; glycan-deficient CHO cell lines (Lec1, Lec2, and Lec8); and P3U1, Y-MESO-14, HSC3, LN229, HEK-293T, Met-5A, and NCI-H226 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). RERF-LC-AI cells were obtained from the RIKEN BioResource Center (Ibaraki, Japan). PC-10 cells were purchased from Immuno-Biological Laboratories Co., Ltd. (Gunma, Japan). Lymphatic endothelial cells-1 and Lymphatic endothelial cells-2 were obtained from Cambrex (Walkersville, MD) and AngioBio (Del Mar, CA), respectively. The human glioblastoma cell line LN319 was donated by Dr. Webster K. Cavenee (Ludwig Institute for Cancer Research, San Diego, CA)27 (link). CHO, Lec1, Lec2, Lec8, LN229, Y-MESO-14, RERF-LC-AI, and HSC3 cells were transfected with human podoplanin plasmids (CHO/hPDPN, Lec1/hPDPN, Lec2/hPDPN, Lec8/hPDPN, LN229/hPDPN, Y-MESO-14/hPDPN, RERF-LC-AI/hPDPN, HSC3/hPDPN) using Lipofectamine 2000 (Life Technologies Corp., Carlsbad, CA) or ScreenFect A (Wako Pure Chemical Industries Ltd., Osaka, Japan), according to the manufacturer's instructions1 (link)24 (link). CHO, Lec1, Lec2, Lec8, P3U1, Y-MESO-14, RERF-LC-AI, HSC3, PC-10, CHO/hPDPN, Y-MESO-14/hPDPN, RERF-LC-AI/hPDPN, and HSC3/hPDPN cells were cultured in RPMI 1640 medium (Wako Pure Chemical Industries, Ltd.) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Life Technologies Corp.), 2 mM L-glutamine (Life Technologies Corp.), 100 units/ml of penicillin, and 100 μg/ml of streptomycin (Life Technologies Corp.) at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. L-proline (0.04 mg/ml) was added for Lec1, Lec2, and Lec8 cells. LN319, LN229, HEK-293T, and Met-5A cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Wako Pure Chemical Industries Ltd.) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 units/ml of penicillin, and 100 μg/ml of streptomycin. MITO + serum extender (Thermo Fisher Scientific Inc., Waltham, MA) was added for Met-5A cells. LEC cells were cultured in endothelial cell medium EGM-2MV supplemented with 5% FBS (Cambrex Corp.). One mg/ml of geneticin (G418; Wako Pure Chemical Industries Ltd.) was added for CHO/hPDPN, Lec1/hPDPN, Lec2/hPDPN, Lec8/hPDPN, LN229/hPDPN, Y-MESO-14/hPDPN, RERF-LC-AI/hPDPN, and HSC3/hPDPN cells. Female BALB/c mice (four-weeks old) were purchased from CLEA Japan (Tokyo, Japan). Animals were housed under pathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved the animal experiments described herein. Tissue microarrays were purchased from Cybrdi, Inc. (Frederick, MD) or BioChain Institute Inc. (Newark, CA).