Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Gasdermin D Drives the Nonexosomal Secretion of Galectin-3, an Insulin Signal Antagonist.

doi: 10.4049/jimmunol.1900212

Figure Lengend Snippet: FIGURE 1. Identification of galectin-3 as an inflammasome-regulated secretory protein. (A) Diagram of secreted proteins in CM from LPS/ATP–treated WT and Nlrp32/2 BMDMs. (B) Two-dimensional PAGE analysis of supernatant from WT and Nlrp3+/R258W BMDMs treated with 500 ng/ml LPS for 4 h. The labeled dots were dug out for MS analysis. (C) Protein list of identified proteins from (B) by MS. The blue-labeled proteins are at least 2-fold lower in Nlrp32/2 CM than that in WT CM of (A). p # 0.05. (D) Volcano plot of secreted proteins in (A). The pink circles represent individual protein at least 2-fold higher or lower in WT CM than that in Nlrp32/2 CM, with p # 0.05. (E) ELISA detection of serum galectin-3 level in WT mice (n = 5), which received i.p. injection of Ac-YVAD (20 mg/kg) or vehicle daily for 3 d. Data are shown as mean 6 SEM. ***p # 0.001. (F) ELISA detection of serum galectin-3 level in WT and Casp1/112/2 mice (n = 4). Data are shown as mean 6 SEM. ***p # 0.001.

Article Snippet: Casp1 inhibitor Ac-YVAD-CMK (catalog no. sc-300323; Santa Cruz Biotechnology) or galectin-3 inhibitor TD139 (catalog no. HY-19940; MedChemExpress) was administrated daily by i.p. injection.

Techniques: Labeling, Enzyme-linked Immunosorbent Assay, Injection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Gasdermin D Drives the Nonexosomal Secretion of Galectin-3, an Insulin Signal Antagonist.

doi: 10.4049/jimmunol.1900212

Figure Lengend Snippet: FIGURE 2. The inflammasome activation drives cellular and systemic secretion of galectin-3. (A) BMDMs from WT or Nlrp3+/R258W mice were stimulated with 500 ng/ml LPS for the indicated times; supernatant (SUP) and whole cell lysate (WCL) were collected for immunoblotting of the indi- cated proteins. Data are representative of at least three independent experiments. (B) LPS-primed WT BMDMs were treated with ATP (5 mM) or nigericin (5 mg/ml) for 30 min, and immunoblotting detection of indicated proteins is shown. Data are representative of at least three independent experiments. (C) LPS-primed peritoneal macrophages were stimulated with ATP (5 mM) for 30 min. Indicated proteins were detected via immunoblotting assay. Data are representative of at least two independent experiments. (D) LPS-primed BMDC were stimulated with ATP (5 mM) for 30 min. Indicated proteins were detected via immunoblotting assay. Data are representative of at least three independent experiments. (E) BMDM were primed with LPS for 10 min, followed by ATP (5 mM) challenge for 30 min. Indicated proteins were detected via immunoblotting assay. Data are representative of at least three in- dependent experiments. (F) Western blotting analysis of SUP and WCL from WT, Nlrp32/2, Asc2/2, or Casp1/112/2 BMDMs stimulated with LPS/ATP. Data are representative of at least three independent experiments. (G) Western blotting analysis of SUP and WCL from LPS-primed THP-1 macrophages stimulated with ATP. Data are representative of at least three independent experiments. (H) Western blotting analysis of SUP and WCL from unprimed WT or Aim22/2 BMDMs transfected with poly(dA:dT) for 2 h. Data are representative of at least two independent experiments. (I) Western blotting analysis of SUP and WCL from unprimed WT or Nlrc42/2 BMDMs infected with S. typhimurium (multiplicity of infection = 10) for 3 h. Data are representative of at least two independent experiments. (J) Western blotting analysis of SUP and WCL from unprimed THP-1 macrophages transfected with poly(dA:dT) for 4 h. Data are representative of at least three independent experiments. (K and L) ELISA analysis of serum galectin-3 (K) and IL-18 (L) level in WT and Nlrp32/2, Aim22/2, Nlrc42/2, and Asc2/2 mice (n = 4); the WT group is the same one used in Fig. 1F. Data are shown as mean 6 SEM. ns: p . 0.05, **p # 0.01, ****p , 0.0001.

Article Snippet: Casp1 inhibitor Ac-YVAD-CMK (catalog no. sc-300323; Santa Cruz Biotechnology) or galectin-3 inhibitor TD139 (catalog no. HY-19940; MedChemExpress) was administrated daily by i.p. injection.

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