Journal: Frontiers in Medicine

Article Title: A Prognostic Signature Consisting of Pyroptosis-Related Genes and SCAF11 for Predicting Immune Response in Breast Cancer

doi: 10.3389/fmed.2022.882763

Figure Lengend Snippet: Identification of seven pyroptosis-related genes with prognostic power in breast cancer (BRCA) patients. (A,B) Seven pyroptosis-related genes with prognostic value were identified by LASSO Cox regression, including CASP9, GPX4, IL18, NLRC4, SCAF11, TIRAP, and TNF. (C–I) Kaplan-Meier survival analysis was employed to evaluate the prognostic capability of these candidate genes, respectively. (J) Time-dependent ROC curves were applied to assess the prognostic value of seven pyroptosis-related gene signature in the TCGA-BRCA cohort.

Article Snippet: Primary antibodies included SCAF11 antibody (1:1,000, 28098-1-AP, proteintech) and GAPDH (1:10,000, 60004-1-Ig, proteintech).

Techniques:

Journal: Frontiers in Medicine

Article Title: A Prognostic Signature Consisting of Pyroptosis-Related Genes and SCAF11 for Predicting Immune Response in Breast Cancer

doi: 10.3389/fmed.2022.882763

Figure Lengend Snippet: The expression of seven pyroptosis-related genes in BRCA tissues and SCAF11 knockdown inhibited the cell proliferation. (A–G) Differential mRNA expression analysis of the seven pyroptosis-related genes in BRCA samples and normal breast tissues. The Xiantao tool was used to perform the expression profiles. (H,I) Knockdown efficacy of siRNA against SCAF11 in two human breast cancer cell lines, BT549 and T47D, was analyzed by western blotting. (J,K) CCK-8 assay indicated the effects of SCAF11 knockdown on cell proliferation rate of BT549 and T47D. (L,M) SCAF11 knockdown inhibited the cell colony formation rate in BT549 and T47D cell lines. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no statistical significance.

Article Snippet: Primary antibodies included SCAF11 antibody (1:1,000, 28098-1-AP, proteintech) and GAPDH (1:10,000, 60004-1-Ig, proteintech).

Techniques: Expressing, Knockdown, Western Blot, CCK-8 Assay

Journal: Frontiers in Medicine

Article Title: A Prognostic Signature Consisting of Pyroptosis-Related Genes and SCAF11 for Predicting Immune Response in Breast Cancer

doi: 10.3389/fmed.2022.882763

Figure Lengend Snippet: The co-expression genes with SCAF11 from the LinkedOmics database and GSEA enrichment analysis from Xiantao tool. (A) Volcano plot showing genes significantly associated with SCAF11 by Spearman test. (B,C) Heatmaps of the top 50 genes positively and negatively correlated with SCAF11, respectively. (D,E) KEGG and GO pathway analysis.

Article Snippet: Primary antibodies included SCAF11 antibody (1:1,000, 28098-1-AP, proteintech) and GAPDH (1:10,000, 60004-1-Ig, proteintech).

Techniques: Expressing

Journal: Frontiers in Medicine

Article Title: A Prognostic Signature Consisting of Pyroptosis-Related Genes and SCAF11 for Predicting Immune Response in Breast Cancer

doi: 10.3389/fmed.2022.882763

Figure Lengend Snippet: The relationships between immune cell infiltration with SCAF11. (A) Immune infiltration cells associated with SCAF11 by Xiantao tool. (B) Validations of partial lymphocytes by the TISIDB database. Associations of SCAF11 expression with immunoinhibitors (C) , immunostimulators (D) , and MHC molecules (E) . (F) The association between SCAF11 with PD-L1, B7-H3, and PDCD1LG2.

Article Snippet: Primary antibodies included SCAF11 antibody (1:1,000, 28098-1-AP, proteintech) and GAPDH (1:10,000, 60004-1-Ig, proteintech).

Techniques: Expressing

Journal: Bioengineered

Article Title: Dendrobine suppresses endoplasmic reticulum stress-induced apoptosis through upregulating microRNA miR-381-3p to decrease caspase-4.

doi: 10.1080/21655979.2021.1956672

Figure Lengend Snippet: Figure 5. miR-381-3p binds to the 3-untranslated regions of caspase-4. (a) Binding sites predicted by starBase. (b) Results of dual- luciferase reporter assay. miR, micoRNA. ***P < 0.001 Vs WT+ Caspase4+ NC mimic.

Article Snippet: For the detection of caspase4 mRNA, PCR quantification was performed using SYBR Green qPCR Master Mix (MedChemExpress).

Techniques: Binding Assay, Luciferase, Reporter 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 ) 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