Extended Data Fig. 7: CTH is essential for sensing cytoplasmic bacterial lipoproteins.
From: Cytosolic CTH senses bacterial lipoproteins and drives noncanonical inflammasome activation
a, Immunoblots of peritoneal macrophage from Cth+/+ and Cth−/− mice primed with 1 μg/ml Pam3CSK4 overnight before infected with live S. aureus at an MOI of 10 for 6 h. Cells cultured in fresh medium containing 100 μg/ml gentamycin. b, Quantification of pyroptosis in peritoneal macrophage from Cth+/+ and Cth−/− mice treated as in a. ELISA assays of IL-1β in supernatants, LDH release assays of cell death in supernatants and ATP assays of cell viability in cell pellets. c, Immunoblots of BMDMs from wild-type and Gbpchr3 mice primed with 1 μg/ml Pam3CSK4 overnight before transfected with DOTAP alone or together with 10 μg/ml Pam3CSK4 for 16 h. d, Quantification of pyroptosis in BMDMs from wild-type and Gbpchr3 mice treated as in c. ELISA assays of IL-1β in supernatants, LDH release assays of cell death in supernatants and ATP assays of cell viability in cell pellets. e, Immunoblots of BMDMs from wild-type and Gbpchr3 mice primed with 1 μg/ml Pam3CSK4 overnight before infected with live S. aureus at an MOI of 10 for 6 h. Cells cultured in fresh medium containing 100 μg/ml gentamycin. f, Quantification of pyroptosis in BMDMs from wild-type and Gbpchr3 mice treated as in e. ELISA assays of IL-1β in supernatants, LDH release assays of cell death in supernatants and ATP assays of cell viability in cell pellets. g-h, Immunoblots and quantification of cleaved caspase-11 in BMDMs from Vcp+/+ and Vcpflox/flox; Lyz2-Cre (Vcp−/−) mice primed with 1 μg/ml Pam3CSK4 overnight before infected with wild-type or ∆EsxA Listeria (g) or S. aureus (h) at an MOI of 10 for 6 h. Cells cultured in fresh medium containing 100 μg/ml gentamycin. Cells lysed and immunoblotted 16 h post infection. Relative band intensity between cleaved caspase-11 and β-actin calculated. i, Immunoblots of BMDMs from Nlrp6+/+ and Nlrp6−/− mice primed with 1 μg/ml Pam3CSK4 overnight before transfected with cell-penetrating peptides (100 ng/μl) alone or together with SA-Lpp (100 ng/μl) for 16 h. j, Immunoblots of human macrophages from NLRP7+/+ and NLRP7−/− human macrophages treated as in i. In b, d, f-h, data are pooled from three independent experiments (n = 3) and shown as means ± SD. Unpaired two-tailed Student’s t-test was used. Exact P values are indicated in the figures. Data are representative of three independent experiments with similar results in a-j.
