Journal: The EMBO Journal

Article Title: Cardiolipin inhibits the non-canonical inflammasome by preventing LPS binding to caspase-4/11

doi: 10.1038/s44318-025-00507-z

Figure Lengend Snippet: ( A ) Human monocyte-derived macrophages (HMDM) from healthy donors or ( B ) Bone marrow-derived macrophages (BMDM) from wild-type (WT) mice were incubated for 4 h with 1 µg/mL Pam 3 CSK 4 . Cell culture medium was replaced with OptiMEM plus HEPES (−), 10 µM CL, or 5 µg/mL of RS-LPS (complexed with LTX) in the absence or presence of 2 µg/mL of LPS complexed with LTX (A) or CTB ( B ). Cells were incubated for 4 h (A) or 18 h ( B ). Cell supernatants were analysed for cleaved IL-1β (ELISA) and LDH (cytotoxicity assay). ( C , D ) BMDM from Nlrp3 −/− mice were incubated for 4 h with 1 µg/mL Pam 3 CSK 4 before cell culture medium was replaced with OptiMEM plus HEPES (Ctrl), or 2 µg/mL of LPS complexed with CTB in the presence of HEPES (−), or 10 µM CL (CL). Cells were incubated for 18 h. GSDMD cleavage and tubulin expression were assessed in mixed supernatants and lysates by western blot ( C ). LDH release was quantified by cytotoxicity assay ( D ). ( E ) HMDM from healthy donors or ( F – H) Human bronchial epithelial cells (HBEC) were incubated for 4 h ( E ) or 18 h ( F – H ) with 1 µg/mL Pam 3 CSK 4 . Cell culture medium was replaced with OptiMEM with vehicle (DMSO) or 10 µM MCC950 (NLRP3 inhibitor), or 10 µM VX765 (CASP1/4 inhibitor). Cells were incubated for 1 h, and then 2 µg/mL of LPS complexed with LTX was added in the presence of HEPES (−) or 10 µM CL (CL). Cells were incubated for 4 h. Cell supernatants were analysed for LDH ( E , F ) (cytotoxicity assay), cleaved IL-1β ( G ) and IL-18 ( H ) (ELISA). Data information: Each symbol is the mean of technical triplicates from an independent biological replicate. Bars are the mean of three or more independent biological replicates ( n = 3–5) ± SEM. Statistical analysis: Data were verified for normality using a Shapiro–Wilk test and analysed by ( A , B , E – H ) one-way ANOVA Šídák’s multiple comparisons test, ( D ) paired t test. P values are reported above bars. Statistical significance was defined as follows: significant difference for P < 0.05 (*), not significant for P ≥ 0.05 (ns). .

Article Snippet: After 4 h, the cell culture medium was replaced with OptiMEM alone or containing 10 μM of the NLRP3 inhibitor MCC950 or the CASP1/4 inhibitor VX765 (MedChem Express).

Techniques: Derivative Assay, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, LDH Cytotoxicity Assay, Expressing, Western Blot, Cytotoxicity Assay