Journal: Cell Death & Disease

Article Title: Suppression of lncRNA NLRP3 inhibits NLRP3-triggered inflammatory responses in early acute lung injury

doi: 10.1038/s41419-021-04180-y

Figure Lengend Snippet: A The heat map lists the top 20 differentially expressed lncRNAs and mRNAs in NR8383 AM after treatment with PBS, LPS for 2 h, and LPS for 9 h. A , B RNA-seq analysis shows the quantified gene expression of lncRNA NLRP3 and NLRP3 in AM cells in the negative control, LPS 2 h, and LPS 9 h groups. C , D Agarose gel electrophoresis analysis shows the quantified expression of lncRNA NLRP3 and NLRP3 in NR8383 cells. β-Actin served as the control. E The conservation of lncRNA NLRP3 was predicted and analysed by the UCSC Genome Browser. F The lncRNA NLRP3 potential protein-coding and binding sites were analysed with RNA 2.0 tools. G The results show that lncRNA NLRP3 has no protein-coding capability. H Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis were used to analyze differentially expressed genes. I The relationship between lncRNA NLRP3 and NLRP3, and the correlation coefficient is listed. * P < 0.05; ** P < 0.01; *** P < 0.001; NS, no statistically significant difference.

Article Snippet: The primary antibodies used in this experiment included rabbit polychonal anti-NLRP3 (1 : 500; Cell NBP2-12446, Novus, USA), anti-GAPDH (1 : 2000; Cell Signaling Technology, USA), and anti-caspase-1 (1 : 2000, ab1872, Abcam, USA).

Techniques: RNA Sequencing Assay, Expressing, Negative Control, Agarose Gel Electrophoresis, Binding Assay

Journal: Cell Death & Disease

Article Title: Suppression of lncRNA NLRP3 inhibits NLRP3-triggered inflammatory responses in early acute lung injury

doi: 10.1038/s41419-021-04180-y

Figure Lengend Snippet: A qRT-PCR assay was used to analyse the mRNA expression of A LncRNA NLRP3, B miR-138-5p, C NLRP3, D Caspase-1, E IL-1β, and F IL-18 in LPS-induced ALI. β-Actin was used as the reference gene. G , H ELISA analysis of the IL-1β and IL-18 levels in the culture supernatant. Cell apoptosis was determined by Hoechst 33342 and PI dual staining assays ( I ) and counted ( J ). The expression of NLRP3 and caspase-1 in the NR8383 AM cells from the four groups was analysed by western blotting ( K ). L Expression trends of lncRNA NLRP3, NLRP3, caspase-1, IL-1β, IL-18, and miR-138-5p in the negative control group and groups treated with LPS for 6, 12, and 24 h. The data are presented as mean ± SE ( n = 6). * P < 0.05; ** P < 0.01; *** P < 0.001; NS, no statistically significant difference.

Article Snippet: The primary antibodies used in this experiment included rabbit polychonal anti-NLRP3 (1 : 500; Cell NBP2-12446, Novus, USA), anti-GAPDH (1 : 2000; Cell Signaling Technology, USA), and anti-caspase-1 (1 : 2000, ab1872, Abcam, USA).

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Negative Control

Journal: Cell Death & Disease

Article Title: Suppression of lncRNA NLRP3 inhibits NLRP3-triggered inflammatory responses in early acute lung injury

doi: 10.1038/s41419-021-04180-y

Figure Lengend Snippet: A qRT-PCR assay was used to analyse the mRNA expression of A lncRNA NLRP3, B NLRP3, C Caspase-1, D IL-18, E IL-1β, and F miR-138-5p in LPS -induced ALI. β-Actin was used as the reference gene. G , H ELISA was used to analyse the IL-1β and IL-18 levels in the culture supernatants. I , J Cell apoptosis was determined by Hoechst 33342 and PI dual staining assays ( I ) and counted ( J ). K Western blotting was used to analyse the protein expression of NLRP3 and caspase-1 after lncRNA NLRP3 overexpression in the cytoplasm. The data are presented as mean ± SE ( n = 6). * P < 0.05; ** P < 0.01; *** P < 0.001; NS, no statistically significant difference.

Article Snippet: The primary antibodies used in this experiment included rabbit polychonal anti-NLRP3 (1 : 500; Cell NBP2-12446, Novus, USA), anti-GAPDH (1 : 2000; Cell Signaling Technology, USA), and anti-caspase-1 (1 : 2000, ab1872, Abcam, USA).

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Over Expression

Journal: Cell Death & Disease

Article Title: Suppression of lncRNA NLRP3 inhibits NLRP3-triggered inflammatory responses in early acute lung injury

doi: 10.1038/s41419-021-04180-y

Figure Lengend Snippet: A LncLocator was used to investigate the distribution of lncRNA NLRP3. B LncRNA NLRP3 was mainly located in the cytoplasm. C Venn diagram of miRDB predicting miR-138-5p and miR-370-3p sponged by lncRNA NLRP3 and NLRP3. D , E The predicted miR-138-5p-binding sites in the lncRNA NLRP3 3′-UTR. F miR-138-5p mimics notably reduced the luciferase activity of the lncRNA NLRP3-Wt group. G silncRNA NLRP3 significantly increased miR-138-5p expression; however, overexpression of lncRNA NLRP3 reduced miR-138-5p expression in LPS-treated NR8383 AM cells. H RIP assays revealed that Ago2-containing beads enriched the expression of miR-138-5p and NLRP3. I The miR-138-5p inhibitor and miR-138-5p mimics had no effects on lncRNA NLRP3 expression in LPS-treated NR8383 AM cells. The data are presented as mean ± SE ( n = 6). J LncRNA NLRP3 expression was negatively correlated with miR-138-5p expression in LPS-treated NR8383 AM cells. * P < 0.05; ** P < 0.01; *** p < 0.001; NS, no statistically significant difference.

Article Snippet: The primary antibodies used in this experiment included rabbit polychonal anti-NLRP3 (1 : 500; Cell NBP2-12446, Novus, USA), anti-GAPDH (1 : 2000; Cell Signaling Technology, USA), and anti-caspase-1 (1 : 2000, ab1872, Abcam, USA).

Techniques: Binding Assay, Luciferase, Activity Assay, Expressing, Over Expression

Journal: Cell Death & Disease

Article Title: Suppression of lncRNA NLRP3 inhibits NLRP3-triggered inflammatory responses in early acute lung injury

doi: 10.1038/s41419-021-04180-y

Figure Lengend Snippet: A The predicted miR-138-5p-binding sites in the NLRP3 mRNA 3′-UTR. B A firefly luciferase reporter containing either wild-type or mutant NLRP3 was cotransfected into NR8383 AM cells with miR-138-5p mimics NC or miR-138-5p mimics. qRT-PCR assays were used to analyse the mRNA expression of C miR-138-5p, D NLRP3, E Caspase-1, F IL-18, and G IL-1β in the NR8383 AM cells ( n = 6). β-Actin was used as a reference gene. H , I ELISA was used to analyse the IL-1β and IL-18 levels in the culture supernatants. J Western blotting assay of the protein expression levels of NLRP3 and Caspase-1. K , L Cell apoptosis was determined by Hoechst 33342 and PI dual staining assays ( K ) and counted ( L ). The data are presented as mean ± SE ( n = 6). * P < 0.05; ** P < 0.01; *** P < 0.001; NS, no statistically significant difference.

Article Snippet: The primary antibodies used in this experiment included rabbit polychonal anti-NLRP3 (1 : 500; Cell NBP2-12446, Novus, USA), anti-GAPDH (1 : 2000; Cell Signaling Technology, USA), and anti-caspase-1 (1 : 2000, ab1872, Abcam, USA).

Techniques: Binding Assay, Luciferase, Mutagenesis, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Staining

Journal: Cell Death & Disease

Article Title: Suppression of lncRNA NLRP3 inhibits NLRP3-triggered inflammatory responses in early acute lung injury

doi: 10.1038/s41419-021-04180-y

Figure Lengend Snippet: miR-138-5p suppression reversed the effects of silncRNA NLRP3 on the mRNA expression of A lncRNA NLRP3, B NLRP3, C Caspase-1, D IL-1β, E IL-18, and F miR-138-5p in NR8383 alveolar macrophage (AMs) cells. β-Actin was used as the reference gene. G , H ELISA analysis of the IL-1β and IL-18 levels in the culture supernatant. I Western blotting assay of the protein expression levels of NLRP3 and caspase-1. J , K Cell apoptosis was determined by Hoechst 33342 and PI dual staining assays ( J ) and counted ( K ). The data are presented as mean ± SE ( n = 6). * P < 0.05; ** P < 0.01; *** P < 0.001; NS, no statistically significant difference.

Article Snippet: The primary antibodies used in this experiment included rabbit polychonal anti-NLRP3 (1 : 500; Cell NBP2-12446, Novus, USA), anti-GAPDH (1 : 2000; Cell Signaling Technology, USA), and anti-caspase-1 (1 : 2000, ab1872, Abcam, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Staining

Journal: Cell Death & Disease

Article Title: Suppression of lncRNA NLRP3 inhibits NLRP3-triggered inflammatory responses in early acute lung injury

doi: 10.1038/s41419-021-04180-y

Figure Lengend Snippet: Rat lungs were injected with PBS in the control group and LPS-treated rats were further treated with si-r-lncRNA NLRP3, Lv-lncRNA NLRP3, agomiR-138-5p, antagomiR-138-5p, Lv-lncRNA NLRP3 + agomiR-138-5p, and si-r-lncRNA NLRP3 + antagomiR-138-5p. A Lung tissue samples were collected 6 h after establishing LPS-induced ALI to analyse the histopathological changes (×200, ×400). The black arrow indicates neutrophil infiltration, pulmonary oedema, alveolar wall thickening, and alveolar haemorrhage. B The lung injury score was determined via H&E staining, a representative histological analysis ( n = 6 animals per group). C ELISA was used to measure the BALF albumin content. D Detection of the lung W/D ratio in rats. E MPO activity in the lung tissues of rats. F , G Immunohistochemical detection of the NLRP3 contents in rat lung tissues (×200, ×400). H The inflammatory response in NR8383 AM cells was suppressed by si-r-lncRNA NLRP3 and miR-138-5p mimics alone or in combination, as shown by the decreased number of cells colabeled with CD68 (green) and NLRP3 (red). LncRNA NLRP3 overexpression, miR-138-5p inhibition, and NLRP3 augmented the inflammatory response in LPS-induced ALI with more NLRP3 and CD68 anchored in the plasma membrane of the AM cells. The data are presented as mean ± SE ( n = 6). * P < 0.05; ** P < 0.01; *** P < 0.001; NS, no statistically significant difference.

Article Snippet: The primary antibodies used in this experiment included rabbit polychonal anti-NLRP3 (1 : 500; Cell NBP2-12446, Novus, USA), anti-GAPDH (1 : 2000; Cell Signaling Technology, USA), and anti-caspase-1 (1 : 2000, ab1872, Abcam, USA).

Techniques: Injection, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Immunohistochemical staining, Over Expression, Inhibition

Journal: Cell Death & Disease

Article Title: Suppression of lncRNA NLRP3 inhibits NLRP3-triggered inflammatory responses in early acute lung injury

doi: 10.1038/s41419-021-04180-y

Figure Lengend Snippet: The lungs of rats were injected with PBS in the control group and LPS-treated rats were further treated with si-r-lncRNA NLRP3, Lv-lncRNA NLRP3, agomiR-138-5p, antagomiR-138-5p, Lv-lncRNA NLRP3 + agomiR-138-5p, and si-r-lnc NLRP3 + antagomiR-138-5p. A Morphometric changes in the appearance of the lungs that had been fixed in 4% paraformaldehyde for 24 h at 25 °C in each group. B , C The protein expression levels of NLRP3 and caspase-1 in rat lung tissues. qRT-PCR assays were used to analyse mRNA expression of D lncRNA NLRP3, E NLRP3, F IL-18, G Caspase-1, H IL-1β, and I miR-138-5p in the lung tissues of rats. ELISA analysis of the IL-1β ( J ) and IL-18 ( K ) levels in the culture supernatant. L Graphical summary of the role of the lncRNA NLRP3/miR-138-5p/NLRP3 ceRNET in acute lung injury. β-Actin was used as the reference. The data are presented as mean ± SE ( n = 6). * P < 0.05; ** P < 0.01; *** P < 0.001; NS, no statistically significant difference.

Article Snippet: The primary antibodies used in this experiment included rabbit polychonal anti-NLRP3 (1 : 500; Cell NBP2-12446, Novus, USA), anti-GAPDH (1 : 2000; Cell Signaling Technology, USA), and anti-caspase-1 (1 : 2000, ab1872, Abcam, USA).

Techniques: Injection, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay