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crid3 mcc950  (Tocris)


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    Tocris crid3 mcc950
    ( A – C ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF, ( D – F ) normal human epidermal keratinocytes (NHEK), ( G – I ) CD14 + monocytes, and ( J ) PMA-differentiated THP-1 macrophages were left untreated or pretreated with IFN-γ overnight. Cells were infected with indicated recombinant VACV strains at an MOI of 5 ( A – F , J ) or MOI 10 ( G – I ), in the presence ( A , C , D , F , G , I , J ) or absence ( B , E , H ) of VX, and where indicated, in the presence of 2.5 µM <t>CRID3.</t> Cells were harvested and fixed 6 h post infection. Infection (EGFP + cells) and C1C-EGFP speck assembly in infected or mock-treated cells was quantified by flow cytometry ( A , D , G , J ); infection data (EGFP + cells) corresponding to the experiment shown in panel ( J ) is displayed in Fig. . Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte live-cell imaging system ( B , E , H ). Scale bar = 100 μm. Samples for confocal microscopy were fixed and stained with 5 μg/mL wheat germ agglutinin (WGA)-Alexa Fluor (AF) 647 and 4 μM Hoechst 33342 ( C , F , I ). Scale bar = 5 μm. ( K ) PMA-differentiated WT or indicated knockout THP-1 cells were left untreated or pretreated with IFN-γ overnight and infected with the indicated reporter VACV strains at an MOI of 5 in the presence of VX. C1C-EGFP speck assembly was quantified as before. The corresponding infection data (EGFP + cells) is shown in Fig. . ( L , M ) NHEK ( L ) or PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV WT or monkeypox virus (MPXV) at an MOI of 5, and where indicated, in the presence of CRID3. Cells were harvested and fixed 6 h post infection. VACV- or MPXV-infected cells were stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies, and measured by flow cytometry. Corresponding infection data (αH5-AF647 + cells) is shown in Fig . C1C-EGFP speck assembly was analyzed in infected or mock-treated cells. Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells). .
    Crid3 Mcc950, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/crid3/pmc12953764-127-0-3?v=Tocris
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    Images

    1) Product Images from "Poxvirus dsDNA genomes differentially activate AIM2 or NLRP3 inflammasomes in human primary cells"

    Article Title: Poxvirus dsDNA genomes differentially activate AIM2 or NLRP3 inflammasomes in human primary cells

    Journal: The EMBO Journal

    doi: 10.1038/s44318-025-00690-z

    ( A – C ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF, ( D – F ) normal human epidermal keratinocytes (NHEK), ( G – I ) CD14 + monocytes, and ( J ) PMA-differentiated THP-1 macrophages were left untreated or pretreated with IFN-γ overnight. Cells were infected with indicated recombinant VACV strains at an MOI of 5 ( A – F , J ) or MOI 10 ( G – I ), in the presence ( A , C , D , F , G , I , J ) or absence ( B , E , H ) of VX, and where indicated, in the presence of 2.5 µM CRID3. Cells were harvested and fixed 6 h post infection. Infection (EGFP + cells) and C1C-EGFP speck assembly in infected or mock-treated cells was quantified by flow cytometry ( A , D , G , J ); infection data (EGFP + cells) corresponding to the experiment shown in panel ( J ) is displayed in Fig. . Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte live-cell imaging system ( B , E , H ). Scale bar = 100 μm. Samples for confocal microscopy were fixed and stained with 5 μg/mL wheat germ agglutinin (WGA)-Alexa Fluor (AF) 647 and 4 μM Hoechst 33342 ( C , F , I ). Scale bar = 5 μm. ( K ) PMA-differentiated WT or indicated knockout THP-1 cells were left untreated or pretreated with IFN-γ overnight and infected with the indicated reporter VACV strains at an MOI of 5 in the presence of VX. C1C-EGFP speck assembly was quantified as before. The corresponding infection data (EGFP + cells) is shown in Fig. . ( L , M ) NHEK ( L ) or PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV WT or monkeypox virus (MPXV) at an MOI of 5, and where indicated, in the presence of CRID3. Cells were harvested and fixed 6 h post infection. VACV- or MPXV-infected cells were stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies, and measured by flow cytometry. Corresponding infection data (αH5-AF647 + cells) is shown in Fig . C1C-EGFP speck assembly was analyzed in infected or mock-treated cells. Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells). .
    Figure Legend Snippet: ( A – C ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF, ( D – F ) normal human epidermal keratinocytes (NHEK), ( G – I ) CD14 + monocytes, and ( J ) PMA-differentiated THP-1 macrophages were left untreated or pretreated with IFN-γ overnight. Cells were infected with indicated recombinant VACV strains at an MOI of 5 ( A – F , J ) or MOI 10 ( G – I ), in the presence ( A , C , D , F , G , I , J ) or absence ( B , E , H ) of VX, and where indicated, in the presence of 2.5 µM CRID3. Cells were harvested and fixed 6 h post infection. Infection (EGFP + cells) and C1C-EGFP speck assembly in infected or mock-treated cells was quantified by flow cytometry ( A , D , G , J ); infection data (EGFP + cells) corresponding to the experiment shown in panel ( J ) is displayed in Fig. . Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte live-cell imaging system ( B , E , H ). Scale bar = 100 μm. Samples for confocal microscopy were fixed and stained with 5 μg/mL wheat germ agglutinin (WGA)-Alexa Fluor (AF) 647 and 4 μM Hoechst 33342 ( C , F , I ). Scale bar = 5 μm. ( K ) PMA-differentiated WT or indicated knockout THP-1 cells were left untreated or pretreated with IFN-γ overnight and infected with the indicated reporter VACV strains at an MOI of 5 in the presence of VX. C1C-EGFP speck assembly was quantified as before. The corresponding infection data (EGFP + cells) is shown in Fig. . ( L , M ) NHEK ( L ) or PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV WT or monkeypox virus (MPXV) at an MOI of 5, and where indicated, in the presence of CRID3. Cells were harvested and fixed 6 h post infection. VACV- or MPXV-infected cells were stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies, and measured by flow cytometry. Corresponding infection data (αH5-AF647 + cells) is shown in Fig . C1C-EGFP speck assembly was analyzed in infected or mock-treated cells. Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells). .

    Techniques Used: Derivative Assay, Infection, Recombinant, Flow Cytometry, Membrane, Live Cell Imaging, Confocal Microscopy, Staining, Knock-Out, Expressing, Virus, Labeling

    ( A – I ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF ( A , D , E ), normal human epidermal keratinocytes (NHEK) ( B , F , G ), or human primary CD14 + monocytes ( C , H , I ), were left untreated or pretreated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP at an MOI of 5 ( A , B ) or MOI 10 ( C ), in the absence of VX, and where indicated in the presence of 2.5 µM CRID3. Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte Live-Cell Imaging system ( A – C ). DRAQ7 uptake normalized to cell confluency are shown. IL-1β in the supernatant was measured by homogeneous time resolved fluorescence (HTRF) ( D , F , H ). Cell death was measured by LDH release and normalized to cells lysed in 1% Triton X-100 ( E , G , I ). ( J , K ) PMA-differentiated WT ( J ) or indicated knockout THP-1 cells ( K ) were left untreated or pretreated with IFN-γ overnight and infected with indicated VACV strains in the presence of VX, and where indicated, CRID3. Infection levels are displayed here; specking data from the same experiments are shown in Fig. . ( L , M ) NHEK ( L ) and PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV or monkeypox virus (MPXV) at an MOI of 5 in the presence of VX, and where indicated, CRID3. Cells were fixed 6 h post infection and stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies. Infected cells (EGFP + ) were analyzed in infected or mock-treated cells by flow cytometry as described before. Infection levels are displayed here; specking data from the same experiments are displayed in Fig. . Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells).
    Figure Legend Snippet: ( A – I ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF ( A , D , E ), normal human epidermal keratinocytes (NHEK) ( B , F , G ), or human primary CD14 + monocytes ( C , H , I ), were left untreated or pretreated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP at an MOI of 5 ( A , B ) or MOI 10 ( C ), in the absence of VX, and where indicated in the presence of 2.5 µM CRID3. Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte Live-Cell Imaging system ( A – C ). DRAQ7 uptake normalized to cell confluency are shown. IL-1β in the supernatant was measured by homogeneous time resolved fluorescence (HTRF) ( D , F , H ). Cell death was measured by LDH release and normalized to cells lysed in 1% Triton X-100 ( E , G , I ). ( J , K ) PMA-differentiated WT ( J ) or indicated knockout THP-1 cells ( K ) were left untreated or pretreated with IFN-γ overnight and infected with indicated VACV strains in the presence of VX, and where indicated, CRID3. Infection levels are displayed here; specking data from the same experiments are shown in Fig. . ( L , M ) NHEK ( L ) and PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV or monkeypox virus (MPXV) at an MOI of 5 in the presence of VX, and where indicated, CRID3. Cells were fixed 6 h post infection and stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies. Infected cells (EGFP + ) were analyzed in infected or mock-treated cells by flow cytometry as described before. Infection levels are displayed here; specking data from the same experiments are displayed in Fig. . Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells).

    Techniques Used: Derivative Assay, Infection, Membrane, Live Cell Imaging, Fluorescence, Knock-Out, Expressing, Virus, Staining, Labeling, Flow Cytometry

    ( A ) Genome structure of recombinant VACV strains expressing C1C-EGFP from the J2R early promoter (pE) and bivalent nanobodies from a synthetic early/late promoter (pE/L). Transgenes were inserted into the VACV TK locus by homologous recombination. ( B ) Scheme of AIM2 inflammasome components indicating the VACV-encoded nanobodies used to perturb inflammasome activation. ( C – E ) Human MDMs differentiated with GM-CSF ( C ), NHEKs ( D ), and CD14 + monocytes ( E ) were left untreated or treated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP expressing the indicated nanobodies at an MOI of 5 ( C , D ) or 10 ( E ), in the presence of VX and, where indicated, CRID3. Six hours post infection, cells were harvested, fixed, and infection and C1C-EGFP speck assembly was analyzed by flow cytometry. Average values (with individual data points) from N = 3 independent donors ± SEM are displayed. .
    Figure Legend Snippet: ( A ) Genome structure of recombinant VACV strains expressing C1C-EGFP from the J2R early promoter (pE) and bivalent nanobodies from a synthetic early/late promoter (pE/L). Transgenes were inserted into the VACV TK locus by homologous recombination. ( B ) Scheme of AIM2 inflammasome components indicating the VACV-encoded nanobodies used to perturb inflammasome activation. ( C – E ) Human MDMs differentiated with GM-CSF ( C ), NHEKs ( D ), and CD14 + monocytes ( E ) were left untreated or treated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP expressing the indicated nanobodies at an MOI of 5 ( C , D ) or 10 ( E ), in the presence of VX and, where indicated, CRID3. Six hours post infection, cells were harvested, fixed, and infection and C1C-EGFP speck assembly was analyzed by flow cytometry. Average values (with individual data points) from N = 3 independent donors ± SEM are displayed. .

    Techniques Used: Recombinant, Expressing, Homologous Recombination, Activation Assay, Infection, Flow Cytometry



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    ( A – C ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF, ( D – F ) normal human epidermal keratinocytes (NHEK), ( G – I ) CD14 + monocytes, and ( J ) PMA-differentiated THP-1 macrophages were left untreated or pretreated with IFN-γ overnight. Cells were infected with indicated recombinant VACV strains at an MOI of 5 ( A – F , J ) or MOI 10 ( G – I ), in the presence ( A , C , D , F , G , I , J ) or absence ( B , E , H ) of VX, and where indicated, in the presence of 2.5 µM CRID3. Cells were harvested and fixed 6 h post infection. Infection (EGFP + cells) and C1C-EGFP speck assembly in infected or mock-treated cells was quantified by flow cytometry ( A , D , G , J ); infection data (EGFP + cells) corresponding to the experiment shown in panel ( J ) is displayed in Fig. . Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte live-cell imaging system ( B , E , H ). Scale bar = 100 μm. Samples for confocal microscopy were fixed and stained with 5 μg/mL wheat germ agglutinin (WGA)-Alexa Fluor (AF) 647 and 4 μM Hoechst 33342 ( C , F , I ). Scale bar = 5 μm. ( K ) PMA-differentiated WT or indicated knockout THP-1 cells were left untreated or pretreated with IFN-γ overnight and infected with the indicated reporter VACV strains at an MOI of 5 in the presence of VX. C1C-EGFP speck assembly was quantified as before. The corresponding infection data (EGFP + cells) is shown in Fig. . ( L , M ) NHEK ( L ) or PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV WT or monkeypox virus (MPXV) at an MOI of 5, and where indicated, in the presence of CRID3. Cells were harvested and fixed 6 h post infection. VACV- or MPXV-infected cells were stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies, and measured by flow cytometry. Corresponding infection data (αH5-AF647 + cells) is shown in Fig . C1C-EGFP speck assembly was analyzed in infected or mock-treated cells. Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells). .

    Journal: The EMBO Journal

    Article Title: Poxvirus dsDNA genomes differentially activate AIM2 or NLRP3 inflammasomes in human primary cells

    doi: 10.1038/s44318-025-00690-z

    Figure Lengend Snippet: ( A – C ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF, ( D – F ) normal human epidermal keratinocytes (NHEK), ( G – I ) CD14 + monocytes, and ( J ) PMA-differentiated THP-1 macrophages were left untreated or pretreated with IFN-γ overnight. Cells were infected with indicated recombinant VACV strains at an MOI of 5 ( A – F , J ) or MOI 10 ( G – I ), in the presence ( A , C , D , F , G , I , J ) or absence ( B , E , H ) of VX, and where indicated, in the presence of 2.5 µM CRID3. Cells were harvested and fixed 6 h post infection. Infection (EGFP + cells) and C1C-EGFP speck assembly in infected or mock-treated cells was quantified by flow cytometry ( A , D , G , J ); infection data (EGFP + cells) corresponding to the experiment shown in panel ( J ) is displayed in Fig. . Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte live-cell imaging system ( B , E , H ). Scale bar = 100 μm. Samples for confocal microscopy were fixed and stained with 5 μg/mL wheat germ agglutinin (WGA)-Alexa Fluor (AF) 647 and 4 μM Hoechst 33342 ( C , F , I ). Scale bar = 5 μm. ( K ) PMA-differentiated WT or indicated knockout THP-1 cells were left untreated or pretreated with IFN-γ overnight and infected with the indicated reporter VACV strains at an MOI of 5 in the presence of VX. C1C-EGFP speck assembly was quantified as before. The corresponding infection data (EGFP + cells) is shown in Fig. . ( L , M ) NHEK ( L ) or PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV WT or monkeypox virus (MPXV) at an MOI of 5, and where indicated, in the presence of CRID3. Cells were harvested and fixed 6 h post infection. VACV- or MPXV-infected cells were stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies, and measured by flow cytometry. Corresponding infection data (αH5-AF647 + cells) is shown in Fig . C1C-EGFP speck assembly was analyzed in infected or mock-treated cells. Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells). .

    Article Snippet: The following small compound inhibitors and reagents were used: CRID3 (MCC950) (Tocris), cycloheximide (Sigma-Aldrich), Cytarabine (AraC) (Abcam), doxycycline (Biomol), G140 (Invivogen), H-151 (Biozol), human IFN-α (PBL Assay Science), human IFN-γ (Immunotools), LPS-EK Ultrapure (Invivogen), PMA (phorbol 12-myristate 13-acetate) (Sigma-Aldrich), and VX-765/belnacasan (Selleckchem).

    Techniques: Derivative Assay, Infection, Recombinant, Flow Cytometry, Membrane, Live Cell Imaging, Confocal Microscopy, Staining, Knock-Out, Expressing, Virus, Labeling

    ( A – I ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF ( A , D , E ), normal human epidermal keratinocytes (NHEK) ( B , F , G ), or human primary CD14 + monocytes ( C , H , I ), were left untreated or pretreated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP at an MOI of 5 ( A , B ) or MOI 10 ( C ), in the absence of VX, and where indicated in the presence of 2.5 µM CRID3. Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte Live-Cell Imaging system ( A – C ). DRAQ7 uptake normalized to cell confluency are shown. IL-1β in the supernatant was measured by homogeneous time resolved fluorescence (HTRF) ( D , F , H ). Cell death was measured by LDH release and normalized to cells lysed in 1% Triton X-100 ( E , G , I ). ( J , K ) PMA-differentiated WT ( J ) or indicated knockout THP-1 cells ( K ) were left untreated or pretreated with IFN-γ overnight and infected with indicated VACV strains in the presence of VX, and where indicated, CRID3. Infection levels are displayed here; specking data from the same experiments are shown in Fig. . ( L , M ) NHEK ( L ) and PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV or monkeypox virus (MPXV) at an MOI of 5 in the presence of VX, and where indicated, CRID3. Cells were fixed 6 h post infection and stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies. Infected cells (EGFP + ) were analyzed in infected or mock-treated cells by flow cytometry as described before. Infection levels are displayed here; specking data from the same experiments are displayed in Fig. . Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells).

    Journal: The EMBO Journal

    Article Title: Poxvirus dsDNA genomes differentially activate AIM2 or NLRP3 inflammasomes in human primary cells

    doi: 10.1038/s44318-025-00690-z

    Figure Lengend Snippet: ( A – I ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF ( A , D , E ), normal human epidermal keratinocytes (NHEK) ( B , F , G ), or human primary CD14 + monocytes ( C , H , I ), were left untreated or pretreated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP at an MOI of 5 ( A , B ) or MOI 10 ( C ), in the absence of VX, and where indicated in the presence of 2.5 µM CRID3. Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte Live-Cell Imaging system ( A – C ). DRAQ7 uptake normalized to cell confluency are shown. IL-1β in the supernatant was measured by homogeneous time resolved fluorescence (HTRF) ( D , F , H ). Cell death was measured by LDH release and normalized to cells lysed in 1% Triton X-100 ( E , G , I ). ( J , K ) PMA-differentiated WT ( J ) or indicated knockout THP-1 cells ( K ) were left untreated or pretreated with IFN-γ overnight and infected with indicated VACV strains in the presence of VX, and where indicated, CRID3. Infection levels are displayed here; specking data from the same experiments are shown in Fig. . ( L , M ) NHEK ( L ) and PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV or monkeypox virus (MPXV) at an MOI of 5 in the presence of VX, and where indicated, CRID3. Cells were fixed 6 h post infection and stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies. Infected cells (EGFP + ) were analyzed in infected or mock-treated cells by flow cytometry as described before. Infection levels are displayed here; specking data from the same experiments are displayed in Fig. . Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells).

    Article Snippet: The following small compound inhibitors and reagents were used: CRID3 (MCC950) (Tocris), cycloheximide (Sigma-Aldrich), Cytarabine (AraC) (Abcam), doxycycline (Biomol), G140 (Invivogen), H-151 (Biozol), human IFN-α (PBL Assay Science), human IFN-γ (Immunotools), LPS-EK Ultrapure (Invivogen), PMA (phorbol 12-myristate 13-acetate) (Sigma-Aldrich), and VX-765/belnacasan (Selleckchem).

    Techniques: Derivative Assay, Infection, Membrane, Live Cell Imaging, Fluorescence, Knock-Out, Expressing, Virus, Staining, Labeling, Flow Cytometry

    ( A ) Genome structure of recombinant VACV strains expressing C1C-EGFP from the J2R early promoter (pE) and bivalent nanobodies from a synthetic early/late promoter (pE/L). Transgenes were inserted into the VACV TK locus by homologous recombination. ( B ) Scheme of AIM2 inflammasome components indicating the VACV-encoded nanobodies used to perturb inflammasome activation. ( C – E ) Human MDMs differentiated with GM-CSF ( C ), NHEKs ( D ), and CD14 + monocytes ( E ) were left untreated or treated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP expressing the indicated nanobodies at an MOI of 5 ( C , D ) or 10 ( E ), in the presence of VX and, where indicated, CRID3. Six hours post infection, cells were harvested, fixed, and infection and C1C-EGFP speck assembly was analyzed by flow cytometry. Average values (with individual data points) from N = 3 independent donors ± SEM are displayed. .

    Journal: The EMBO Journal

    Article Title: Poxvirus dsDNA genomes differentially activate AIM2 or NLRP3 inflammasomes in human primary cells

    doi: 10.1038/s44318-025-00690-z

    Figure Lengend Snippet: ( A ) Genome structure of recombinant VACV strains expressing C1C-EGFP from the J2R early promoter (pE) and bivalent nanobodies from a synthetic early/late promoter (pE/L). Transgenes were inserted into the VACV TK locus by homologous recombination. ( B ) Scheme of AIM2 inflammasome components indicating the VACV-encoded nanobodies used to perturb inflammasome activation. ( C – E ) Human MDMs differentiated with GM-CSF ( C ), NHEKs ( D ), and CD14 + monocytes ( E ) were left untreated or treated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP expressing the indicated nanobodies at an MOI of 5 ( C , D ) or 10 ( E ), in the presence of VX and, where indicated, CRID3. Six hours post infection, cells were harvested, fixed, and infection and C1C-EGFP speck assembly was analyzed by flow cytometry. Average values (with individual data points) from N = 3 independent donors ± SEM are displayed. .

    Article Snippet: The following small compound inhibitors and reagents were used: CRID3 (MCC950) (Tocris), cycloheximide (Sigma-Aldrich), Cytarabine (AraC) (Abcam), doxycycline (Biomol), G140 (Invivogen), H-151 (Biozol), human IFN-α (PBL Assay Science), human IFN-γ (Immunotools), LPS-EK Ultrapure (Invivogen), PMA (phorbol 12-myristate 13-acetate) (Sigma-Aldrich), and VX-765/belnacasan (Selleckchem).

    Techniques: Recombinant, Expressing, Homologous Recombination, Activation Assay, Infection, Flow Cytometry

    ( A – C ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF, ( D – F ) normal human epidermal keratinocytes (NHEK), ( G – I ) CD14 + monocytes, and ( J ) PMA-differentiated THP-1 macrophages were left untreated or pretreated with IFN-γ overnight. Cells were infected with indicated recombinant VACV strains at an MOI of 5 ( A – F , J ) or MOI 10 ( G – I ), in the presence ( A , C , D , F , G , I , J ) or absence ( B , E , H ) of VX, and where indicated, in the presence of 2.5 µM CRID3. Cells were harvested and fixed 6 h post infection. Infection (EGFP + cells) and C1C-EGFP speck assembly in infected or mock-treated cells was quantified by flow cytometry ( A , D , G , J ); infection data (EGFP + cells) corresponding to the experiment shown in panel ( J ) is displayed in Fig. . Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte live-cell imaging system ( B , E , H ). Scale bar = 100 μm. Samples for confocal microscopy were fixed and stained with 5 μg/mL wheat germ agglutinin (WGA)-Alexa Fluor (AF) 647 and 4 μM Hoechst 33342 ( C , F , I ). Scale bar = 5 μm. ( K ) PMA-differentiated WT or indicated knockout THP-1 cells were left untreated or pretreated with IFN-γ overnight and infected with the indicated reporter VACV strains at an MOI of 5 in the presence of VX. C1C-EGFP speck assembly was quantified as before. The corresponding infection data (EGFP + cells) is shown in Fig. . ( L , M ) NHEK ( L ) or PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV WT or monkeypox virus (MPXV) at an MOI of 5, and where indicated, in the presence of CRID3. Cells were harvested and fixed 6 h post infection. VACV- or MPXV-infected cells were stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies, and measured by flow cytometry. Corresponding infection data (αH5-AF647 + cells) is shown in Fig . C1C-EGFP speck assembly was analyzed in infected or mock-treated cells. Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells). .

    Journal: The EMBO Journal

    Article Title: Poxvirus dsDNA genomes differentially activate AIM2 or NLRP3 inflammasomes in human primary cells

    doi: 10.1038/s44318-025-00690-z

    Figure Lengend Snippet: ( A – C ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF, ( D – F ) normal human epidermal keratinocytes (NHEK), ( G – I ) CD14 + monocytes, and ( J ) PMA-differentiated THP-1 macrophages were left untreated or pretreated with IFN-γ overnight. Cells were infected with indicated recombinant VACV strains at an MOI of 5 ( A – F , J ) or MOI 10 ( G – I ), in the presence ( A , C , D , F , G , I , J ) or absence ( B , E , H ) of VX, and where indicated, in the presence of 2.5 µM CRID3. Cells were harvested and fixed 6 h post infection. Infection (EGFP + cells) and C1C-EGFP speck assembly in infected or mock-treated cells was quantified by flow cytometry ( A , D , G , J ); infection data (EGFP + cells) corresponding to the experiment shown in panel ( J ) is displayed in Fig. . Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte live-cell imaging system ( B , E , H ). Scale bar = 100 μm. Samples for confocal microscopy were fixed and stained with 5 μg/mL wheat germ agglutinin (WGA)-Alexa Fluor (AF) 647 and 4 μM Hoechst 33342 ( C , F , I ). Scale bar = 5 μm. ( K ) PMA-differentiated WT or indicated knockout THP-1 cells were left untreated or pretreated with IFN-γ overnight and infected with the indicated reporter VACV strains at an MOI of 5 in the presence of VX. C1C-EGFP speck assembly was quantified as before. The corresponding infection data (EGFP + cells) is shown in Fig. . ( L , M ) NHEK ( L ) or PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV WT or monkeypox virus (MPXV) at an MOI of 5, and where indicated, in the presence of CRID3. Cells were harvested and fixed 6 h post infection. VACV- or MPXV-infected cells were stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies, and measured by flow cytometry. Corresponding infection data (αH5-AF647 + cells) is shown in Fig . C1C-EGFP speck assembly was analyzed in infected or mock-treated cells. Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells). .

    Article Snippet: CRID3 (MCC950) , Tocris , 5479.

    Techniques: Derivative Assay, Infection, Recombinant, Flow Cytometry, Membrane, Live Cell Imaging, Confocal Microscopy, Staining, Knock-Out, Expressing, Virus, Labeling

    ( A – I ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF ( A , D , E ), normal human epidermal keratinocytes (NHEK) ( B , F , G ), or human primary CD14 + monocytes ( C , H , I ), were left untreated or pretreated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP at an MOI of 5 ( A , B ) or MOI 10 ( C ), in the absence of VX, and where indicated in the presence of 2.5 µM CRID3. Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte Live-Cell Imaging system ( A – C ). DRAQ7 uptake normalized to cell confluency are shown. IL-1β in the supernatant was measured by homogeneous time resolved fluorescence (HTRF) ( D , F , H ). Cell death was measured by LDH release and normalized to cells lysed in 1% Triton X-100 ( E , G , I ). ( J , K ) PMA-differentiated WT ( J ) or indicated knockout THP-1 cells ( K ) were left untreated or pretreated with IFN-γ overnight and infected with indicated VACV strains in the presence of VX, and where indicated, CRID3. Infection levels are displayed here; specking data from the same experiments are shown in Fig. . ( L , M ) NHEK ( L ) and PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV or monkeypox virus (MPXV) at an MOI of 5 in the presence of VX, and where indicated, CRID3. Cells were fixed 6 h post infection and stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies. Infected cells (EGFP + ) were analyzed in infected or mock-treated cells by flow cytometry as described before. Infection levels are displayed here; specking data from the same experiments are displayed in Fig. . Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells).

    Journal: The EMBO Journal

    Article Title: Poxvirus dsDNA genomes differentially activate AIM2 or NLRP3 inflammasomes in human primary cells

    doi: 10.1038/s44318-025-00690-z

    Figure Lengend Snippet: ( A – I ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF ( A , D , E ), normal human epidermal keratinocytes (NHEK) ( B , F , G ), or human primary CD14 + monocytes ( C , H , I ), were left untreated or pretreated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP at an MOI of 5 ( A , B ) or MOI 10 ( C ), in the absence of VX, and where indicated in the presence of 2.5 µM CRID3. Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte Live-Cell Imaging system ( A – C ). DRAQ7 uptake normalized to cell confluency are shown. IL-1β in the supernatant was measured by homogeneous time resolved fluorescence (HTRF) ( D , F , H ). Cell death was measured by LDH release and normalized to cells lysed in 1% Triton X-100 ( E , G , I ). ( J , K ) PMA-differentiated WT ( J ) or indicated knockout THP-1 cells ( K ) were left untreated or pretreated with IFN-γ overnight and infected with indicated VACV strains in the presence of VX, and where indicated, CRID3. Infection levels are displayed here; specking data from the same experiments are shown in Fig. . ( L , M ) NHEK ( L ) and PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV or monkeypox virus (MPXV) at an MOI of 5 in the presence of VX, and where indicated, CRID3. Cells were fixed 6 h post infection and stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies. Infected cells (EGFP + ) were analyzed in infected or mock-treated cells by flow cytometry as described before. Infection levels are displayed here; specking data from the same experiments are displayed in Fig. . Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells).

    Article Snippet: CRID3 (MCC950) , Tocris , 5479.

    Techniques: Derivative Assay, Infection, Membrane, Live Cell Imaging, Fluorescence, Knock-Out, Expressing, Virus, Staining, Labeling, Flow Cytometry

    ( A ) Genome structure of recombinant VACV strains expressing C1C-EGFP from the J2R early promoter (pE) and bivalent nanobodies from a synthetic early/late promoter (pE/L). Transgenes were inserted into the VACV TK locus by homologous recombination. ( B ) Scheme of AIM2 inflammasome components indicating the VACV-encoded nanobodies used to perturb inflammasome activation. ( C – E ) Human MDMs differentiated with GM-CSF ( C ), NHEKs ( D ), and CD14 + monocytes ( E ) were left untreated or treated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP expressing the indicated nanobodies at an MOI of 5 ( C , D ) or 10 ( E ), in the presence of VX and, where indicated, CRID3. Six hours post infection, cells were harvested, fixed, and infection and C1C-EGFP speck assembly was analyzed by flow cytometry. Average values (with individual data points) from N = 3 independent donors ± SEM are displayed. .

    Journal: The EMBO Journal

    Article Title: Poxvirus dsDNA genomes differentially activate AIM2 or NLRP3 inflammasomes in human primary cells

    doi: 10.1038/s44318-025-00690-z

    Figure Lengend Snippet: ( A ) Genome structure of recombinant VACV strains expressing C1C-EGFP from the J2R early promoter (pE) and bivalent nanobodies from a synthetic early/late promoter (pE/L). Transgenes were inserted into the VACV TK locus by homologous recombination. ( B ) Scheme of AIM2 inflammasome components indicating the VACV-encoded nanobodies used to perturb inflammasome activation. ( C – E ) Human MDMs differentiated with GM-CSF ( C ), NHEKs ( D ), and CD14 + monocytes ( E ) were left untreated or treated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP expressing the indicated nanobodies at an MOI of 5 ( C , D ) or 10 ( E ), in the presence of VX and, where indicated, CRID3. Six hours post infection, cells were harvested, fixed, and infection and C1C-EGFP speck assembly was analyzed by flow cytometry. Average values (with individual data points) from N = 3 independent donors ± SEM are displayed. .

    Article Snippet: CRID3 (MCC950) , Tocris , 5479.

    Techniques: Recombinant, Expressing, Homologous Recombination, Activation Assay, Infection, Flow Cytometry

    ( A ) Scheme (created in BioRender: A. Rifat (2025); https://BioRender.com/jx63a07 ) illustrating the workflow for determining IL-1β levels from supernatants of brain slices upon purinergic stimulation (5 mM ATP, 3 hours) to simulate acute brain damage. ( B ) Mean effects on IL-1β release by inhibition of CLIC1 and NLRP3 with IAA-94 and MCC950, respectively ( n = 4, 8, 7, and 4). ( C ) Mean effect on IL-1β release by inhibition of CLIC1 in Cl − -free extracellular medium ( n = 4 each). ( D ) Schematic illustrating the predicted direction of Cl − flux under the given experimental conditions for nonactivated and ATP/P2X7-activated microglia. Blue arrows indicate the direction of the electrochemical gradient for Cl − per condition. E Cl , Cl − equilibrium potential; V m , membrane potential. ( E ) Mean effect of GSTO1-IN-1 on IL-1β release, with no additional effect upon coapplication with IAA-94 ( n = 6, 4, 4, and 4). Data information: Data indicate means ± SEM and are normalized to control condition. Dots on bars show number of slices. Data are from four mice per experimental condition. P values are from Welch’s t test (B), unpaired Student’s t tests (C), and Welch’s ANOVA with Games-Howell post hoc test (E). h, hours.

    Journal: Science Advances

    Article Title: The chloride intracellular channel 1 (CLIC1) is essential for microglial morphodynamics and neuroinflammation

    doi: 10.1126/sciadv.ads9181

    Figure Lengend Snippet: ( A ) Scheme (created in BioRender: A. Rifat (2025); https://BioRender.com/jx63a07 ) illustrating the workflow for determining IL-1β levels from supernatants of brain slices upon purinergic stimulation (5 mM ATP, 3 hours) to simulate acute brain damage. ( B ) Mean effects on IL-1β release by inhibition of CLIC1 and NLRP3 with IAA-94 and MCC950, respectively ( n = 4, 8, 7, and 4). ( C ) Mean effect on IL-1β release by inhibition of CLIC1 in Cl − -free extracellular medium ( n = 4 each). ( D ) Schematic illustrating the predicted direction of Cl − flux under the given experimental conditions for nonactivated and ATP/P2X7-activated microglia. Blue arrows indicate the direction of the electrochemical gradient for Cl − per condition. E Cl , Cl − equilibrium potential; V m , membrane potential. ( E ) Mean effect of GSTO1-IN-1 on IL-1β release, with no additional effect upon coapplication with IAA-94 ( n = 6, 4, 4, and 4). Data information: Data indicate means ± SEM and are normalized to control condition. Dots on bars show number of slices. Data are from four mice per experimental condition. P values are from Welch’s t test (B), unpaired Student’s t tests (C), and Welch’s ANOVA with Games-Howell post hoc test (E). h, hours.

    Article Snippet: IAA-94 (I117, Sigma-Aldrich), NSC-305787 hydrochloride (HY-18931A, MedChemExpress), NSC-23766 hydrochloride (HY-15723A, MedChemExpress), A9C (10449010, Thermo Fisher Scientific), DIDS (4523, Tocris), NPPB (0593, Tocris), 2-fluoro- N -[2-(2-methyl-1H-indol-3-yl)ethyl]-benzamide (CK-666; SML0006, Sigma-Aldrich), oridonin (09639, Sigma-Aldrich), GSTO1-IN-1 (HY-111530, MedChemExpress), and MCC950 (5479, Tocris) were dissolved and stored according to the manufacturer’s instructions.

    Techniques: Inhibition, Membrane, Control

    ( A ) Illustration of xenotransplantation model used in (B) to (F) . ( B ) Images of brain slices stained with Isolectin GS-IB4 and Tomatolectin to distinguish mouse (Isolectin + ) from human (Tomatolectin + /Isolectin − ) microglia . ( C ) Superimposed images before and with IAA-94. ( D ) Time course of surveillance before and with 50 μM IAA-94. ( E and F ) Effects of IAA-94 on (E) surveillance index and (F) motility index of human and murine microglia ( n = 11 and 20). ( G ) Scheme depicting the utilization of acutely resected surgical tissue from human temporal lobe. ( H ) Representative images showing microglia in the human neocortex in the absence (control) and presence of 40-min exposure to IAA-94. ( I ) Comparison of microglial ramification without and with IAA-94. ( J ) Effects of IAA-94 ( n = 39 and 29) on number of intersections (left), total process length (middle), and number of processes (right). ( K ) Effect of IAA-94 on the surveillance territory of microglia, normalized to control ( n = 39 and 29). ( L ) Comparison of ATP-evoked IL-1β release in dependence of IAA-94 and MCC950 in three individual patients (left; patient 1: n = 4, 11, 4, and 4; patient 2: n = 4, 3, 4, and 4; patient 3: n = 6, 7, and 4) and pooled for all patients (very right; n = 22, 12, and 8). Individual data are normalized to control condition or to the ATP condition for pooled data. Data indicate means ± SEM. Dots on bars show number of cells [(E), (F), and (J)] or number of slices (L). Data are from three mice [(B) to (F)] and three individual patients [(H) to (L)]. P values are from paired [(E) and (F): murine microglia] and unpaired [(L): patients 1 and 3] Student’s t tests, Wilcoxon signed-rank test [(F): human microglia], Welch’s t test [(L): patient 2], and Mann-Whitney U tests [(J) to (L): combined data]. Illustrations are created in BioRender: A. Rifat (2025); https://BioRender.com/9mwwjbn and https://BioRender.com/8xo9x45 .

    Journal: Science Advances

    Article Title: The chloride intracellular channel 1 (CLIC1) is essential for microglial morphodynamics and neuroinflammation

    doi: 10.1126/sciadv.ads9181

    Figure Lengend Snippet: ( A ) Illustration of xenotransplantation model used in (B) to (F) . ( B ) Images of brain slices stained with Isolectin GS-IB4 and Tomatolectin to distinguish mouse (Isolectin + ) from human (Tomatolectin + /Isolectin − ) microglia . ( C ) Superimposed images before and with IAA-94. ( D ) Time course of surveillance before and with 50 μM IAA-94. ( E and F ) Effects of IAA-94 on (E) surveillance index and (F) motility index of human and murine microglia ( n = 11 and 20). ( G ) Scheme depicting the utilization of acutely resected surgical tissue from human temporal lobe. ( H ) Representative images showing microglia in the human neocortex in the absence (control) and presence of 40-min exposure to IAA-94. ( I ) Comparison of microglial ramification without and with IAA-94. ( J ) Effects of IAA-94 ( n = 39 and 29) on number of intersections (left), total process length (middle), and number of processes (right). ( K ) Effect of IAA-94 on the surveillance territory of microglia, normalized to control ( n = 39 and 29). ( L ) Comparison of ATP-evoked IL-1β release in dependence of IAA-94 and MCC950 in three individual patients (left; patient 1: n = 4, 11, 4, and 4; patient 2: n = 4, 3, 4, and 4; patient 3: n = 6, 7, and 4) and pooled for all patients (very right; n = 22, 12, and 8). Individual data are normalized to control condition or to the ATP condition for pooled data. Data indicate means ± SEM. Dots on bars show number of cells [(E), (F), and (J)] or number of slices (L). Data are from three mice [(B) to (F)] and three individual patients [(H) to (L)]. P values are from paired [(E) and (F): murine microglia] and unpaired [(L): patients 1 and 3] Student’s t tests, Wilcoxon signed-rank test [(F): human microglia], Welch’s t test [(L): patient 2], and Mann-Whitney U tests [(J) to (L): combined data]. Illustrations are created in BioRender: A. Rifat (2025); https://BioRender.com/9mwwjbn and https://BioRender.com/8xo9x45 .

    Article Snippet: IAA-94 (I117, Sigma-Aldrich), NSC-305787 hydrochloride (HY-18931A, MedChemExpress), NSC-23766 hydrochloride (HY-15723A, MedChemExpress), A9C (10449010, Thermo Fisher Scientific), DIDS (4523, Tocris), NPPB (0593, Tocris), 2-fluoro- N -[2-(2-methyl-1H-indol-3-yl)ethyl]-benzamide (CK-666; SML0006, Sigma-Aldrich), oridonin (09639, Sigma-Aldrich), GSTO1-IN-1 (HY-111530, MedChemExpress), and MCC950 (5479, Tocris) were dissolved and stored according to the manufacturer’s instructions.

    Techniques: Staining, Control, Comparison, MANN-WHITNEY