Fig. 4. TNFR1 and TRIF signalling are required for macrophage RIPK1-driven pyroptosis, but only TRIF signalling affects cytokine production in macrophages.

BMDMs were left A unstimulated or primed with B 100 ng/ml LPS, C 100 ng/ml IFN-γ or D both LPS and IFN-γ for 3 h before infecting with Y. pseudotuberculosis (Yp) (MOI 5). A–D Percentage SYTOX Green uptake was quantified over 8 h. E BMDMs were primed 100 ng/ml LPS for 3 h and infected with Y. pseudotuberculosis (Yp) (MOI 5). Mixed supernatant and cell extracts were examined by immunoblotting at the indicated time points. Two membranes were used for immunoblotting (i, ii). F–H BMDMs were left unstimulated or primed with 100 ng/ml LPS, 100 ng/ml IFN-γ or both LPS and IFN-γ for 3 h before infecting with Y. pseudotuberculosis (Yp) (MOI 5) for 4 h. F TNF, G IL-1β and H IL-1α in the cell culture supernatant were quantified by ELISA. A–D, F–H Data are represented as mean + SEM cell stimulation from three independent experiments. *P < 0.05, **P < 0.01.