Journal: JACC: Basic to Translational Science

Article Title: NLRP3 Inflammasome Inhibition by the Novel Bispecific Antibody InflamAb Attenuates Atherosclerosis in Apolipoprotein E–Deficient Mice

doi: 10.1016/j.jacbts.2024.12.012

Figure Lengend Snippet: InflamAb Inhibits NLRP3 Inflammasome-Mediated IL-1β Release In Vitro and In Vivo (A) The NLRP3 inflammasome (using LPS and alum)-induced IL-1β secretion by BMDMs was significantly inhibited by both 2.5 and 25 ng/mL InflamAb: n = 4-5 replicates per condition, ordinary 1-way analysis of variance. (B) InflamAb significantly inhibited the NLRP3 inflammasome-induced IL-1β response in WTD-fed Apoe -/- mice, compared with isotype control (left, n = 4 both unpaired t- test). (C) InflamAb inhibited the NLRP3 inflammasome (using LPS and Nigericin; LPS + Nig)-induced IL-1β secretion by THP-1 cells. Single mIL-1R1 or ScFvNLRP3 antibodies, as well as a bispecific but inactive Il-1R1 ScFvNLRP3 (inIL-1R1 ScFvNLRP3) control, did not significantly affect IL1β secretion. (D) LPS and Nigericin-induced caspase-1 activity was significantly inhibited by InflamAb but not by the bispecific control antibody. (E) InflamAb, but not a control antibody, significantly binds to recombinant human NLRP3 protein. Mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ### P < 0.001. IL = interleukin; LPS = lipopolysaccharides.

Article Snippet: NLRP3 (NALP3) , APC , Miltenyi Biotec , Order no. : 130-111-397 , 1:50.

Techniques: In Vitro, In Vivo, Control, Activity Assay, Recombinant

Journal: International journal of oral science

Article Title: Circadian disruption by simulated shift work aggravates periodontitis via orchestrating BMAL1 and GSDMD-mediated pyroptosis.

doi: 10.1038/s41368-024-00331-x

Figure Lengend Snippet: Fig. 3 BMAL1 functions in the regulation of GSDMD-mediated pyroptosis. a, f The representative images of cell morphology in indicated groups examined by SEM (scale bar: Low magnification = 50 μm; High magnification = 10 μm) b, g Quantification of LDH activity in mGFs treated with P. gingivalis LPS and Bmal1 siRNA (n = 5) or overexpression plasmid (n = 4). c, h The representative images of PI staining in indicated groups were examined by LCFM (scale bar: 100 μm). d, i Relative mRNA expressions of genes related to GSDMD-mediated pyroptosis in indicated groups measured by qRT-PCR (n = 3). e, j Relative levels of proteins related to NLRP3 inflammasome signaling in indicated groups detected by western blot (n = 3). Data are represented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations: LPS lipopolysaccharide, LDH lactate dehydrogenase, PI propidium iodide, SEM scanning electron microscope, LCFM laser confocal fluorescence microscopy

Article Snippet: The PVDF membranes were incubated overnight at 4 °C with antibodies against BMAL1 (ABclonal, A17334, Wuhan, China), CLOCK (ABclonal, A7265), CRY1 (ABclonal, A6890), PER2 (ABclonal, A13168), NR1D1 (ABclonal, A20452), NLRP3 (R&D Systems, MAB7578, Minneapolis, MN, USA), CASPASE1 (Proteintech, 22915-1-AP, Wuhan, China), GSDMD (Santa Cruz Biotechnology, sc-393581, Santa Cruz, CA, USA), IL-1β (ImmunoWay Biotechnology, YT5201, Plano, TX, USA), β-Actin (ImmunoWay Biotechnology, YM3029).

Techniques: Activity Assay, Over Expression, Plasmid Preparation, Staining, Quantitative RT-PCR, Western Blot, Microscopy

Journal: International journal of oral science

Article Title: Circadian disruption by simulated shift work aggravates periodontitis via orchestrating BMAL1 and GSDMD-mediated pyroptosis.

doi: 10.1038/s41368-024-00331-x

Figure Lengend Snippet: Fig. 4 BMAL1 regulates Gsdmd transcription independent of NR1D1. a Relative mRNA expressions of Bmal1, Nr1d1, and genes related to NLRP3 inflammasome signaling in indicated groups measured by qRT-PCR (n = 3). b Relative expressions of BMAL1, NR1D1, and proteins related to NLRP3 inflammasome signaling in indicated groups detected by western blot (n = 3). c The representative images of PI staining in indicated groups examined by LCFM (scale bar: 100 μm). d Quantification of LDH activity in indicated groups (n = 4). e Sequence logos of consensus DNA binding sites for genes regulated by BMAL1. f Verification of the binding between BMAL1 and the Gsdmd promoter region by ChIP assay. g The binding sites of BMAL1 and Gsdmd, and the design for luciferase reporters. h Normalized luciferase activity after co- transfection of pcDNA or Bmal1 plasmid together with GSDMD-WT or GSDMD-MUT luciferase reporters measured by dual luciferase assay (n = 3). Data are represented as the mean ± SD. ns, no significance; **P < 0.01; ***P < 0.001. Abbreviations: LPS lipopolysaccharide, LCFM laser confocal fluorescence microscopy, PI propidium iodide, LDH lactate dehydrogenase; WT wild type, MUT mutant type

Article Snippet: The PVDF membranes were incubated overnight at 4 °C with antibodies against BMAL1 (ABclonal, A17334, Wuhan, China), CLOCK (ABclonal, A7265), CRY1 (ABclonal, A6890), PER2 (ABclonal, A13168), NR1D1 (ABclonal, A20452), NLRP3 (R&D Systems, MAB7578, Minneapolis, MN, USA), CASPASE1 (Proteintech, 22915-1-AP, Wuhan, China), GSDMD (Santa Cruz Biotechnology, sc-393581, Santa Cruz, CA, USA), IL-1β (ImmunoWay Biotechnology, YT5201, Plano, TX, USA), β-Actin (ImmunoWay Biotechnology, YM3029).

Techniques: Quantitative RT-PCR, Western Blot, Staining, Activity Assay, Sequencing, Binding Assay, Luciferase, Cotransfection, Plasmid Preparation, Microscopy, Mutagenesis

Journal: International journal of oral science

Article Title: Circadian disruption by simulated shift work aggravates periodontitis via orchestrating BMAL1 and GSDMD-mediated pyroptosis.

doi: 10.1038/s41368-024-00331-x

Figure Lengend Snippet: Fig. 6 BMAL1-upregulation alleviates circadian disruption-accelerated periodontitis via lessening GSDMD-mediated pyroptosis. a Representative images and quantitative analysis of BMAL1, NR1D1, and proteins related to NLRP3 inflammasome signaling in indicated groups detected by western blot (n = 3). b, c Representative images (scale bar: 100 μm) and quantification of GSDMD expression in indicated groups by immunofluorescence staining (n = 3). d The representative sagittal tridimensional and bidimensional views of the maxillary molars in indicated groups scanned by micro-CT (scale bar: 500 μm). e Quantification of the BMD, the BV/TV, and the distance from the CEJ to the ABC in indicated groups (n = 6). f Representative images of H&E staining of the gingiva in indicated groups (scale bar: 500 μm; 75 μm). Data are represented as the mean ± SD. ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations: BMD bone mineral density, BV/TV bone volume fraction, CEJ cement-enamel junction, ABC alveolar bone crest, H&E hematoxylin and eosin

Article Snippet: The PVDF membranes were incubated overnight at 4 °C with antibodies against BMAL1 (ABclonal, A17334, Wuhan, China), CLOCK (ABclonal, A7265), CRY1 (ABclonal, A6890), PER2 (ABclonal, A13168), NR1D1 (ABclonal, A20452), NLRP3 (R&D Systems, MAB7578, Minneapolis, MN, USA), CASPASE1 (Proteintech, 22915-1-AP, Wuhan, China), GSDMD (Santa Cruz Biotechnology, sc-393581, Santa Cruz, CA, USA), IL-1β (ImmunoWay Biotechnology, YT5201, Plano, TX, USA), β-Actin (ImmunoWay Biotechnology, YM3029).

Techniques: Disruption, Western Blot, Expressing, Staining, Micro-CT