Host Responses to Intracellular Pathogens

Human foreskin fibroblast cells were seeded in 12-well plate and stimulated with 100 U/ml IFN-γ for 24 h, or transfected with 0.5 μg pcDNA3.1-TRIM21-HA for 24 h, or 80 nmol of si-TRIM21 (combining of si-TRIM21-1, si-TRIM21-2, and si-TRIM21-3) or si-NC for 48 h (the sequence of si-TRIM21 are shown in Supplementary Table 2), and then stimulated with 100 U/ml IFN-γ for 24 h. After these treatments, the HFF cells were infected with RH tachyzoites (MOI = 3) for 18 h, or CEP tachyzoites (MOI = 3) for 24 h. Subsequently, cell monolayers were subjected to immunofluorescence assay (IFA) to determine the average number of parasites within 100 PVs or the number of PVs containing 1, 2, 4, or 8 tachyzoites from 25 separated fields of view (He et al., 2017 (link)). Anti-SAG1 mouse monoclonal antibody (Abcam, Cambridge, United Kingdom), and Goat anti-Mouse IgG (H + L) highly cross-adsorbed secondary antibody and Alexa Fluor Plus 488 (Invitrogen, United States) were used. Experiments were repeated three times for statistical analysis.