Journal: EMBO Reports

Article Title: Promyelocytic leukemia protein targets MK2 to promote cytotoxicity

doi: 10.15252/embr.202052254

Figure Lengend Snippet:

Article Snippet: LDH Cytotoxicity Detection Kit , Clontech , Cat # MK401 , .

Techniques: Generated, Transgenic Assay, Recombinant, Plasmid Preparation, Blocking Assay, Transfection, Antibody Labeling, Software, Cell Viability 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: ( A , B ) WT BMDM were incubated for 4 h with 1 µg/mL Pam 3 CSK 4 . Cell culture medium was then replaced with OptiMEM plus 20 µg/mL CTB, 2 µg/mL LPS, or 20 µg/mL CTB complexed with 2 µg/mL LPS (CTB/LPS), in the absence or presence of 10 µM saturated 16:0 CL, saturated 18:0 CL or unsaturated 18:2 CL. Cells were incubated for 18 h. Cleaved IL-1β was quantified in cell supernatants by ELISA ( A ). LDH release was quantified by cytotoxicity assay ( B ). ( C , D ) HMDM were incubated for 4 h with 0.1 µg/mL LPS. Cell culture medium was then replaced with OptiMEM or 20 µg/mL LTX complexed with 2 µg/mL LPS (LTX/LPS) in OptiMEM, in the absence or presence of 10 µM saturated 16:0 CL or unsaturated 18:2 CL. Cells were incubated for 4 h. Cleaved IL-1β was quantified in cell supernatants by ELISA ( C ). LDH release was quantified by cytotoxicity assay ( D ). Data information: Each symbol is the mean of three technical replicates from an independent biological replicate. Bars are the mean of three independent biological replicates ( n = 3) ± SEM. Statistical analysis: Data were verified for normality using a Shapiro–Wilk test and analysed by 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: Cytotoxicity Detection Kit (LDH) , Merck , , 11644793001.

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: ( 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: Cytotoxicity Detection Kit (LDH) , Merck , , 11644793001.

Techniques: Derivative Assay, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, LDH Cytotoxicity Assay, Expressing, Western Blot, 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: 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: Cytotoxicity Detection Kit (LDH) , Merck , , 11644793001.

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: Cytotoxicity Detection Kit (LDH) , Merck , , 11644793001.

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 BMDM ( A , B ) and HMDM ( C ) were primed for 4 h with 1 µg/mL Pam 3 CSK 4 . Cell culture medium was then replaced with 10 µM nigericin, 200 µg/mL silica, or 5 μg/ml of poly(dA:dT) in the presence of 10 μM CL (+) or HEPES vehicle (−). Cells were incubated for 1 h (nigericin), 18 h (silica), or 4 h (poly(dA:dT). Cell supernatants were analysed for cleaved IL-1β (ELISA) and LDH (cytotoxicity assay). 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. Statistical analysis: Data were verified for normality using a Shapiro–Wilk test and analysed by 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: Cytotoxicity Detection Kit (LDH) , Merck , , 11644793001.

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