Journal: Frontiers in Immunology

Article Title: SARS-COV-2 viroporins activate the NLRP3-inflammasome by the mitochondrial permeability transition pore

doi: 10.3389/fimmu.2023.1064293

Figure Lengend Snippet: Expression of 2-E+2-3a induces mROS production by elevating mitochondrial Ca ++ levels. (A, B) Schematics of our LV-EV and LV-E3a vectors. (C) 293T cells were transduced with LV-EV or LV-E3a, and samples analyzed by immunoblot with a mouse monoclonal antibody against FLAG-Tag, to detect the FLAG-tagged 2-E+2-3a viroporins. GAPDH was a loading control. (D, E) 24 hrs post-transduction cells were stained with Fura-Red to measure cytosolic Ca ++ levels, (F, G) and with Rhod2 & Mitotracker Deep Red (MTDR) to measure mitochondrial Ca ++ levels . (E, G, J) data from confocal microscopy or (D, F) data from plate reader assays. (H) 24 hrs post-transduction with LV-EV or LV-E3a 293T cells were treated with or without 10 μM MCUi11, and then 2.5 μM TG and stained with Rhod2 to measure mitochondrial Ca ++ levels by plate reader assays. (I) Cells, 24 hrs post-transduction with LV-EV or LV-E3a, were analyzed for mean mitochondrial NADH lifetime (t mean ) indicating (NAD + /NADH ratio) using FLIM. (J) Representative images of Fura-Red, Rhod2-stained, and NADH lifetime imaged cells. Scale bar = 30 μm. Error bars represent SEM from 3 independent experiments; statistically significant data is indicated with asterisks (*).

Article Snippet: 293T & THP1 cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Transduction, Western Blot, FLAG-tag, Staining, Confocal Microscopy

Journal: Frontiers in Immunology

Article Title: SARS-COV-2 viroporins activate the NLRP3-inflammasome by the mitochondrial permeability transition pore

doi: 10.3389/fimmu.2023.1064293

Figure Lengend Snippet: Expression of mCAT or treatment with the mROS scavenger MnTBAP antioxidant defenses blocks 2-E+2-3a induced mROS. (A–C) 24 hrs post-transduction cells were stained with MitoSOX and MTDR to measure mROS levels via (A) data from plate reader assays or (B, C) data from confocal microscopy. (D) 24 hrs post-transduction with LV-EV or LV-E3a 293T, cells were treated with or without 10 μM MCUi11, and then 2.5 μM TG and stained with MitoSOX to measure mROS levels by plate reader assays. (E, F) Schematics of p- p-mCAT and EV vectors. (G) Catalase assay through cleavage of H 2 O 2 in 293T cell lysates collected 24 hrs post-transfection with p-EV or p-mCAT. (H-M) 24 hrs post-transfection levels of mROS assessed using MitoSOX and MTDR fluorescence 293T cells transduced with LV-EV or LV-E3a and transfected (H, I, L) with p-mCAT or its respective control p-EV or (J, K, M) cultured in the presence or absence of 50 μM MnTBAP, DMSO used as a negative control. MTDR fluorescence was used to normalize for mitochondrial content with mROS expressed as the ratio of MitoSOX/MTDR, by (H, J) plate reader assays or (I, K, L, M) confocal microscopy. (C, L, M) Representative images of MitoSOX-stained cells. Scale bar = 30 μm. Error bars represent SEM from 3 independent experiments; statistically significant data is indicated with asterisks (*).

Article Snippet: 293T & THP1 cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Transduction, Staining, Confocal Microscopy, Transfection, Fluorescence, Cell Culture, Negative Control

Journal: Frontiers in Immunology

Article Title: SARS-COV-2 viroporins activate the NLRP3-inflammasome by the mitochondrial permeability transition pore

doi: 10.3389/fimmu.2023.1064293

Figure Lengend Snippet: mROS and the mtPTP are required for activation of the NLRP3-I by the 2-E+2-3a viroporins. (A, B) Experimental design used to assess NLRP3-activated by IL-1β in cell-free supernatants quantified by ELISA. (A) 293T cells with an NLRP3-I reconstitution system (NLRP3, ASC, pro-CASP1, pro-IL-1β) and (B) THP1 differentiated into macrophages and primed with LPS + nigericin. (C) 293T cells transfected with LV-EV or LV-E3a were transfected with the NLRP3-I plasmids and p-mCAT or its control plasmid p-EV, or cultured in the presence or absence of 50 μM MnTBAP. (D, F, G) THP1 cells were transduced with LV-EV or LV-E3a, differentiated into macrophages, treated with LPS + nigericin, and treated with 5 μM MCC950, 100 μM MnTBAP or 10 μM NIM811, the supernatants analyzed for IL-1β by ELISA. (E) THP1 cells stably expressing LV-mCAT or control plasmid were infected with LV-EV or LV-E3a, differentiated into macrophages, and supernatant IL-1β levels determined via ELISA. Error bars represent SEM from 3 independent experiments; statistically significant data is indicated with asterisks (*).

Article Snippet: 293T & THP1 cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Cell Culture, Transduction, Stable Transfection, Expressing, Infection

Journal: Frontiers in Immunology

Article Title: SARS-COV-2 viroporins activate the NLRP3-inflammasome by the mitochondrial permeability transition pore

doi: 10.3389/fimmu.2023.1064293

Figure Lengend Snippet: 2-E+2-3a viroporins induced mROS is required for the release of mtDNA from the mitochondrion into the cytosol via the mtPTP. (A) Representative Figure. (B, C) 293T cells were transduced with LV-EV or LV-E3a. (B) 24 hrs post-transduction cells were treated with or without 50 uM MnTBAP or 10 μM NIM811. 10 hrs post-treatment, cells were lysed, cytosolic-enriched fractions collected, DNA isolated from the cytosolic-enriched fractions, and levels of cytosolic mtDNA determined by RT-PCR with a probe specific for MT-ND5 . (C) 6 hrs post-transduction with LV-EV or LV-E3a, 293T cells were transfected with p-mCAT or its control plasmid p-EV. 24 hrs post-transfection, levels of cytosolic mtDNA were determined as described above. (D) 293T or 293T-ρ 0 cells transduced with LV-EV or LV-E3a were transfected with the NLRP3-I plasmids the supernatants analyzed for IL-1β by ELISA. Error bars represent SEM from 3 independent experiments; statistically significant data is indicated with asterisks (*).

Article Snippet: 293T & THP1 cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Transduction, Isolation, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Dentistry Journal

Article Title: Immunomodulatory Effects of Endodontic Sealers: A Systematic Review

doi: 10.3390/dj11020054

Figure Lengend Snippet: Summary of immunomodulatory effect of endodontic sealers in vitro studies.

Article Snippet: THP1 human monocytic cells (ATCC TIB 202) , AH-Plus-Jet, Pulp Canal Sealer, MTA-type sealers, ProRoot White MTA, and an experimental calcium silicate-based sealer , IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-15, IFN-γ, TGF-β1, TNF-α, TNF-β, VEGF , Cytokine Array based on ELISA , ↓ IL-2, IL-6, IL-10, and IL-15 ↑ IL-1α and IL-1β , [ ] .

Techniques: In Vitro, Marker, Agarose Gel Electrophoresis, CCK-8 Assay, MTT Assay, Nucleic Acid Electrophoresis, Enzyme-linked Immunosorbent Assay, Inhibition, ROS Assay, Activity Assay, Nitric Oxide Assay, Fluorescence, Staining, Laser-Scanning Microscopy, WST Assay, Immunohistochemistry, Immunofluorescence, Western Blot, Flow Cytometry, Multiplex Assay, Cytokine Assay, Immunostaining

Journal: bioRxiv

Article Title: The tetrapeptide sequence of IL-1β regulates its recruitment and activation by inflammatory caspases

doi: 10.1101/2023.02.16.528859

Figure Lengend Snippet: ( A , B ) THP1 cells were terminally differentiated into macrophages with phorbol 12-myristate 12-acetate (50 ng/mL) for 24 h and primed with LPS (5 μg/mL) for another 24 h. Where indicated, THP1 macrophages were treated with ZVAD (40 μM) or VX765 (40 μM) 30 minutes before LPS transfections. Cells were then transfected with LPS (25 μg/mL). 24 h after LPS transfection, samples were analyzed for LDH release ( A ) and immunoblotting ( B ). ( C , D ) THP1 cells were terminally differentiated into macrophages with phorbol 12-myristate 12-acetate (40 ng/mL) for 48 h. Cells were then treated with Legionella pneumophila (MOI = 20) for 7 h before the supernatants were analyzed for LDH release ( C ) and then combined with the lysates for immunoblotting ( D ). € Caco-2 cells were primed with 100 ng/ml Pam3CSK4 for 3 h, then infected with Salmonella Typhimurium (MOI = 60) for 6 h. Cells and their supernatants were collected separately, samples were precipitated, and analyzed by immunoblotting. Data are means ± SEM of three biological replicates. ***P < 0.001 and **P < 0.01 by two-sided Student’s t -test compared with control. *Represents non-specific bands in immunoblot. Data are representative of two or more independent experiments.

Article Snippet: HEK 293T cells, HeLa cells, Caco-2 cells, and THP1 cells were purchased from ATCC.

Techniques: Transfection, Western Blot, Infection