Journal: The EMBO Journal

Article Title: The human disease-associated gene ZNFX1 controls inflammation through inhibition of the NLRP3 inflammasome

doi: 10.1038/s44318-024-00236-9

Figure Lengend Snippet: ( A , B ) LPS-primed THP-1-derived macrophages or iBMDM were treated with nigericin for the indicated time. Cells were lysed, and the supernatant and pellet of the cell lysate were separated by centrifugation as described in the material and methods. Proteins were detected by immunoblot using the indicated antibodies. ( C , D ) LPS-primed WT or ZNFX1 KO THP-1-derived macrophages were treated with nigericin with or without MCC950 or caspase-1 inhibitor (Ac-YVAD-cmk) pretreatment. Proteins from cell lysate or culture medium supernatants were analyzed by immunoblotting. ( E , F ) Quantification of ZNFX1 levels using ImageJ from ( C , D ). n = 2 biological replicates for ( E ). n = 3 biological replicates for ( F ). Data were presented as mean ± s.d., two-sided Student’s t -test. ( G ) LPS-primed iBMDM cells were treated with nigericin, with or without MCC950 or caspase-1 inhibitor pretreatment. Proteins from cell lysate or culture medium supernatant were detected by immunoblotting. ( H ) Quantification of ZNFX1 levels using ImageJ from ( G ). n = 3 biological replicates. Data were presented as mean ± s.d., two-sided Student’s t -test. For each biological replicate in ( E , F , H ), band intensity was measured using ImageJ. The WT control was set to 1, and the ratio for ZNFX1 KO cells was calculated by dividing their intensity by the corresponding WT control intensity. .

Article Snippet: Ac-YVAD-cmk , MedChemExpress , HY-16990.

Techniques: Derivative Assay, Centrifugation, Western Blot, Control

Journal: The EMBO Journal

Article Title: The human disease-associated gene ZNFX1 controls inflammation through inhibition of the NLRP3 inflammasome

doi: 10.1038/s44318-024-00236-9

Figure Lengend Snippet: ( A , B ) LPS-primed THP-1-derived macrophages ( A ) or iBMDM ( B ) were treated with gramicidin for the indicated time. Proteins from medium supernatant, cell lysate supernatant, and pellet were detected by immunoblot. ( C ) LPS-primed THP-1 derived macrophages were transfected with poly (dA:dT). Proteins from medium supernatant, cell lysate supernatant, and pellet post-transfection were detected by immunoblot. ( D – F ) THP-1 derived macrophages were primed with LPS and treated with 10 μM nigericin for the indicated time, the protein level of ZNFX1 ( D ), mRNA level of ZNFX1 ( E ), and mRNA level of IL-1β ( F ) and TNF-α ( F ) were measured by immunoblotting or quantitative reverse transcription PCR (qRT-PCR). n = 3 biological replicates, mean ± s.d., Student’s t -test, two-tailed. ( G , H ) LPS-primed THP-1 derived macrophages were pretreated with proteasome inhibitor MG132 (10 μM), autophagy inhibitor 3-MA (20 mM), or CQ (100 μM) for 4 h, and then stimulated with nigericin. Proteins from medium supernatant and cell lysate were detected by immunoblot ( G ). Quantification of ZNFX1 protein was performed using ImageJ ( H ). n = 3 biological replicates. Error bars represent ± s.d. Student’s t -test, two-tailed. For each biological replicate, band intensity was measured using ImageJ. The WT control was set to 1, and the ratio for ZNFX1 KO cells was calculated by dividing their intensity by the corresponding WT control intensity. ( I – K ) Primed WT THP-1 derived macrophages ( I – J ) or iBMDM ( K ) were subjected to LPS or nigericin treatment with or without MCC950 or caspase-1 inhibitor (Ac-YVAD-cmk) pretreatment. Proteins from culture medium supernatant were detected by ELISA. n = 3 biological replicates, mean ± s.d., Student’s t -test, two-tailed. ( L ) Indicated proteins were co-expressed in HeLa cells. Proteins from medium supernatant and cell lysate were detected by immunoblot. ( M ) Proteins from cell lysate of LPS + nigericin-treated THP-1 cells were detected by immunoblot. The red arrow indicates putative cleaved ZNFX1 fragments. ( N ) Purified 3xFLAG-mEGFP-ZNFX1 with or without potential caspase-1 cleavage sites mutated were incubated with caspase-1 protein. Products were detected by immunoblot using α-FLAG antibody. This is an independent biological replicate related to Fig. . are available online for this figure.

Article Snippet: Ac-YVAD-cmk , MedChemExpress , HY-16990.

Techniques: Derivative Assay, Western Blot, Transfection, Reverse Transcription, Quantitative RT-PCR, Two Tailed Test, Control, Enzyme-linked Immunosorbent Assay, Purification, Incubation

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