Journal: Frontiers in Immunology

Article Title: NLRP3 Inflammasome Activation of Mast Cells by Estrogen via the Nuclear-Initiated Signaling Pathway Contributes to the Development of Endometriosis

doi: 10.3389/fimmu.2021.749979

Figure Lengend Snippet: NLRP3 expression was increased by estrogen stimulation via ER-α in MCs. (A) Bar plot of differentially expressed genes (DEGs) from the KEGG pathway enrichment analysis; a comparison between β-estradiol treated P815 cells and the control. (B) A volcano plot was used for visualization of the differentially expressed genes. The gray point in the plot represents genes with no statistical differences (-1< log 2 (FC) < 1, P > 0.05), the red point represents upregulated genes (log 2 (FC) > 1, P < 0.05), and the blue point represents downregulated genes (log 2 (FC) < -1, P < 0.05) with statistical significance. (C) Quantitative PCR analysis of ESR1 in P815 cells after ESR1-specific shRNA transfection. (D) Western blot analysis of ER-α in P815 cell nuclei after stable transfection of ESR1-specific shRNA. (E) Relative quantification of NLRP3 mRNA level in P815 was measured by FPKM reads using RNA-seq. (F) Quantitative PCR analysis NLRP3 mRNA levels in P815 cells treated with β-estradiol after knockdown of ESR1 compared with the negative control. (G) Representative images of immunofluorescence staining for NLRP3 (red) in mast cell line P815 incubated with or without 100 pmol/mL β-estradiol for 24 h Data in (C, D, F) represent three independent experiments. Data are expressed as mean ± SEM. Statistics: Unpaired Student’s t-test. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, statistically not significant.

Article Snippet: Mouse mast cell line P815 from ScienCell Research Laboratories (CA, USA) was cultured with RPMI-1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (PS) (Thermo Fisher).

Techniques: Expressing, Real-time Polymerase Chain Reaction, shRNA, Transfection, Western Blot, Stable Transfection, RNA Sequencing Assay, Negative Control, Immunofluorescence, Staining, Incubation

Journal: Frontiers in Immunology

Article Title: NLRP3 Inflammasome Activation of Mast Cells by Estrogen via the Nuclear-Initiated Signaling Pathway Contributes to the Development of Endometriosis

doi: 10.3389/fimmu.2021.749979

Figure Lengend Snippet: ERE sites in the NLRP3 promoter mediated estrogen-induced NLRP3 transcription. (A) ChIP analysis was performed using anti-ER-α or anti-Histone H3 antibody to ascertain the existence of the ERE in the promoter of the NLRP3 gene. The PCR results showed that a 159-bp fragment containing the presumed ERE could be precipitated after P815 cells were treated with β-estradiol for 24 h. (B) The pulled-down band was excised from the gel and sequenced. (C) Schematic diagram of luciferase reporter constructs. Basic-Luc: pGL4-basic plasmid; NLRP3-Luc: pGL4-basic plasmid with the NLRP3 promoter fragment including presumed ERE-like sequence; delNLRP3-Luc: pGL4-basic plasmid with the mutant NLRP3 promoter fragment, with the presumed ERE-like sequence deleted. (D) Luciferase activities of three report systems with or without ESR1 plasmid co-transfection in 293T cells were compared with each other. Renilla luciferase plasmid was used to normalize transfection efficiencies. The experiments were repeated three times and data are presented as means ± SEM. Statistics: Unpaired Student’s t-test. * P < 0.05, ** P < 0.01.

Article Snippet: Mouse mast cell line P815 from ScienCell Research Laboratories (CA, USA) was cultured with RPMI-1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (PS) (Thermo Fisher).

Techniques: Luciferase, Construct, Plasmid Preparation, Sequencing, Mutagenesis, Cotransfection, Transfection

Journal: Frontiers in Immunology

Article Title: NLRP3 Inflammasome Activation of Mast Cells by Estrogen via the Nuclear-Initiated Signaling Pathway Contributes to the Development of Endometriosis

doi: 10.3389/fimmu.2021.749979

Figure Lengend Snippet: The NLRP3 inflammasome pathway was activated by estrogen via ER-α. (A) Western blot analysis of NLRP3, cleaved caspase-1, caspase-1 precursor, cleaved IL-1β, IL-1β precursor, and ASC in P815 cells treated with 100 pmol/mL β-estradiol for 1 h, 3 h, 6 h, 12 h, and 24 h. (B) Quantified results of western blot assays. (C) Intracellular concentration of K + was determined by the ratio of the fluorescence intensities obtained by exciting PBFI at 340/380 nm wavelengths while monitoring emission at 500 nm, and presented as percent of untreated control. (D) The concentration of IL-1β in the P815 cell culture supernatant was tested using ELISA. Data in (B–D) represent three independent experiments. Data are expressed as mean ± SEM. Statistics: ANOVA followed by Dunnett t-test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Mouse mast cell line P815 from ScienCell Research Laboratories (CA, USA) was cultured with RPMI-1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (PS) (Thermo Fisher).

Techniques: Western Blot, Concentration Assay, Fluorescence, Cell Culture, Enzyme-linked Immunosorbent Assay