G0200

The previously described lines RHΔku80Δhxgprt^{93 (link)} and RHΔku80Δhxgprt/TIR1^{58 (link)}, referred to as RH^ku80and TIR1, were used to generate the transgenic lines (T. gondii tachyzoites) reported here. The parental lines and derived lines (Supplementary Data 1) were grown in human foreskin fibroblasts HFF-1 (ATCC SCRC-1041) in D5 media composed of Dulbecco’s modified Eagle medium (DMEM) (12800-082, Thermo Fisher Scientific), supplemented with 5% heat-inactivated fetal bovine serum (10099-141, Gibco), 2 mM glutamine (G0200, Solarbio Biotech) and 100 units penicillin-streptomycin (P1410, Solarbio Biotech) at 37 with 5% CO_2. HFF-1 cells were cultured in the same media and conditions, and were all maintained as mycoplasma negative^{94 (link)}. The TIR1 parental line, and their derived AID lines were cultured in HFF monolayers with 500 mM auxin (I2886, Sigma-Aldrich) (+IAA) or 0.1% ethanol alone (−IAA) for phenotypic assays, as previously described^{46 (link),58 (link)}. Parasites were allowed to naturally egress or were mechanically lysed by passing through 22 g needles, and harvested by filtration through 3.0 micron polycarbonate membranes, from which extracellular parasites were used for experimental assays.

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.