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 ) HMDM or ( B ) WT BMDM were incubated for 4 h with cell culture medium (unprimed, UP) or 1 µg/mL Pam 3 CSK 4 (all other conditions). Cell culture medium was then replaced with OptiMEM (Ctrl), FuGENE HD (Fu) 0.5% v/v, LTX 0.25% v/v, 10 µM CL, or 2 µg/mL of LPS complexed with 0.25% v/v LTX ( A ) or 0.5% FuGENE HD ( B ) in the presence of HEPES vehicle (iLPS) or 10 µM CL (iLPS + CL). Cells were incubated for 4 h. GSDMD, CASP1, CASP4, CASP11, IL-1β, and tubulin expression and cleavage were assessed in mixed supernatants and lysates by western blot. ( C ) Fixed-Airyscan confocal imaging of WT BMDM primed for 4 h with 1 µg/mL Pam 3 CSK 4 and incubated with HEPES (Control) or 10 µM of CL liposomes containing 1% (w/w) TopFluor CL (BODIPY-CL) for 18 h. Macrophages were immunostained with TOMM20 (red), BODIPY-CL (green), and DAPI (grey). Images are maximum intensity projections of Z-stack acquisitions. ( D ) WT BMDM were incubated for 4 h with 1 µg/mL Pam 3 CSK 4 . Cells were then lysed and incubated with HEPES (−), 100 or 200 µg/mL CL or 100 µg/mL LPS for 1 h. 1 µg/mL of biotinylated LPS (biotin-LPS), was added, and lysates were incubated for 2 h. Biotinylated LPS with bound proteins was purified using magnetic streptavidin beads. Both purified (bound) and unbound fractions were immunoblotted for CASP11 and GAPDH by western blot. ( E , F ) Upper panels: Schematic of full-length ( E ) and ΔCARD ( F ) CASP4 constructs with the DmrB dimerisation system, permitting controlled dimerisation by AP20187 (AP). All DmrB constructs were N-terminally V5-tagged. ( E , F ) Lower panels: HEK293T cells were transfected with the native or the CASP4 catalytic cysteine mutant (C258A) of V5-DmrB-CASP4 (full length, ( E )) or V5-DmrB-ΔCARD-CASP4 ( F ) constructs. 24 h post-transfection, cells were harvested, plated, and 4 h later treated with AP for 50 min in the absence or presence of 30 µM CL (1 h pre-incubation). CASP4 auto-processing was analysed by western blot of cell lysates. ( G ) Recombinant CASP4-CARD-eGFP (1 µM) was incubated for 1 h at 37 °C with HEPES (Ctrl), or 1.67 mM of liposomes containing phosphatidylcholine (PC) or CL alone (CL) or increasing CL:PC molar ratio (5:95, 25:75, 50:50 and 75:25). Samples were centrifuged and the eGFP fluorescence was measured in supernatant (unbound CARD in non-liposome fraction) and resuspended pellet (liposome-bound CARD). The Y axis represents the percentage of overall fluorescence in the pellet. Data information: Each symbol is the mean of technical triplicates from an independent biological replicate. Bars are the mean of three independent biological replicates ( n = 3) ± SEM. Blots and images are representative of three or four independent experiments ( n = 3–4). Scale bar = 10 µm and 5 µm in inset panels. Statistical analysis: Data were verified for normality using a Shapiro–Wilk test and analysed by one-way ANOVA, Dunnett’s multiple comparisons 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: C57BL/6J mice Casp11 −/− , The Jackson Laboratory , , RRID:IMSR_JAX:024698.

Techniques: Incubation, Cell Culture, Expressing, Western Blot, Imaging, Control, Liposomes, Purification, Construct, Transfection, Mutagenesis, Recombinant, Fluorescence

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: HMDM from healthy donors ( A ), and BMDM from wild-type ( B ) or Tlr4 −/− ( C – E ) mice were incubated for 4 h with 1 µg/mL Pam 3 CSK 4 . Cell culture medium was then replaced with OptiMEM or the noncanonical inflammasome activator LPS complexed with 0.25 % v/v LTX ( A ) or 20 µg/mL CTB ( B – E ) (iLPS) in the presence of HEPES (−) or 10 µM CL. Released TNF and cleaved IL-1β were measured in supernatants by ELISA ( A , B , D ). CASP11, GSDMD, IL-1β cleavage, and tubulin expression were assessed by western blot ( C ). LDH release was quantified by cytotoxicity assay ( E ). Data information: The blot is representative of three independent biological replicates ( n = 3). Each symbol is the mean of three technical replicates from an independent biological replicate. Bars are the mean of three or more independent biological replicates ( n = 3 to 4) ± SEM. Statistical analysis: Data were verified for normality using a Shapiro–Wilk test and analysed by ( A , E ) Mann–Whitney test, ( B , D ) unpaired 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: C57BL/6J mice Casp11 −/− , The Jackson Laboratory , , RRID:IMSR_JAX:024698.

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Cytotoxicity Assay, MANN-WHITNEY

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 – D ) BMDM from WT mice were incubated for 4 h with 1 µg/mL Pam 3 CSK 4 . Cell culture medium was then replaced with OptiMEM, or 2 µg/mL of the noncanonical inflammasome activator LPS from Escherichia coli B4 strain (EC-B4) complexed with 0.5% FuGENE HD ( A , B ) or 1.2% Xfect ( C , D ) (iLPS) in the presence of HEPES (−) or 10 µM CL. Cells were incubated for 18 h ( A , B ) or 4 h ( C , D ). Cleaved IL-1β was quantified in cell supernatants by ELISA. LDH release was quantified by cytotoxicity assay. ( E , F ) BMDM from wild-type or Casp11 −/− mice were incubated for 4 h with 1 µg/mL Pam 3 CSK 4 . Cell culture medium was then replaced with OptiMEM in the presence of HEPES (−), 10 µM CL, or 2 µg/mL of LPS. Cells were left untouched or electroporated ( + EP), then fresh OptiMEM was added, and cells were incubated for 4 h. Cleaved IL-1β was quantified in cell supernatants by ELISA. LDH release was quantified by cytotoxicity assay. 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 to 4) ± SEM. Statistical analysis: Data were verified for normality using a Shapiro–Wilk test and analysed by ( A – D ) unpaired t test, ( E , F ) one-way ANOVA Šídák’s multiple comparisons 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: C57BL/6J mice Casp11 −/− , The Jackson Laboratory , , RRID:IMSR_JAX:024698.

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay

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: WT and Casp11 −/− mice were weighed and injected intraperitoneally (i.p.) with HEPES or 25 µg/g CL. 10 min later, mice were i.p. challenged with PBS or 10 µg/g LPS. After 6 h, animal weight was recorded and blood was collected. Body weight loss was calculated as a percentage of the initial weight ( A ). IFNγ ( B ), IL-6 ( C ), TNF ( D ), and cleaved IL-1β ( E ) were quantified in sera by ELISA. Data information: Violin plot of data from four different cohorts of mice (individual mice from each cohort shown as colour-matched data points). WT PBS and WT PBS + CL n = 3; WT HEPES + LPS n = 13; WT CL + LPS n = 13; and Casp11 −/− HEPES + LPS n = 17. Statistical analysis: Data were verified for normality using a Shapiro–Wilk test and analysed by ( A , E ) one-way ANOVA Sidak’s multiple comparisons test, ( B – D ) Kruskal–Wallis with Dunn’s multiple comparisons 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: C57BL/6J mice Casp11 −/− , The Jackson Laboratory , , RRID:IMSR_JAX:024698.

Techniques: Injection, Enzyme-linked Immunosorbent Assay