Journal: Frontiers in Immunology

Article Title: Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome

doi: 10.3389/fimmu.2018.01920

Figure Lengend Snippet: CVL reduced NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 5 h with LPS (1 μg/ml) (LPS priming) followed by incubation for 0.5 h with CVL. Cells were then incubated with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), ATP (5 mM, 0.5 h), nigericin (10 μM, 0.5 h), and nano-SiO 2 (100 μg/ml, 24 h). (B) LPS-primed J774A.1 macrophages were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), and ATP (5 mM, 0.5 h). (C) LPS-primed BMDM were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml) for an additional 24 h. (D) LPS-primed or Pam3CSK4-primed (for LPS transfection only) cells were incubated for 0.5 h with CVL followed by transfection with poly(dA/dT) (2 μg/ml, 6 h), FLA-ST (1 μg/ml, 6 h), MDP (10 μg/ml, 6 h), or LPS (2 μg/ml, 6 h). The levels of IL-1β, IL-18, NLRP3, ASC, and caspase-1 in the culture medium were measured by Western blot. The IL-1β levels in the supernatants were measured by ELISA. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. *, **, and *** indicate a significant difference at the level of p < 0.05, p < 0.01, and p < 0.001, respectively, compared to activator-treated cells.

Article Snippet: ELISA kit for active caspase-1 was purchased from IBL-America (Minneapolis, MN).

Techniques: Activation Assay, Incubation, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome

doi: 10.3389/fimmu.2018.01920

Figure Lengend Snippet: CVL reduced caspase-1-dependent pyroptosis. J774A.1 macrophages were incubated for 5 h with LPS (1 μg/ml) followed by incubation for 0.5 h with CVL (20 μM). Cells were then incubated for 0.5 h with ATP (5 mM). (A) Cell viability was assayed by the AlamarBlue cell viability assay kit. (B) LDH release was assayed by the CytoTox 96 Non-radioactive Cytotoxicity assay kit. (C) The membrane integrity was measured by staining the cells with 40 μg/ml of PI, and analyzed by flow cytometry. (D) The cell size was determined by calculating 20 representative cells using ImageJ software. The data are expressed as the means ± SD for three separate experiments. * and ** indicates a significant difference at the level of P < 0.05 and P < 0.01, respectively.

Article Snippet: ELISA kit for active caspase-1 was purchased from IBL-America (Minneapolis, MN).

Techniques: Incubation, Viability Assay, Cytotoxicity Assay, Staining, Flow Cytometry, Software

Journal: Frontiers in Immunology

Article Title: Repositioning of the β-Blocker Carvedilol as a Novel Autophagy Inducer That Inhibits the NLRP3 Inflammasome

doi: 10.3389/fimmu.2018.01920

Figure Lengend Snippet: CVL reduced inflammation in a mouse model of MSU-mediated peritonitis. (A) Neutrophil influx was quantified by Gr-1 and CD45 staining. (B) The levels of IL-1β, active caspase-1, IL-6, TNF-α, MCP-1, and CXCL1 in the peritoneal lavage fluids were measured by ELISA. (C) The levels of IL-18 and ASC in the peritoneal lavage fluids and NLRP3 and HO-1 in the peritoneal cells were measured by Western blot. The Western blot results are representative of different experiments and the histogram shows the quantification expressed as the mean ± SD. The ELISA data are expressed as the mean ± SD of three separate experiments. *, **, and *** indicate a significant difference at the level of p < 0.05, p < 0.01, and p < 0.001, respectively, compared to MSU-injected mice. Control group: n = 3; MSU group: n = 6; CVL+MSU group: n = 6; CVL group: n = 3.

Article Snippet: ELISA kit for active caspase-1 was purchased from IBL-America (Minneapolis, MN).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Injection