Figure 6

Overexpressed ChTLR15 upregulated the expression levels of molecules in the ChTLR15/ChNF-κB-ChNLRP3/ChIL-1β signaling pathway. A pCMV-ChTLR15 plasmid was transfected into DF1 monolayer cells cultured in 6-well plates using Lipofectamine 2000 diluted according to the manufacturer’s protocol. ChTLR15 protein levels in transfected cells were highly significantly upregulated (p < 0.01) at 36 h post-transfection compared to other time points. B At 36 h post-transfection with pCMV-ChTLR15, DF1 cells in each group were stimulated by purified E. tenella sporozoites (4 × 10⁵ sporozoites per well). Cells transfected with empty plasmid pCMV and stimulated with (pCMV + Stim) or without (pCMV + Unstim) sporozoites were used as controls. At 0, 2, 4, 6, and 8 h post-stimulation, cells in each group were collected to quantify the mRNA levels of ChTLR15, ChMyD88, ChNF-κB, ChNLRP3, ChCaspase-1, ChIL-18 and ChIL-1β by quantitative real-time PCR (qPCR). C Protein expression levels of ChTLR15 and ChNLRP3 in DF1 cells were detected by Western blot at 0, 2, 4, 6, and 8 h post-stimulation. D Protein expression levels of ChIL-1β in culture supernatant were analyzed using an ELISA kit (Jiancheng Bioengineering Institute, Nanjing, China) at 0, 2, 4, 6, and 8 h post-stimulation.