Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A, B March5 cKO mice are resistant to P. aeruginosa infection (A) survival rates ( n = 8) and (B) changes in the body weight of March5 fl/fl and March5 fl/fl;Lyz‐Cre ( n = 8) mice after intraperitoneal injection with 1 × 10 7 CFU Pseudomonas aeruginosa . C–F TNF‐α (C), IL‐6 (D), IL‐1β (E) and IL‐18 (F) from serum, spleen homogenate and peritoneal fluid collected from mice ( n = 8) that were sacrificed 12 and 24 h following the bacterial infection. Each cytokine was analyzed by ELISA. Data information: Values, * P < 0.05, ** P < 0.01 (two‐tailed Student's t ‐test or Mantel–Cox test). Data were expressed as the mean ± SEM. See also Fig . Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Infection, Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A, B (A) Survival rates ( n = 12–14) and (B) variation of body weight of March5 fl/fl and March5 fl/fl;Lyz‐Cre ( n = 7–9) mice after intraperitoneal injection with 28 mg/kg body weight of LPS. C–F ELISA of a TNF‐α (C), IL‐6 (D), IL‐1β (E) and IL‐18 (F) from serum, spleen homogenate and peritoneal fluid from mice ( n = 5), sacrificed 12 and 24 h after LPS injection. Values, * P < 0.05 (two‐tailed Student's t ‐test or Mantel–Cox test.). Data were expressed as the mean ± SEM. Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A–C (A) Caspase‐1, (B) IL‐1β secretion and (C) LDH release were measured in the supernatants of March5 fl/fl and March5 fl/fl;Lyz‐Cre BMDMs subjected to the indicated stimuli followed by material method. Values are the mean ± SD. Experiments (A–C) were performed in triplicate and repeated at least three times. *** P < 0.001 (two‐tailed Student's t ‐test) D–F MARCH5 mediates activation of the NLRP3 pathway in response to bacterial infection. March5 fl/fl and March5 fl/fl;Lyz‐Cre BMDMs were infected with (D) Citrobacter rodentium (20 MOI, 40 MOI and 80 MOI), (E) Salmonella typhimurium (1 MOI, 5 MOI and 10 MOI), and (F) Pseudomonas aeruginosa (1 MOI, 10 MOI and 20 MOI). Secretion of IL‐1β, IL‐18, IL‐6 and TNF‐α in BMDMs infected for 12 h was measured by ELISA. Values, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (two‐tailed Student's t‐ test). Data are expressed as the mean ± SEM. Experiments (D–F) were performed in triplicate and repeated three times. See also Fig . Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Two Tailed Test, Activation Assay, Infection, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A–C (A) Activated caspase‐1, (B) IL‐1β, and (C) LDH release were measured in the supernatants of siControl (siCtrl) or siMARCH5 THP‐1 cells and were subjected to the indicated stimuli. Independent experiments were repeated at least three times. Values are the mean ± SD. ** P < 0.01, *** P < 0.001 (two‐tailed student's t ‐test). D–G March5 fl/fl and March5 fl/fl;Lyz‐Cre BMDMs were infected with (D) Citrobacter rodentium (20 MOI, 40 MOI and 80 MOI), (E) Salmonella typhimurium (1 MOI, 5 MOI and 10 MOI), and (F) Psedumonas aeruginosa (1 MOI, 10 MOI and 20 MOI). Secretions of caspase‐1 in BMDMs infected for 12 h were measured (D–F), and the cell pellet was used for western blotting to detect the activation of NLRP3 inflammasome (G) in response to bacterial infection. Values, *** P < 0.001, **** P < 0.0001 (two‐tailed Student's t ‐test). Data were expressed as the mean ± SEM. Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Two Tailed Test, Infection, Western Blot, Activation Assay

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A MARCH5 WT and KO HEK293T cells were transfected with FLAG‐NLRP3 and HA‐ub. After the cells were stimulated with LPS (200 ng/ml for 4 h) and nigericin (15 μM for 60 min), the cell lysates were immunoprecipitated with FLAG‐M2 beads. Ubiquitinated NLRP3 was detected by anti‐HA antibody. B FLAG‐NLRP3 and HA‐ub K48R mutant plasmids were transfected into MARCH5 KO HEK293T cells with or without Myc‐MARCH5 2KR. Then, the cells were stimulated with 200 ng/ml LPS followed by 15 μM nigericin for the indicated durations. The cell lysates were immunoprecipitated with FLAG‐M2 beads and immunoblotted by using indicated antibodies. C MARCH5 KO HEK293T cells were transfected with FLAG‐NLRP3, Myc‐MARCH5 2KR, and each HA ‐ ubiquitin, shown by the number of the remaining single Lys residue with the other Lys changed to Arg. For stimulation, the cells were treated with 200 ng/ml LPS for 4 h, followed by 15 μM nigericin for 30–60 min. Cell lysates were immunoprecipitated with FLAG‐M2 beads and analyzed by immunoblotting with the indicated antibodies. D The NLRP3 inflammasome was reconstituted in HEK293T MARCH5 KO cells expressing ASC, pro‐caspase‐1, and IL‐1β with HA‐ub WT, K27 only (K27O) mutant, or K27R mutant. Additionally, cells were cotransfected with or without Myc‐MARCH5 2KR or the Myc‐MARCH5 H43W mutant. Following stimulation with LPS for 4 h and nigericin for 30 min, IL‐1β secretion was quantitated by ELISA. Values are the mean ± SD. ** P < 0.01, *** P < 0.001 (two‐tailed Student's t ‐test). All the experiments were carried out in triplicate three times. E HEK293T MARCH5 K/O cells were transfected with FLAG‐NLRP3, Myc‐MARCH5 (2KR), HA‐ubiquitin and GFP‐YOD1. Transfected cells were stimulated with 200 ng/ml LPS for 4 h, followed by 15 μM nigericin for 60 min. Cell lysates were immunoprecipitated with FLAG‐M2 beads and analyzed by ubiquitination with HA‐antibody. The other proteins were detected by indicated antibodies. F Schematic representation of NLRP3 Lys mutants (Upper). HEK293T MARCH5 KO cells were cotransfected with FLAG‐NLRP3 WT or indicated Lys point mutants, HA‐ubiquitin, and Myc‐MARCH5 (2KR). Cells were stimulated with 200 ng/ml LPS for 4 h followed by 15 μM nigericin treatment for 30 min. The cell lysates were immunoprecipitated with FLAG‐M2 beads. NLRP3 ubiquitination was assessed via western blotting using anti‐HA. And each protein was detected by indicated antibodies (Lower). G FLAG‐NLRP3 WT or indicated Lys point mutants, HA‐ub K27 only mutant and Myc‐MARCH5 2KR were transfected into MARCH5 KO HEK293T cells. Cell lysates were immunoprecipitated with FLAG‐M2 beads. Ubiquitinated NLRP3 was detected by immunoblotting using an HA antibody. Representative data are shown from independent experiments that were repeated at least three times. See also Fig . Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Transfection, Immunoprecipitation, Mutagenesis, Residue, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A March5 fl/fl and March5 fl/fl;Lyz‐Cre BMDMs were primed with LPS for 4 h and were stimulated without or with 5 mM ATP at the indicated time points. Whole‐cell lysates were analyzed via western blotting using the indicated antibodies. B MARCH5 KO HEK293T cells were cotransfected with HA‐ub (K48R) mutant and NLRP3 without or with Myc‐MARCH5 2KR or Myc‐MARCH5 H43W. After treatment with nigericin for 30 min of the LPS treatment for 4 h, the cell lysates were subjected to immunoprecipitation with anti‐FLAG M2 beads. An HA antibody was used to assess NLRP3 ubiquitination. C HEK293T MARCH5 KO cells were cotransfected with HA‐ub (K48R) and NLRP3 WT or NLRP3 truncated mutants with or without SFB‐MARCH5 (2KR). After treatment with LPS and nigericin, the cell lysates were subjected to immunoprecipitation with an anti‐Myc antibody overnight, and NLRP3 ubiquitination was assessed via western blotting by using an anti‐HA antibody. D HEK293T MARCH5 KO cells were transfected HA‐ub (K27O) with NLRP3 WT and truncated mutants. After stimulation with LPS and nigericin, cell lysates were immunoprecipitated with anti‐Myc antibody. The HA antibody detected ubiquitination in the subjects. E The NLRP3 inflammasome was reconstituted in HEK293T MARCH5 KO cells expressing ASC, pro‐caspase 1, and IL‐1β with HA‐ub WT, K27O mutant, or K27R mutant NLRP3. Additionally, cells were cotransfected with or without Myc‐MARCH5 2KR or the Myc‐MARCH5 H43W mutant. After stimulation with LPS for 4 h and nigericin for 30 min, IL‐1β secretion was quantitated using ELISA, and the lysates were detected by western blotting with the indicated antibodies. Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Western Blot, Mutagenesis, Immunoprecipitation, Transfection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A HEK293T cells were transfected with NLRP3 WT or indicated Lys mutants and ASC. Cells were stimulated with 200 ng/ml LPS for 4 h followed by 15 μM nigericin for 30 min. The percentage of cells with an ASC speck was quantified after Confocal microscopy. At least 100 cells were analyzed. Values are the mean ± SD. Values, ** P < 0.01, *** P < 0.001 (two‐tailed Student's t ‐test). Bar, 10 μm. B HEK293T cells were transfected with ASC and NLRP3 (WT or individual NLRP3 mutants). Cells were treated for 4 h with 200 ng/ml LPS and 30 min with 15 μM nigericin. Harvested cell lysates were incubated with 2 mM DSS for cross‐linking. Triton X‐100 insoluble pellets and soluble fraction lysates were detected by immunoblotting with the indicated antibodies. C NLRP3 inflammasome‐reconstituted HEK293T cells with NLRP3 WT or indicated mutants were stimulated for 4 h with 200 ng/ml LPS and 30 min with 15 μM nigericin. Harvested cells were detected by immunoblotting with the indicated antibodies. D Culture supernatants were obtained from (C) and subjected to ELISA to quantify secreted IL‐1β. Representative data are shown from independent experiments that were repeated at least three times. Values are the mean ± SD. * P < 0.05, *** P < 0.001 (two‐tailed Student's t ‐test) E Schematic diagram of NLRP3 inflammasome activation by MARCH5. Upon activation by a priming signal, NLRP3 associates with MAVS on mitochondria ①. MARCH5 recognizes and ubiquitinates two lysine sites, K324 and K430, on the NLRP3 NACHT domain via K27‐linked polyubiquitination ②. NLRP3 ubiquitination by MARCH5 promotes the recruitment of NEK7 and enables self‐oligomerization ③. As a result, ASC and pro‐caspase‐1 are recruited to oligomerized NLRP3 ④ for NLRP3 inflammasome assembly ⑤ and trigger the activation of caspase‐1 and pro‐cytokine cleavage ⑥. Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Transfection, Confocal Microscopy, Two Tailed Test, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet:

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Recombinant, Sequencing, Software, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cardiovascular Translational Research

Article Title: Bufalin Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Macrophage Pyroptosis via P62 Pathway

doi: 10.1007/s12265-024-10577-9

Figure Lengend Snippet: Bufalin attenuated macrophage pyroptosis and reduced IL-1β release into the myocardium and serum during I/R injury. A-C The RNA sequencing data were extracted from GSE 126772. The mRNA levels of Nlrp3 , Pycard ( ASC ) and Il-1b in macrophages isolated from heart after MI were shown as fold change compared to the Pre group (control group, n = 2,3). D Macrophages were identified and isolated from the heart by flow cytometry. E–F Immunoblotting was performed to assess the expression levels of NLRP3, GSDMD and IL-1β in macrophages after I/R injury with or without bufalin treatment. Each target band was normalized to the β-actin band and the relative expression was calculated as the fold change compared to the Sham + PBS group (control group, n = 3). G Representative IL-1β expression and localization in murine hearts are shown by IHC analysis in longitudinal sections of whole hearts. For each mouse, 5 representative zones in the left ventricle were chosen randomly to determine the percentage of IL-1β-positive ratio by ImageJ ( n = 3). H IL-1β levels in the peripheral blood of mice that were subjected to I/R injury and were treated with or without bufalin were measured by ELISA ( n = 6). IHC (immunohistochemistry). Data are expressed as the mean ± SEM. P values were determined by ordinary one-way ANOVA with Dunnett’s multiple comparisons test ( A - C ) and two-way ANOVA with Tukey’s multiple comparisons test ( F and H )

Article Snippet: For interleukin-1β (IL-1β) treatment, recombinant mouse IL-1β (HY-P7073, MedChemExpress, New Jersey, USA) was added to HL-1 cells at different concentrations (0, 100 pg/ml, 300 pg/ml, 1000 pg/ml) and incubated for 24 h. For anakinra treatment, the recombinant human interleukin-1 receptor (IL-1R) antagonist anakinra (HY-108841, MedChemExpress) was added to cardiomyocytes every 6 h for 24 h when they were cocultured with normoxia-treated or H/R-treated macrophages.

Techniques: RNA Sequencing, Isolation, Control, Flow Cytometry, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Journal of Cardiovascular Translational Research

Article Title: Bufalin Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Macrophage Pyroptosis via P62 Pathway

doi: 10.1007/s12265-024-10577-9

Figure Lengend Snippet: Bufalin attenuated H/R-treated macrophage pyroptosis in vitro. Macrophages were subjected to 1 h of hypoxia and 24 h of reoxygenation treated with PBS or bufalin. A-B Proteins were extracted from cells and immunoblotting analysis was performed. The expressions of NLRP3, GSDMD, and IL-1β were normalized to that of β-actin and then compared with the Sham + PBS group (control group, n = 3). C PI staining was used to examine the plasma membrane integrity of macrophages. Positive cells were counted and normalized to the total number of cells ( n = 5). D Cell viability was assessed by measuring the quantity of ATP in macrophages subjected to different treatments ( n = 3, 6). E IL-1β levels in the culture medium of macrophages were measured by ELISA ( n = 3). Data are presented as the mean ± SEM. P values were calculated by two-way ANOVA with Tukey’s multiple comparisons test ( B , C , and E ) or Šídák’s multiple comparisons test ( D )

Article Snippet: For interleukin-1β (IL-1β) treatment, recombinant mouse IL-1β (HY-P7073, MedChemExpress, New Jersey, USA) was added to HL-1 cells at different concentrations (0, 100 pg/ml, 300 pg/ml, 1000 pg/ml) and incubated for 24 h. For anakinra treatment, the recombinant human interleukin-1 receptor (IL-1R) antagonist anakinra (HY-108841, MedChemExpress) was added to cardiomyocytes every 6 h for 24 h when they were cocultured with normoxia-treated or H/R-treated macrophages.

Techniques: In Vitro, Western Blot, Control, Staining, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cardiovascular Translational Research

Article Title: Bufalin Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Macrophage Pyroptosis via P62 Pathway

doi: 10.1007/s12265-024-10577-9

Figure Lengend Snippet: Bufalin restored autophagic flux by suppressing P62 pathway in H/R-induced macrophages. Macrophages were transfected with the p62-overexpression plasmid for 24 h and then subjected to H/R with or without bufalin. A - B The protein levels of P62 were measured by immunoblotting. The bands were normalized to the β-actin band, and the relative expressions were calculated as the fold changes compared to the control group ( n = 3). C The mRNA levels of p62 were measured by qRT‒PCR. The Cq results were normalized to Mus-18 s and then compared with the control group ( n = 3). D-E Proteins were extracted from H/R-treated macrophages that were infected with vector or OE-p62 plasmid in advance and treated with bufalin or PBS. Immunoblotting was performed to measure the expressions of NLRP3, GSDMD and IL-1β. Each band was normalized to the β-actin band and the relative expression was calculated as the fold change compared to the Con + PBS group (control group, n = 3). F PI staining was used to assess pyroptosis in macrophages. Positive ratios were calculated as the percentage of positive cells compared with total number of cells ( n = 5). G IL-1β levels in the culture medium of macrophages were measured by ELISA ( n = 3). Data are presented as the mean ± SEM. P values were calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons test

Article Snippet: For interleukin-1β (IL-1β) treatment, recombinant mouse IL-1β (HY-P7073, MedChemExpress, New Jersey, USA) was added to HL-1 cells at different concentrations (0, 100 pg/ml, 300 pg/ml, 1000 pg/ml) and incubated for 24 h. For anakinra treatment, the recombinant human interleukin-1 receptor (IL-1R) antagonist anakinra (HY-108841, MedChemExpress) was added to cardiomyocytes every 6 h for 24 h when they were cocultured with normoxia-treated or H/R-treated macrophages.

Techniques: Transfection, Over Expression, Plasmid Preparation, Western Blot, Control, Infection, Expressing, Staining, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: High-Level Replication of Human Immunodeficiency Virus in Thymocytes Requires NF-?B Activation through Interaction with Thymic Epithelial Cells

doi:

Figure Lengend Snippet: TNF and, to a lesser extent, IL-1 are involved in the permanently induced NF-κB activity present in thymocytes during the coculture with TEC. Thymocytes were freshly isolated (lanes 1 and 2) or cultured for 45 h (lanes 3 to 11) either alone (lane 3 [Control thymocytes]) or in the presence of TEC under conditions avoiding contact (lanes 4 to 9) or allowing contact (lanes 10 and 11). During the coculture, the cells were either left untreated (lane 4 [Control coculture, 45 hours] and lane 10 [Control coculture]) or were treated at the start of the coculture with antibodies respectively raised against TNF (lane 5), IL-6 (lane 6), and IL-1β (lane 8), with the antagonist of the IL-1 receptor (lane 7), or with αTNF plus IL-1 ra plus αIL-6 (lanes 9 and 11). Whole-cell extracts from these various samples were incubated with a 32P-labeled oligonucleotide representing the HIV LTR-derived κB motif. Competition (comp [lane 1]) with a 40-fold molar excess of unlabeled oligonucleotide was used to confirm the specificity of the DNA-binding activity detected. NF-κB activity is indicated. The positions of two nonspecific (n.s 1 and n.s 2) binding activities are indicated. The positions of two nonspecific (n.s 1 and n.s 2) binding activities are indicated. This experiment is representative of three independent experiments, each carried out on a different thymus.

Article Snippet: MAb against IL-1β (B-A15) was purchased from Diaclone Research (Besançon, France).

Techniques: Activity Assay, Isolation, Cell Culture, Incubation, Labeling, Derivative Assay, Binding Assay

Journal:

Article Title: High-Level Replication of Human Immunodeficiency Virus in Thymocytes Requires NF-?B Activation through Interaction with Thymic Epithelial Cells

doi:

Figure Lengend Snippet: TNF and, to a lesser extent, IL-1 induce NF-κB activity in thymocytes, and IL-7 is required for this effect. Whole-cell extracts were prepared from freshly isolated thymocytes (panel A, lane 1) or from thymocytes cultivated for 30, 60, or 45 h. (A) During the indicated culture times, thymocytes were either left untreated (lane 1, control for freshly isolated; lane 2, control 30 h; and lane 5, control 60 h) or were stimulated with either TNF (lanes 3 and 6), IL-1β (lanes 4 and 7), or TNF in the presence of TEC CM (TEC CM + TNF) (lane 9). TEC conditioned medium was also added alone (lane 8 [TEC CM]). The data shown here are representative of three independent experiments carried out on three thymuses. (B) Thymocytes were cultured for 45 h either untreated (lane 1, control 45 h), with TNF (lane 2), with TEC CM (lane 3), or with TNF in the presence of TEC CM (TEC CM + TNF) preincubated (lane 5) or not (lane 4) with antibody against IL-7. In lanes 6 to 8, the thymocytes were left untreated for 6 h (lane 6) or incubated with an antibody against IL-7Rα (lane 7) or with an IgG1 control serum (lane 8) before being cocultured with TEC for 45 h. The data shown here are representative of two independent experiments carried out on two thymuses. (C) Cells were left untreated for the 45 h of the culture (lane 1, control 45 h) or treated with IL-1β (lane 2), TNF (lane 3), IL-1β plus TNF plus IL-6 plus GM-CSF (lane 4), IL-7 (lane 5), IL-7 plus IL-1β (lane 6), or IL-7 plus TNF (lane 7). This experiment is representative of three independent experiments carried out on three thymuses. (D) Thymocytes were left untreated (lane 1, control) or stimulated with IL-7 (lane 2) or IL-7 in presence of anti-TNF and Il-1ra (lane 3). Whole-cell extracts were incubated with a 32P-labeled oligonucleotide representing κB motif. NF-κB activity is indicated. This experiment is representative of two independent experiments, each carried out on a different thymus.

Article Snippet: MAb against IL-1β (B-A15) was purchased from Diaclone Research (Besançon, France).

Techniques: Activity Assay, Isolation, Cell Culture, Incubation, Labeling

Journal: Stem Cell Research & Therapy

Article Title: Interleukin-1 receptor antagonist overexpression in mesenchymal stem cells improves hemorrhagic cystitis outcomes via HtrA serine peptidase 3

doi: 10.1186/s13287-025-04443-x

Figure Lengend Snippet: oeIL-1Ra-MSCs attenuated the inflammatory response in bladder tissues from HC rats. A CD80 staining of rat bladder tissues and CD80-positive cell counts (n = 3). B CD206 staining of rat bladder tissues and CD206-positive cell counts (n = 3). C – E mRNA expression levels of IL-1β, IL-6, and TNF-α in rat bladder tissues (n = 3). Scale bar = 50 μm. Data are presented as the mean ± SEM of three independent experiments. ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: oeIL-1Ra-MSCs were pre-treated with JNK-activator Anisomycin (5 μM, HY-18982, MedChem Express, USA), recombinant IL-1β protein (10 ng/mL [ , ], HZ-1164, Proteintech, Wuhan, China), JNK inhibitor SP600125 (20 μM, HY-12041, MedChem Express, USA), and Erk inhibitor Ravoxertinib (GDC-0994, MedChem Express, USA).

Techniques: Staining, Expressing

Journal: Frontiers in Pharmacology

Article Title: Scutellarein ameliorates dextran sulfate sodium-induced ulcerative colitis by inhibiting colonic epithelial cell proinflammation and barrier disruption

doi: 10.3389/fphar.2024.1479441

Figure Lengend Snippet: Scu attenuated colonic inflammation in DSS-treated mice. (A) MPO activity in colon tissues from Veh- and DSS-treated mice, administered with Scu and 5-ASA. (B–F) Quantitative RT-PCR analysis of mRNA levels of Tnf-α (B) , Il-1β (C) , Il-1α (D) , Cxcl1 (E) , and Il-6 (F) , respectively, in colon tissues from different groups of mice. Data are presented as the mean ± SEM. N = 6 mice; **, p < 0.01 vs. Veh; # , p < 0.05, ## , p < 0.01 vs. DSS. One-way ANOVA with Bonferroni’s multiple comparisons tests.

Article Snippet: BAY11-7085 (Cat# HY-10257), 5-aminosalicylic acid (5-ASA, Cat# HY-15027), and IL-1β (Cat# HY-P78459A) were obtained from MedChemExpress (Monmouth Junction, NJ, United States).

Techniques: Activity Assay, Quantitative RT-PCR

Journal: Frontiers in Pharmacology

Article Title: Scutellarein ameliorates dextran sulfate sodium-induced ulcerative colitis by inhibiting colonic epithelial cell proinflammation and barrier disruption

doi: 10.3389/fphar.2024.1479441

Figure Lengend Snippet: Scu inhibited IL-1β-induced mRNA expression of inflammatory mediators and barrier damage of HT-29 cells. (A) HT-29 cell viability measured by the MTT assay after being exposed to different concentrations of Scu for 24 h (B, C) Quantitative RT-PCR analysis of mRNA levels of IL-6 (B) and IL-8 (C) in HT-29 cells treated with IL-1β (10 ng/mL) for 12 h in the absence and presence of Scu (10 μM). (D) Change in the TEER of HT-29 monolayer treated with IL-1β (10 ng/mL, 24 h) in the absence and presence of Scu (10 μM). (E) Representative immunofluorescence images of E-cadherin (upper panel), occludin (middle panel), and ZO-1 (lower panel) in HT-29 cells after being exposed to IL-1β (10 ng/mL) for 24 h in the absence and presence of Scu (10 μM). Nuclei were stained with Hoechst 33342. Scale bar = 10 μm. (F) Representative Western blots of E-cadherin, occludin, and ZO-1 in HT-29 cells exposed to IL-1β (10 ng/mL) for 24 h in the absence and presence of Scu (10 μM). (G) Quantification of E-cadherin, occludin, and ZO-1 protein expression in HT-29 cells after being exposed to IL-1β (10 ng/mL) for 24 h in the absence and presence of Scu (10 μM). Data are expressed as the mean ± SEM. N = 5–6; **, p < 0.01 vs. Veh (0.1% DMSO); # , p < 0.05, ## , p < 0.01 vs. IL-1β. One-way ANOVA with Bonferroni’s multiple comparisons tests.

Article Snippet: BAY11-7085 (Cat# HY-10257), 5-aminosalicylic acid (5-ASA, Cat# HY-15027), and IL-1β (Cat# HY-P78459A) were obtained from MedChemExpress (Monmouth Junction, NJ, United States).

Techniques: Expressing, MTT Assay, Quantitative RT-PCR, Immunofluorescence, Staining, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Scutellarein ameliorates dextran sulfate sodium-induced ulcerative colitis by inhibiting colonic epithelial cell proinflammation and barrier disruption

doi: 10.3389/fphar.2024.1479441

Figure Lengend Snippet: Scu inhibited IL-1β-induced phosphorylation of NF-κB and its nuclear translocation in HT-29 cells. (A) Representative Western blots and (B) quantification of phosphorylated NF-κB in HT-29 cells. HT-29 cells were exposed to IL-1β (10 ng/mL) in the absence and presence of Scu (10 μM). (C) Representative immunofluorescence images of stained with NF-κB in HT-29 cells. Cells were treated with IL-1β (10 ng/mL) for 30 min in the absence and presence of Scu (10 μM). Nuclei were stained with Hoechst 33342. Scale bar = 10 μm. (D, E) Quantitative RT-PCR analysis of mRNA levels of IL-6 (D) and IL-8 (E) in HT-29 cells treated with IL-1β (10 ng/mL) for 12 h in the absence and presence of BAY11-7082 (3 μM). (F) Changes in the TEER of HT-29 monolayer treated with IL-1β (10 ng/mL) for 24 h in the absence and presence of BAY11-7082 (3 μM). (G) Representative immunofluorescence images of E-cadherin, occludin, and ZO-1 in HT-29 cells exposed to IL-1β (10 ng/mL) for 24 h in the absence and presence of BAY11-7085 (3 μM). Nuclei were stained with Hoechst 33342. Scale bar = 10 μm. N = 3–6. **, p < 0.01 vs. Veh; # , p < 0.05, ## , p < 0.01 vs. IL-1β. One-way ANOVA with Bonferroni’s multiple comparisons tests.

Article Snippet: BAY11-7085 (Cat# HY-10257), 5-aminosalicylic acid (5-ASA, Cat# HY-15027), and IL-1β (Cat# HY-P78459A) were obtained from MedChemExpress (Monmouth Junction, NJ, United States).

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR