Journal: MedComm

Article Title: High‐Throughput Screening Reveals That CeeNU Acts as a New NLRP3 Inflammasome Inhibitor

doi: 10.1002/mco2.70695

Figure Lengend Snippet: CeeNU remarkably represses pyroptosis and IL‐1β release dependent on NLRP3 in human macrophages. (A, B, D, and E) PMA‐differentiated human macrophage THP‐1 cells were either treated with LPS for 3 h plus CeeNU for 1 h, followed by stimulation with nigericin (A and B) or MSU (D and E), the supernatant was collected for ELISA of IL‐1β (A and D) and LDH assay (B and E). (C and F) Western blotting analysis of cleaved IL‐1β p17 and caspase‐1 p20 levels in cell supernatant and pro‐IL‐1β, procaspase‐1, NLRP3, GSDMD, GSDMD‐NT, and GAPDH in cell lysates of PMA‐differentiated THP‐1 cells primed with LPS for 4 h in the presence of CeeNU or control, prior to stimulation with nigericin (C) or MSU(F). (G) PMA‐differentiated THP‐1 cells were treated sequentially: first with LPS for 3 h, then with CeeNU for an additional hour. Following this, cells were stimulated with nigericin for 1 h. Subsequently, cells were stained with propidium iodide (PI, 2 µg/mL) to label dying cells and DAPI (1 µg/mL) to label all nuclei for 15 min. Imaging was performed using a 10× objective on an Olympus confocal microscope. Merged images include the corresponding bright‐field view. (H) The number of PI‐positive cells was counted in five randomly chosen microscopic fields. Lytic cell death was defined as the ratio of PI‐positive cells to the total number of DAPI‐positive nuclei and is expressed as a percentage. (I) Scanning electron microscopy of cell surface perforations caused by GSDMD in THP‐1 (Bar = 3 µm). * p < 0.05, ** p < 0.01, *** p < 0.001. Data are mean ± SEM.

Article Snippet: Cytotoxicity (LDH) assay kit ( C10007 ), Caspase‐1 activity assay kit (C1102), and Flagellin (P7388) were from Beyotime (Shanghai, China).

Techniques: Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Western Blot, Control, Staining, Imaging, Microscopy, Electron Microscopy

Journal: MedComm

Article Title: High‐Throughput Screening Reveals That CeeNU Acts as a New NLRP3 Inflammasome Inhibitor

doi: 10.1002/mco2.70695

Figure Lengend Snippet: CeeNU inhibits pyroptotic cell death in mouse macrophages. LPS‐primed BMDM or iBMDMs treated with CeeNU for 1 h before stimulation with ATP for 30 min or nigericin for 1 h. (A) BMDMs were treated with different doses of CeeNU for 24 h, and cell viability was assessed by the CCK‐8 assay. (B) ELISA of IL‐1β in the supernatant of LPS+Nigericin‐stimulated BMDMs. (C and F) LDH release from BMDMs supernatant stimulated with LPS+ATP (C), or LPS+Nigericin (F). (D and G) ELISA of IL‐1β in the supernatant of BMDMs stimulated with LPS+ATP (D), or LPS+Nigericin (G). (E and H) Western blotting analysis of cleaved IL‐1β p17 and caspase‐1 p20 levels in cell supernatant and pro‐IL‐1β, procaspase‐1, NLRP3, GSDMD, GSDMD‐NT, and GAPDH in cell lysates of BMDMs stimulated with LPS+ATP (E), or LPS+Nigericin (H). (I) LDH release from BMDMs supernatant stimulated with LPS+ATP. (J) ELISA of IL‐1β in the supernatant of BMDMs stimulated with LPS+ATP. (K) Western blotting analysis of cleaved IL‐1β p17 and caspase‐1 p20 levels in cell supernatant and pro‐IL‐1β, procaspase‐1, NLRP3, GSDMD, GSDMD‐NT, and GAPDH in cell lysates of iBMDMs stimulated with LPS+ATP. (L) iBMDMs were treated with LPS and CeeNU prior to ATP stimulation. Cells were then stained with calcein AM (green; viability dye) and PI (red; death marker) to distinguish live and dead populations, respectively. Merged images include fluorescent signals overlaid on the corresponding bright‐field image. Scale bars, 50 µm. (M) PI‐positive and calcein AM‐positive cells were counted in four random fields per sample. Lytic cell death was defined as the ratio of PI‐positive cells to the total cell count (sum of PI‐positive and calcein AM‐positive cells), expressed as a percentage. * p < 0.05, *** p < 0.001. Data are mean ± SEM.

Article Snippet: Cytotoxicity (LDH) assay kit ( C10007 ), Caspase‐1 activity assay kit (C1102), and Flagellin (P7388) were from Beyotime (Shanghai, China).

Techniques: CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Marker, Cell Characterization

Journal: MedComm

Article Title: High‐Throughput Screening Reveals That CeeNU Acts as a New NLRP3 Inflammasome Inhibitor

doi: 10.1002/mco2.70695

Figure Lengend Snippet: CeeNU is a specific inhibitor of the NLRP3 inflammasome. (A and C) Production of cleaved‐IL‐1β from LPS‐primed BMDMs treated with CeeNU before transfected with poly(dA:dT) (A), flagellin (C). (B and D) LDH release from LPS‐primed BMDMs treated with various doses of CeeNU before transfected with poly(dA:dT) (B), flagellin (D). (E–H) Western blotting analysis of cleaved IL‐1β p17 and caspase‐1 p20 levels in cell supernatant (SN) and pro‐IL‐1β, procaspase‐1, NLRP3, and GAPDH in cell lysates of BMDMs (E and F) and THP‐1 (G and H), LPS‐primed BMDMs and THP‐1 cells treated with CeeNU for 1 h before transfected with poly(dA:dT) for 16 h (E and G), flagellin for 16 h (F and H). (I and K) Production of cleaved‐IL‐1β from LPS‐primed THP‐1 cells treated with CeeNU before transfected with poly(dA:dT) (I), flagellin (K). (J and L) LDH release from LPS‐primed THP‐1 cells treated with various doses of CeeNU before transfected with poly(dA:dT) (J), flagellin (L). (M) BMDMs were treated with or without LPS, and plus different concentrations of CeeNU for 4 h, cell lysates were collected for Western blotting. (N) Western blotting analysis of p20 in SNs, pro‐casp1, and NLRP3 in input from LPS‐primed wild type, nlrp3 knockout, and gsdmd knockout BMDMs treated with 50 µM CeeNU, and then stimulated with ATP for 30 min. (O) ELISA of IL‐1β in SNs from LPS‐primed wild type, nlrp3 knockout, and gsdmd knockout BMDMs treated with 50 µM CeeNU, and then stimulated with ATP for 30 min. (P) LDH release from SN of LPS‐primed wild type, nlrp3 knockout, and gsdmd knockout BMDMs treated with 50 µM CeeNU, and then stimulated with ATP for 30 min. *** p < 0.001. Data are mean ± SEM.

Article Snippet: Cytotoxicity (LDH) assay kit ( C10007 ), Caspase‐1 activity assay kit (C1102), and Flagellin (P7388) were from Beyotime (Shanghai, China).

Techniques: Transfection, Western Blot, Knock-Out, Enzyme-linked Immunosorbent Assay

Journal: MedComm

Article Title: High‐Throughput Screening Reveals That CeeNU Acts as a New NLRP3 Inflammasome Inhibitor

doi: 10.1002/mco2.70695

Figure Lengend Snippet: CeeNU significantly alleviates MSU‐induced peritonitis and gouty arthritis. Eight‐week‐old male C57BL/6 mice were intraperitoneally injected with 100 µL vehicle control or 100 µL CeeNU before intraperitoneal injection with 100 µL PBS or 1 mg MSU (dissolved in 100 µL PBS). (A) Flow cytometry analysis of neutrophils (CD11b + and Ly6G + ) and monocytes (CD11b + and Ly6C + ) in peritoneal lavage fluid. Statistical analysis of flow cytometry results of neutrophil percentage in white blood cells (B), neutrophil number (C), monocyte percentage in white blood cells (D), and monocyte number (E). (F) ELISA of IL‐1β levels in the supernatants of peritoneal lavage fluid. (G) ELISA of IL‐6 levels in the supernatants of peritoneal lavage fluid. (H) The photograph of mice joint swelling level. (I) Mice joint thickness after injection of MSU (0.8 mg in 20 µL PBS). (J) H&E staining of the joint tissue. (K) Caspase‐1 activity in the foot joints of mice. (L) IL‐1β levels in the foot joints of mice. * p < 0.05, *** p < 0.001. Data are mean ± SEM.

Article Snippet: Cytotoxicity (LDH) assay kit ( C10007 ), Caspase‐1 activity assay kit (C1102), and Flagellin (P7388) were from Beyotime (Shanghai, China).

Techniques: Injection, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Activity Assay