Journal: International journal of molecular sciences

Article Title: Inhibition of the NF-κB Signaling Pathway Alleviates Pyroptosis in Bladder Epithelial Cells and Neurogenic Bladder Fibrosis.

doi: 10.3390/ijms241311160

Figure Lengend Snippet: Figure 6. Bioinformatics analysis of the transcriptome sequencing results of cells. (A,B) Heatmap analysis of IRGs in cellular DEGs. (C–E) Expression levels of the pyroptosis proteins NLRP3 and ASC in SV-HUC-1 cells were detected by immunofluorescence staining (scale bar = 100 µm). (F) SEM showed TGF-β1-induced epithelial cell swelling, cell membrane pore formation, and py- roptotic body generation. Scale bar = 5 µm. (G) TEM showed cell membrane disruption, disruption of organelle structures such as mitochondria and the endoplasmic reticulum, and vacuolization. Scale bar = 2 µm and scale bar = 500 nm. (H) Protein expression levels of NLRP3, GSDMD, ASC, and IL1-β were detected by Western blotting in vitro. The data are presented as the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The pyroptosis inhibitor MCC950 (10 μM, MCE, HY-12815A) was used to inhibit pyroptosis in SV-HUC-1 cells for 2 h before TGF-β1 stimulation [37,38].

Techniques: Sequencing, Expressing, Staining, Membrane, Disruption, Western Blot, In Vitro

Journal: Frontiers in Stroke

Article Title: NLRP3 inflammasome inhibition protects against intracranial aneurysm rupture and alters the phenotype of infiltrating macrophages

doi: 10.3389/fstro.2023.1202137

Figure Lengend Snippet: MCC950 treatment reduces the aneurysm rupture rate and extends symptom-free survival. (A) Aneurysm formation rates, (B) Aneurysm rupture rate, * p < 0.05 by Fisher's exact test. (C) Cumulative symptom-free survival, and (D) Mean arterial pressure, p = n.s. at all-time points.

Article Snippet: MCC950 was obtained from Selleckchem (Catalog No.S7809, Houston, TX) and diluted in 0.9% NaCl in water.

Techniques:

Journal: Frontiers in Stroke

Article Title: NLRP3 inflammasome inhibition protects against intracranial aneurysm rupture and alters the phenotype of infiltrating macrophages

doi: 10.3389/fstro.2023.1202137

Figure Lengend Snippet: MCC950 treatment alters macrophage phenotype ratio in cerebral vasculature after aneurysm induction. (A) Representative images of M1-type (top) and M2-type (bottom) macrophages in saline-treated (left) and MCC950-treated (right) mice, scale bar = 50 μm. (B) Ratio of M1:M2 macrophage phenotypes in the vessel wall, * p < 0.05, n = 5 each. (C) Total number of F4/80 + cell per high-powered field, n = 5 each. All plots presented as median + 95% CI.

Article Snippet: MCC950 was obtained from Selleckchem (Catalog No.S7809, Houston, TX) and diluted in 0.9% NaCl in water.

Techniques: Saline

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: Sequences of primers.

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques:

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: Elevated expression of molecules in TMJ synovium. (a, b) Western blotting results of NLRP3, ASC, cleaved caspase-1, IL-1 β , and GSDMD in the sham and OA synovitis rats. (c) mRNA levels of NLRP3, ASC, caspase-1, IL-1 β , and GSDMD expressed in TMJ synovium between the sham and OA rats. (d) Immunohistochemistry of TMJ slices from the sham or OA rats using anti-NLRP3, anti-caspase-1, anti-GSDMD, and anti-IL-1 β antibodies, scale bar = 50 μ m. Positive cells are mainly around synovial membrane and vessels (black arrow). (e) Percentage of positive cells. Data in this figure are analyzed by independent samples t -test and presented as mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques: Expressing, Western Blot, Immunohistochemistry, Membrane

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: The biological effect of AYC and MCC950 on inflammasome- and pyroptosis-related molecules. (a, b) Western blotting results showing the protein levels of NLRP3, ASC, cleaved caspase-1, IL-1 β , and GSDMD among the sham, OA, and AYC groups. (a, c) Levels of the above proteins among the sham, OA, and MCC950 groups. (d, e) TUNEL assay indicated that positive nuclei were increased markedly in the OA synovial tissue compared with the sham-treated group. Positive cells were reduced in the Ac-YVAD-cmk and MCC950 groups. Data in this figure are presented as mean ± SD and analyzed by parametric test. Statistical significance is determined with one-way ANOVA followed by Bonferroni's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques: Western Blot, TUNEL Assay

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: LPS+ATP-treated macrophages. (a) Altered morphology of THP-1 cells under PMA stimulation. Scale bar = 50 μ m. (b, c) Western blotting results of NLRP3, ASC, cleaved caspase-1, IL-1 β , and GSDMD changes on protein levels in LPS-treated macrophages. (d) The results of qRT-PCR showed changes on mRNA levels among the control, LPS+ATP, LPS+ATP+MCC950, and LPS+ATP+AYC groups. Data are presented as mean ± SD and analyzed with one-way ANOVA followed by Bonferroni's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques: Western Blot, Quantitative RT-PCR, Control

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: LPS+ATP stimulated NLRP3 inflammasome- and pyroptosis-related molecules in the cultured FLS cells alone and cocultured system. (a, b) When FLS cells were cultured alone, lysate was collected for western blot after being treated with LPS+ATP. (c) Total RNAs of FLS cells were extracted for detecting mRNAs of NLRP3, ASC, caspase-1, GSDMD, and IL-1 β . Statistical analysis is run by independent samples t -test. (d, e) Secretion of IL-1 β and IL-18 in the supernate increased when FLS cell was cocultured with active THP-1 cell. (f–h) Western blotting and qRT-PCR results of cell lysate indicated the growing expression of NLRP3, ASC, caspase-1, GSDMD, and IL-1 β on protein and mRNA levels. Both the upregulation trend of the above tests after LPS+ATP intervention could be inhibited by MCC950 and AYC in the coculture system. Data are presented as mean ± SD; one-way ANOVA followed by Bonferroni's post hoc test was adopted for analysis. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques: Cell Culture, Western Blot, Quantitative RT-PCR, Expressing

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: Detection of pyroptosis in fibroblast-like synovial cells of coculture system. Cocultured FLS cells were challenged with LPS for 12 h and ATP for 2 h. Cell exposed to saline was as the control group. The MCC950 group and Ac-YVAD-cmk group were simultaneously added inhibitors to FLS cells. (a) PI- and Hoechst 33342-stained FLS cells in the control and LPS+ATP groups. (b) Quantification of PI-positive cells. Independent samples t -test. (c) The release of LDH was measured among the control, LPS+ATP, LPS+ATP+MCC950, and LPS+ATP+AYC groups. (d, e) Cells double stained by PI and kFluor488 for flow cytometry. One-way ANOVA followed by Bonferroni's post hoc test was used. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (f) Representative transmission electron micrographs of FLS cells. Compared with control group, FLS cells exhibited pyroptotic features: extensive membrane bubbles and pores (red arrow); chromatin margination and condensation (yellow arrow); and swollen mitochondria with collapsed cristae (blue arrow).

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques: Saline, Control, Staining, Flow Cytometry, Transmission Assay, Membrane

Journal: Frontiers in Immunology

Article Title: A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages

doi: 10.3389/fimmu.2023.1136669

Figure Lengend Snippet: Both NLRP3- and NLRC4-inflammasome activation contributes to rFlaA:Betv1 induced IL-1β and pro-inflammatory cytokine secretion from THP-1 macrophages. PMA-differentiated wild-type (WT), NLRP3-, NLRC4-, or ASC-deficient THP-1 macrophages were stimulated with either LPS as a positive control, or equimolar amounts of rFlaA + rBet v 1, rFlaA:Betv1, rFlaA *D1 :Betv1, or rFlaA ΔDC0 :Betv1 for 24 h (A) . Supernatants were collected and checked for the secretion of IL-1β, IL-6, IL-12, and TNF-α by ELISA (B) . Data are the mean results of three independent experiments ± SD, and statistical significances are indicated as ns: p-value > 0.05, *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001.

Article Snippet: PMA-differentiated, THP-1 macrophages were pre-incubated with the indicated amounts of either the IKK-β inhibitors TPCA-1 (Abcam, Cambridge, UK) or BMS-345541 (Abcam), the unspecific inflammasome inhibitor VX-765 ( Invivo Gen), which inhibits caspase-1 activity, the specific NLRP3-inflammasome inhibitor MCC950 ( Invivo Gen), or the MAPK inhibitors SP600125 (SAP/JNK MAPK inhibitor, Invivo Gen), SB202190 (p38α/β MAPK inhibitor, Invivo Gen), or U0126 (ERK MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands) for 90 min and subsequently stimulated with rFlaA:Betv1 for 24 h. The target molecules of the used inhibitors are summarized in .

Techniques: Activation Assay, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages

doi: 10.3389/fimmu.2023.1136669

Figure Lengend Snippet: NFκB- and SAP/JNK MAP kinase-signaling pathways contribute to inflammasome activation by regulating pro-Caspase-1, pro-IL-1β-, and NLRP3-expression in rFlaA:Betv1-stimulated THP-1 macrophages. PMA-differentiated THP-1 macrophages were pre-treated with the indicated inhibitors (500 nM TPCA-1, 5 µM BMS-345541, 25 µM SP600125, or 10 µM SB202190) for 90 minutes, followed by stimulation with 27.4 µg/mL rFlaA:Betv1 for additional 24 h (A) . Proteins in supernatant (Sup) and cell lysate (Lysate) were examined by Western Blot (B) . The intensity of Western Blot bands from three independent experiments was analyzed, first normalized to the loading control β-Tubulin and then again normalized to the unstimulated group (shown as value of “1”) (C) . Data are either representative (B) or mean results of three independent experiments ± SD (C) . Statistical significances are indicated as ns: p-value > 0.05, *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001.

Article Snippet: PMA-differentiated, THP-1 macrophages were pre-incubated with the indicated amounts of either the IKK-β inhibitors TPCA-1 (Abcam, Cambridge, UK) or BMS-345541 (Abcam), the unspecific inflammasome inhibitor VX-765 ( Invivo Gen), which inhibits caspase-1 activity, the specific NLRP3-inflammasome inhibitor MCC950 ( Invivo Gen), or the MAPK inhibitors SP600125 (SAP/JNK MAPK inhibitor, Invivo Gen), SB202190 (p38α/β MAPK inhibitor, Invivo Gen), or U0126 (ERK MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands) for 90 min and subsequently stimulated with rFlaA:Betv1 for 24 h. The target molecules of the used inhibitors are summarized in .

Techniques: Activation Assay, Expressing, Western Blot

Journal: Veterinary Research

Article Title: Staphylococcus aureus mediates pyroptosis in bovine mammary epithelial cell via activation of NLRP3 inflammasome

doi: 10.1186/s13567-022-01027-y

Figure Lengend Snippet: NLRP3 inflammasome regulates ASC speck formation during S. aureus . Fluorescence microscopy images of MAC-T cells immunoassayed for ASC (red) with or without MCC950 after 4 h of S. aureus treatment.

Article Snippet: Caspase-1 inhibitor VX765 and NLRP3 inflammasome inhibitor MCC950 were obtained from Glpbio (USA) and used at final concentrations of 100 and 15 μM.

Techniques: Fluorescence, Microscopy

Journal: Veterinary Research

Article Title: Staphylococcus aureus mediates pyroptosis in bovine mammary epithelial cell via activation of NLRP3 inflammasome

doi: 10.1186/s13567-022-01027-y

Figure Lengend Snippet: NLRP3 inflammasome activation by S. aureus is essential for the generation of GSDMD-N and the release of IL-1β and IL-18. A Activated caspase-1 and B GSDMD-N or released C IL-1β and D IL-18 of MAC-T cells treated with or without NLRP3 inhibitor MCC950 or caspase-1 inhibitor VX765 for 1.5 h prior to treatment with S. aureus for 4 h. E Levels of IL-1β and F IL-18 released after S. aureus treatment of MAC-T cells for 4 h in the presence or absence of 25, 50, or 75 mM KCl.

Article Snippet: Caspase-1 inhibitor VX765 and NLRP3 inflammasome inhibitor MCC950 were obtained from Glpbio (USA) and used at final concentrations of 100 and 15 μM.

Techniques: Activation Assay

Journal: Frontiers in Immunology

Article Title: Zika Virus Infection Induces Acute Kidney Injury Through Activating NLRP3 Inflammasome Via Suppressing Bcl-2

doi: 10.3389/fimmu.2019.01925

Figure Lengend Snippet: ZIKV infection induced NLRP3 inflammasome activation in the kidneys. (A,B) The relative mRNA levels (vs. mock group) of NLRP1, NLRP3, IPAF, and AIM2 in the kidneys of mock and ZIKV-infected newborn (A) and adult (B) mice at 7 days post-infection were detected by qRT-PCR. (C–I) Expression levels of NLRP3 (D) , ASC (E) , pro-caspase-1 (F) , cleaved-caspase-1 (G) , pro-IL-1β (H) , and mature IL-1β (I) in the kidneys of newborn and adult mice at 7 days post-infection were assessed by western blot. (J,K) Examination of mature IL-1β release in the supernatants of mouse kidneys at 7 days post-infection ( n = 6 per group) were determined by an enzyme-linked immunoadsorbent assay (ELISA). Data were presented as mean ± SEM ( n = 6), * p < 0.05 vs. mock group; # p < 0.05 vs. ZIKV group ( *** p < 0.001 vs. mock group or ZIKV+MCC950 group in Figure J and K).

Article Snippet: To inhibit NLRP3 and IL-1β activation, mice were i.p. injected with 10 μg MCC950 per gram body weight (BW) or 35 μg/g BW Anakinra (143090-92-0, ChemLeader, CN) or PBS (vehicle control) ever 2 days beginning from 2 days post-infection until tissue harvest.

Techniques: Infection, Activation Assay, Quantitative RT-PCR, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Zika Virus Infection Induces Acute Kidney Injury Through Activating NLRP3 Inflammasome Via Suppressing Bcl-2

doi: 10.3389/fimmu.2019.01925

Figure Lengend Snippet: Overexpression of Bcl-2 attenuated ZIKV-induced NLRP3 inflammasome activation in HK-2 cells. (A–C) Expression level of Bcl-2 in the kidneys of newborn (A,B) and adult mice (A,C) at 7 days post-infection were assessed by western blot. (D–K) HK-2 cells were transfected with control vector or pSG5-FLAG-Bcl-2 plasmid (2 μg/ml) for 24 h or pre-treated with MCC950 (10 μM) for 30 min followed by incubation with PBS (mock group) or ZIKV (MOI = 2), and cultured with complete medium in presence of MCC950 or PBS at 37°C for 24 h. The protein expression levels of Bcl-2, NLRP3, ASC, pro-caspase-1, cleaved-caspase-1, pro-IL-1β, and mature IL-1β protein were assessed by western blot. (L) The mRNA expression level of NLRP3 was detected by qRT-PCR. Data were presented as mean ± SEM, * p < 0.05 vs. Control group; # p < 0.05 vs. mock group.

Article Snippet: To inhibit NLRP3 and IL-1β activation, mice were i.p. injected with 10 μg MCC950 per gram body weight (BW) or 35 μg/g BW Anakinra (143090-92-0, ChemLeader, CN) or PBS (vehicle control) ever 2 days beginning from 2 days post-infection until tissue harvest.

Techniques: Over Expression, Activation Assay, Expressing, Infection, Western Blot, Transfection, Control, Plasmid Preparation, Incubation, Cell Culture, Quantitative RT-PCR