Cell Cultures and Lentiviral Vector Transfection

The multiple myeloma cell line U266 was provided by Central Laboratory of the Peking University Third Hospital. Cell STR authentication was made by Beijing MDHS Testing technology Co., Ltd before we conducted the following experiments as below. Cells were cultured at 37°C/5% CO₂ in RPMI1640 containing 10% fetal bovine serum, supplemented with 1% penicillin/streptomycin. The lentiviral shRNAs were constructed by Generay Biotech Co., Ltd (Shanghai, China). The siRNA sequences for S1RP2 were: 5′-CAACAAGGTCCAGGAACACTATAAT-3′ and the siRNA sequences for NC were: 5′-TTCTCCGAACGTGTCACGTAA-3′. The plasmids including negative control (NC) short-hairpin RNA (shRNA) and S1PR2 gene shRNA were constructed by Hanbio (Shanghai, China). The U266 cells were inoculated to 24 well-plates (2.5⨰10^5/mL) and cultured for 24 h. When the cells reached 50–70% confluence they were infected with S1PR2-shRNA lentivirus or NC-shRNA lentivirus using transfection agents. After 48 h, quantitative polymerase chain reaction (qPCR) and Western blot assays were performed to estimate the transfection efficiency.