Journal: Neural Regeneration Research

Article Title: Upregulation of circ0000381 attenuates microglial/macrophage pyroptosis after spinal cord injury

doi: 10.4103/1673-5374.386399

Figure Lengend Snippet: Antibody information

Article Snippet: NLRP3 , A synthetic peptide corresponding to residues surrounding Ala306 of mouse NLRP3 protein , Cell Signaling Technology , Rabbit mAb , 1:1000 , 15101 , AB_562331 , Rabbit IgG , WB.

Techniques: Concentration Assay, Control, Synthesized, Derivative Assay, Recombinant

Journal: Neural Regeneration Research

Article Title: Upregulation of circ0000381 attenuates microglial/macrophage pyroptosis after spinal cord injury

doi: 10.4103/1673-5374.386399

Figure Lengend Snippet: Primers used for polymerase chain reaction

Article Snippet: NLRP3 , A synthetic peptide corresponding to residues surrounding Ala306 of mouse NLRP3 protein , Cell Signaling Technology , Rabbit mAb , 1:1000 , 15101 , AB_562331 , Rabbit IgG , WB.

Techniques:

Journal: Neural Regeneration Research

Article Title: Upregulation of circ0000381 attenuates microglial/macrophage pyroptosis after spinal cord injury

doi: 10.4103/1673-5374.386399

Figure Lengend Snippet: Microglia/macrophages play a key role in pathological progression after SCI. (A–D) Representative double immunofluorescence staining for Iba1 (green) and different pyroptosis makers: NLRP3 (A), caspase-1 (B), GSDMD (C), or IL-1β (all red) (D) in the spinal cord at 7 days after SCI or sham surgery. DAPI (blue) represents the cell nucleus. Pyroptosis makers in the SCI group showed marked upregulation compared with the sham group. Arrows represent typical microglia/macrophages. Scale bar: 25 μm. (E–H) Quantification of percentages of double-labeled NLRP3 + /Iba1 + (E), GSDMD + /Iba1 + (F), caspase-1 + /Iba1 + (G), and IL-1β + /Iba1 + (H) cells in total Iba1 + cells. Data are presented as mean ± SEM ( n = 6/group). *** P < 0.001, vs . sham group (Student’s t -test). (I) Spinal cord sections co-stained for different pyroptosis makers: NLRP3, caspase-1, GSDMD, or IL-1β (all red), and microglia/macrophage maker: Iba1 (green). The box represents the region from where a–d were captured. DAPI: 4′,6-Diamidino-2-phenylindole; GSDMD: gasdermin D; Iba1: ionized calcium binding adapter molecule 1; IL-1β: interleukin-1β; NLRP3: NOD-like receptor 3; SCI: spinal cord injury.

Article Snippet: NLRP3 , A synthetic peptide corresponding to residues surrounding Ala306 of mouse NLRP3 protein , Cell Signaling Technology , Rabbit mAb , 1:1000 , 15101 , AB_562331 , Rabbit IgG , WB.

Techniques: Double Immunofluorescence Staining, Labeling, Staining, Binding Assay

Journal: Neural Regeneration Research

Article Title: Upregulation of circ0000381 attenuates microglial/macrophage pyroptosis after spinal cord injury

doi: 10.4103/1673-5374.386399

Figure Lengend Snippet: SCI induces cell pyroptosis in the spinal cord. (A) mRNA levels of NLRP3, caspase-1, GSDMD, IL-1β, and IL-18 in the spinal cord were measured by real-time polymerase chain reaction on day 7 after SCI or sham surgery. (B) Representative western blots and quantification of NLRP3, caspase-1, and GSDMD protein levels in the spinal cord on day 7 after SCI or sham surgery. (C) Concentrations of IL-1β and IL-18 in the spinal cord were measured by enzyme-linked immunosorbent assay on day 7 after SCI or sham surgery ( n = 6/group). Data are presented as mean ± SEM ( n = 6/group). *** P < 0.001, vs . sham group (Student’s t -test). GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; GSDMD: gasdermin D; IL: interleukin; mRNA: messenger RNA; NLRP3: NOD-like receptor 3; SCI: spinal cord injury.

Article Snippet: NLRP3 , A synthetic peptide corresponding to residues surrounding Ala306 of mouse NLRP3 protein , Cell Signaling Technology , Rabbit mAb , 1:1000 , 15101 , AB_562331 , Rabbit IgG , WB.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Neural Regeneration Research

Article Title: Upregulation of circ0000381 attenuates microglial/macrophage pyroptosis after spinal cord injury

doi: 10.4103/1673-5374.386399

Figure Lengend Snippet: Microglia/macrophage pyroptosis in vitro . (A) mRNA expression of markers of pyroptosis in HAPI cells treated with 1000 ng/mL LPS for 24 hours. (B) Western blot analysis and levels of NLRP3, caspase-1, and GSDMD in HAPI cells treated with 100 and 1000 ng/mL LPS for 24 hours. Data are expressed as mean ± SEM from at least three independent experiments. ** P < 0.01, *** P < 0.001, vs . control group, Student’s t -test (A) or one-way analysis of variance followed by Tukey’s post hoc analysis (B). Con: Control; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSDMD: gasdermin D; IL: interleukin; LPS: lipopolysaccharide; NLRP3: NOD-like receptor 3.

Article Snippet: NLRP3 , A synthetic peptide corresponding to residues surrounding Ala306 of mouse NLRP3 protein , Cell Signaling Technology , Rabbit mAb , 1:1000 , 15101 , AB_562331 , Rabbit IgG , WB.

Techniques: In Vitro, Expressing, Western Blot, Control

Journal: Neural Regeneration Research

Article Title: Upregulation of circ0000381 attenuates microglial/macrophage pyroptosis after spinal cord injury

doi: 10.4103/1673-5374.386399

Figure Lengend Snippet: Knockdown of circ0000381 expression enhances microglial/macrophage pyroptosis. (A) Transduction with circ0000381-siRNA in HAPI cells enhanced NLRP3 expression induced by LPS. Cells were transfected with circ0000381-siRNA for 24 hours and then treated with LPS (1000 ng/mL) for 12 hours. *** P < 0.001, vs . siRNA-Control treated with vehicle control; ### P < 0.001, vs . siRNA-Control treated with LPS. (B) Expression levels of circ0000381 by real-time PCR. *** P < 0.001, vs . Control (0). (C) Expression levels of NLRP3 by western blot analysis. *** P < 0.001, vs . Control (0). Data are presented as mean ± SEM of three independent experiments and were analyzed by one-way analysis of variance followed by Tukey’s post hoc analysis. Con: Control; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; LPS: lipopolysaccharide; NLRP3: NOD-like receptor 3; PCR: polymerase chain reaction; siRNA: small interference RNA.

Article Snippet: NLRP3 , A synthetic peptide corresponding to residues surrounding Ala306 of mouse NLRP3 protein , Cell Signaling Technology , Rabbit mAb , 1:1000 , 15101 , AB_562331 , Rabbit IgG , WB.

Techniques: Knockdown, Expressing, Transduction, Transfection, Control, Real-time Polymerase Chain Reaction, Western Blot, Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Upregulation of circ0000381 attenuates microglial/macrophage pyroptosis after spinal cord injury

doi: 10.4103/1673-5374.386399

Figure Lengend Snippet: Knockdown of circ0000381 expression enhances microglial/macrophage pyroptosis by targeting miR-423-3p in vitro . (A) miR-423-3p expression was analyzed by real-time polymerase chain reaction. Left, spinal cord on day 7 after SCI or sham surgery ( n = 6/group). Right, HAPI cells were treated with 1000 ng/mL LPS or vehicle (control) for 24 hours. Data were from at least three independent experiments. (B) The bioinformatics program, RNAhybrid, predicts that Circ0000381 contains one site complementary to miR-423-3p. (C) Luciferase activity in HEK-293T cells co-transfected with luciferase reporters containing circ0000381 sequences with wild-type (WT) or mutated (MUT) miR-423-3p binding sites and mimics of miR-423-3p or controls. (D) Co-localization of circ0000381 and miR-423-3p in the cytoplasm of HAPI cells by fluorescence in situ hybridization analysis. Red, circ0000381; Green, miR-423-3p; Blue, DAPI. Scale bars: 25 μm. (E) Transduction with circ0000381-siRNA significantly enhanced NLRP3 expression induced by miR-423-3p inhibitor in HAPI cells. Data were from at least three independent experiments. Data are presented as mean ± SEM. * P < 0.05, vs . NC+circ0000381-WT group; *** P < 0.001, vs . sham group or miR-Control transduced with circ-Control; ## P < 0.01, ### P < 0.001, vs . control group or miR-423-3p transduced with circ-Control group, Student’s t -test (A left, C) or one-way mixed model analysis of variance followed by Tukey’s post hoc analysis (A right, E). DAPI: 4′,6-Diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; LPS: lipopolysaccharide; miR: microRNA; NLRP3: NOD-like receptor 3; SCI: spinal cord injury.

Article Snippet: NLRP3 , A synthetic peptide corresponding to residues surrounding Ala306 of mouse NLRP3 protein , Cell Signaling Technology , Rabbit mAb , 1:1000 , 15101 , AB_562331 , Rabbit IgG , WB.

Techniques: Knockdown, Expressing, In Vitro, Real-time Polymerase Chain Reaction, Control, Luciferase, Activity Assay, Transfection, Binding Assay, Fluorescence, In Situ Hybridization, Transduction