Caco 2 cells

Human colon cancer cell line Caco-2 cells and human IECs (HIECs) were purchased from ATCC (California, USA). Caco-2 cells were cultured in MEM medium (KGM41500-500, Keygen biotech, Nanjing, China) supplemented with the mixture of penicillin and streptomycin (S G0200, olarbio, Beijing, China) and 10% FBS (10,099–141, GibcoTM, Grand Island, USA). HIECs were cultured in 1640 medium (1065–018, Keygen biotech, Nanjing, China) supplemented with the mixture of penicillin and streptomycin (G0200, Solarbio, Beijing, China) and 10% FBS (10,099–141, GibcoTM, Grand Island, USA). Cell supernatants and cells were collected and analyzed for the presence of cytokine proteins and gene expression. In some experiments, TNF-α (0332078JQ27, Novoprotein Biotech Co., Suzhou, China), the S1PR2-specific inhibitor (JTE-013, 49,951, MCE), the S1PR2-specific agonist (CYM5520, C5098, and APEXBIO), and S1P (S9666, sigma) were added to the culture immediately before the start of the culture, or the siRNA targeting S1PR2 vector and plasmid mediated S1PR2 overexpression vector were added simultaneously, or scramble RNA and empty vector were added simultaneously. The siRNA targeting S1PR2 vector, plasmid mediated S1PR2 overexpression vector, scramble RNA and empty vector were all purchased from Zhonghong Boyuan (Shenzhen) Biotechnology Co., Ltd. and performed according to the manufacturer’s instructions.

For tissue lysate preparation, 2-cm distal intestine(ileum) was homogenized with a hand-held homogenizer in lysis buffer. For cell culture lysate preparation, cells on plates were rinsed with cold PBS and lysed in the same lysis buffer used for tissue lysate preparation. Total protein were extracted from the ileum tissue and Caco-2 cells respectively (keyGEN BioTECH, China). Protein concentrations were determined by Pierce BCA assay. Proteins were loaded on polyacrylamide gels and the proteins in the gels were transferred to PVDF membrane (Bio-Rad, USA). For immunodetection the following antibodies were used: anti-claudin-3, anti-occludin and anti -ZO-1(1:400, Invitrogen, USA), and then incubated with an HRP-conjugated anti-rabbit secondary antibody(1:2000, Cell Signaling Technology, USA) (room temperature, 1h). The protein bands were visualized with a G-BOX imaging system(Syngene, UK) using an ECL assay kit (Pierce, USA).