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a549 asc gfp ace2  (InvivoGen)


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    InvivoGen a549 asc gfp ace2
    P. aeruginosa triggers human NLRP1 inflammasome activation in corneal and nasal epithelial cells. (A) Cell lysis (LDH) and IL-1β/IL-18 release evaluation in pHCECs and pHNECs upon P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. When specified, the pan Caspase inhibitor (Z-VAD, 20 µM), Caspase-1 inhibitor (Z-YVAD, 20 µM), Caspase-3/7 inhibitor (Z-DEVD, 20 µM), and Caspase-8 inhibitor (Z-IETD, 20 µM) were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. (B) Immunoblotting examination of NLRP1, NLRP3, and Tubulin in resting, PAO1-exposed as in A or LPS-primed pHCECs and pHNECs or in pHCECs and pHNECs genetically invalidated for NLRP1 using CRISPR-Cas9. PMA (100 µg/ml)- or LPS (100 ng/ml)-primed THP1 monocytic cell line was used as a positive control for NLRP3 expression. Immunoblots show lysates from one experiment performed three times. (C) Florescence microscopy and associated quantifications of <t>ASC-GFP</t> specks in <t>A549</t> NLRP1+/ASC-GFP and A549 NLRP1−/ASC-GFP reporter cell lines exposed to P. aeruginosa (PAO1, 1.10 5 bacteria) for 24 h. ASC-GFP (green) pictures were taken in the dish after the infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (D) Immunoblotting characterization of genetic invalidation of NLRP1 in pHCECs and pHNECs population using CRISPR-Cas9 and microscopy visualization of plasma membrane permeabilization (PI incorporation, orange) in pHCECs co-cultured with PAO1 (1.10 5 bacteria) for 24 h. (E) sgRNA CD8 (SgCD8) was used as control and served as WT cells during subsequent experiments described in E. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 20 µm. Cell lysis (LDH), IL-18 release, and CFU evaluation in WT (SgCD8, D) or NLRP1 -deficient pHCECs and pHNECs, upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. For CFU analysis 1 × 10 4 (MOI 1) or 1 × 10 5 (MOI 10) bacteria were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. Source data are available for this figure: .
    A549 Asc Gfp Ace2, supplied by InvivoGen, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption"

    Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20230104

    P. aeruginosa triggers human NLRP1 inflammasome activation in corneal and nasal epithelial cells. (A) Cell lysis (LDH) and IL-1β/IL-18 release evaluation in pHCECs and pHNECs upon P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. When specified, the pan Caspase inhibitor (Z-VAD, 20 µM), Caspase-1 inhibitor (Z-YVAD, 20 µM), Caspase-3/7 inhibitor (Z-DEVD, 20 µM), and Caspase-8 inhibitor (Z-IETD, 20 µM) were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. (B) Immunoblotting examination of NLRP1, NLRP3, and Tubulin in resting, PAO1-exposed as in A or LPS-primed pHCECs and pHNECs or in pHCECs and pHNECs genetically invalidated for NLRP1 using CRISPR-Cas9. PMA (100 µg/ml)- or LPS (100 ng/ml)-primed THP1 monocytic cell line was used as a positive control for NLRP3 expression. Immunoblots show lysates from one experiment performed three times. (C) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1−/ASC-GFP reporter cell lines exposed to P. aeruginosa (PAO1, 1.10 5 bacteria) for 24 h. ASC-GFP (green) pictures were taken in the dish after the infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (D) Immunoblotting characterization of genetic invalidation of NLRP1 in pHCECs and pHNECs population using CRISPR-Cas9 and microscopy visualization of plasma membrane permeabilization (PI incorporation, orange) in pHCECs co-cultured with PAO1 (1.10 5 bacteria) for 24 h. (E) sgRNA CD8 (SgCD8) was used as control and served as WT cells during subsequent experiments described in E. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 20 µm. Cell lysis (LDH), IL-18 release, and CFU evaluation in WT (SgCD8, D) or NLRP1 -deficient pHCECs and pHNECs, upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. For CFU analysis 1 × 10 4 (MOI 1) or 1 × 10 5 (MOI 10) bacteria were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. Source data are available for this figure: .
    Figure Legend Snippet: P. aeruginosa triggers human NLRP1 inflammasome activation in corneal and nasal epithelial cells. (A) Cell lysis (LDH) and IL-1β/IL-18 release evaluation in pHCECs and pHNECs upon P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. When specified, the pan Caspase inhibitor (Z-VAD, 20 µM), Caspase-1 inhibitor (Z-YVAD, 20 µM), Caspase-3/7 inhibitor (Z-DEVD, 20 µM), and Caspase-8 inhibitor (Z-IETD, 20 µM) were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. (B) Immunoblotting examination of NLRP1, NLRP3, and Tubulin in resting, PAO1-exposed as in A or LPS-primed pHCECs and pHNECs or in pHCECs and pHNECs genetically invalidated for NLRP1 using CRISPR-Cas9. PMA (100 µg/ml)- or LPS (100 ng/ml)-primed THP1 monocytic cell line was used as a positive control for NLRP3 expression. Immunoblots show lysates from one experiment performed three times. (C) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1−/ASC-GFP reporter cell lines exposed to P. aeruginosa (PAO1, 1.10 5 bacteria) for 24 h. ASC-GFP (green) pictures were taken in the dish after the infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (D) Immunoblotting characterization of genetic invalidation of NLRP1 in pHCECs and pHNECs population using CRISPR-Cas9 and microscopy visualization of plasma membrane permeabilization (PI incorporation, orange) in pHCECs co-cultured with PAO1 (1.10 5 bacteria) for 24 h. (E) sgRNA CD8 (SgCD8) was used as control and served as WT cells during subsequent experiments described in E. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 20 µm. Cell lysis (LDH), IL-18 release, and CFU evaluation in WT (SgCD8, D) or NLRP1 -deficient pHCECs and pHNECs, upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. For CFU analysis 1 × 10 4 (MOI 1) or 1 × 10 5 (MOI 10) bacteria were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. Source data are available for this figure: .

    Techniques Used: Activation Assay, Lysis, Bacteria, Co-Culture Assay, Western Blot, CRISPR, Positive Control, Expressing, Microscopy, Infection, Membrane, Cell Culture, Control

    P. aeruginosa –activated hNLRP1 inflammasome requires proteasome activity. (A) Fluorescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to 1 × 10 5 P. aeruginosa clinical isolates from patients with infected lung (strains 3039 1533 and 2348 4390) or with infected cornea (strain 0236 1921) for 24 h. ASC-GFP (green) pictures were taken in the dish after infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from each experiment were analyzed. Values are expressed as mean ± SEM. One-way ANOVA. (B) Schematic drawing of P. aeruginosa co-culture experiments performed with human corneal epithelial cells. (C) Immunoblotting of NLRP1, Gasdermin-D, and Tubulin in pHCECs upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h in presence/absence of proteasome inhibitor bortezomib. Immunoblots show lysates from one experiment performed at least three times. (D) Cell lysis (LDH) and IL-1B release evaluation in pHCECs and pHNECs, upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h in presence/absence of proteasome inhibitor bortezomib. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM from one experiment (in triplicate) performed at least three times. Source data are available for this figure: .
    Figure Legend Snippet: P. aeruginosa –activated hNLRP1 inflammasome requires proteasome activity. (A) Fluorescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to 1 × 10 5 P. aeruginosa clinical isolates from patients with infected lung (strains 3039 1533 and 2348 4390) or with infected cornea (strain 0236 1921) for 24 h. ASC-GFP (green) pictures were taken in the dish after infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from each experiment were analyzed. Values are expressed as mean ± SEM. One-way ANOVA. (B) Schematic drawing of P. aeruginosa co-culture experiments performed with human corneal epithelial cells. (C) Immunoblotting of NLRP1, Gasdermin-D, and Tubulin in pHCECs upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h in presence/absence of proteasome inhibitor bortezomib. Immunoblots show lysates from one experiment performed at least three times. (D) Cell lysis (LDH) and IL-1B release evaluation in pHCECs and pHNECs, upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h in presence/absence of proteasome inhibitor bortezomib. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM from one experiment (in triplicate) performed at least three times. Source data are available for this figure: .

    Techniques Used: Activity Assay, Fluorescence, Microscopy, Infection, Co-Culture Assay, Western Blot, Bacteria, Lysis

    P. aeruginosa EEF2-inactivating EXOA promotes NLRP1 inflammasome response. (A) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to 1 × 10 5 P. aeruginosa (PAO1) and associated isogenic mutants for various secretion systems (PAO1 ΔT3SS , PAO1 ΔT2SS , PAO1 ΔT1SS ) for 24 h. ASC-GFP (green) pictures were taken in the dish after infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (B) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to 1 × 10 5 P. aeruginosa (PAO1) and associated isogenic mutants for various T2SS virulence effectors (PAO1 ΔPLCN , PAO1 ΔPLCH , PAO1 ΔLASB , and PAO1 ΔEXOA ) for 24 h. ASC-GFP (green) pictures were taken in the dish after infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nulcei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (C) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to EXOA (10 ng/ml) or its catalytically dead mutant EXOA H426A (500 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (D) Schematic mechanism of P. aeruginosa EXOA and related toxins at mediating EEF2 ribosylation and inactivation and subsequent ribosome inactivation. (E) Immunoblotting characterization of genetic invalidation of DPH1 in A549 NLRP1+/ASC-GFP cells using CRISPR-Cas9. The red arrow shows the selected KO cells for subsequent experiments. (F) Fluorescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/ DPH1− reporter cell lines exposed to VbP (15 µM), EXOA (10 ng/ml), cholix toxin (CT, 10 ng/ml), and diphtheria toxin (DT, 20 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (G) Plasma membrane permeabilization determination over time using PI incorporation in WT or NLRP1 -deficient pHCECs exposed to VbP (15 µM), EXOA (10 ng/ml) or EXOA H426A (10 ng/ml) for indicated times. ***P ≤ 0.001, T test. Values are expressed as mean ± SEM from one experiment (in triplicate) performed at least three times. Source data are available for this figure: .
    Figure Legend Snippet: P. aeruginosa EEF2-inactivating EXOA promotes NLRP1 inflammasome response. (A) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to 1 × 10 5 P. aeruginosa (PAO1) and associated isogenic mutants for various secretion systems (PAO1 ΔT3SS , PAO1 ΔT2SS , PAO1 ΔT1SS ) for 24 h. ASC-GFP (green) pictures were taken in the dish after infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (B) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to 1 × 10 5 P. aeruginosa (PAO1) and associated isogenic mutants for various T2SS virulence effectors (PAO1 ΔPLCN , PAO1 ΔPLCH , PAO1 ΔLASB , and PAO1 ΔEXOA ) for 24 h. ASC-GFP (green) pictures were taken in the dish after infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nulcei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (C) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to EXOA (10 ng/ml) or its catalytically dead mutant EXOA H426A (500 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (D) Schematic mechanism of P. aeruginosa EXOA and related toxins at mediating EEF2 ribosylation and inactivation and subsequent ribosome inactivation. (E) Immunoblotting characterization of genetic invalidation of DPH1 in A549 NLRP1+/ASC-GFP cells using CRISPR-Cas9. The red arrow shows the selected KO cells for subsequent experiments. (F) Fluorescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/ DPH1− reporter cell lines exposed to VbP (15 µM), EXOA (10 ng/ml), cholix toxin (CT, 10 ng/ml), and diphtheria toxin (DT, 20 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (G) Plasma membrane permeabilization determination over time using PI incorporation in WT or NLRP1 -deficient pHCECs exposed to VbP (15 µM), EXOA (10 ng/ml) or EXOA H426A (10 ng/ml) for indicated times. ***P ≤ 0.001, T test. Values are expressed as mean ± SEM from one experiment (in triplicate) performed at least three times. Source data are available for this figure: .

    Techniques Used: Microscopy, Infection, Mutagenesis, Western Blot, CRISPR, Fluorescence, Membrane

    Multiple EEF2-targeting toxins activate the hNLRP1 inflammasome in a ZAKα-dependent manner. (A) Schematic mechanism of EXOA and related toxin-mediated translation inhibition. tRNA, transfer RNA; E, exit; P, peptidyl; A, aminoacyl. (B) Determination of ribosome inactivation in A549 NLRP1+/ASC-GFP and A549 NLRP1−/ASC-GFP reporter cell lines exposed to EXOA (10 ng/ml) for 2 and 6 h by measuring ribosome polysome accumulation and puromycin incorporation. Images are representative of one experiment performed at least three times. (C) Immunoblotting of ADP-ribosylated proteins, EEF2, and Tubulin in A549 NLRP1+/ASC-GFP cell lysates treated or not with VbP (15 µM) or EXOA (10 ng/ml) in the presence of Nicotinamide adenine dinucleotide-Biotin (NAD-Biot). Immunoblots show lysates from one experiment performed at least three times. (D) Immunoblotting of NLRP1, Tubulin, and phosphorylated P38 and JNK in A549 NLRP1+ and A549 NLRP1− reporter cell lines exposed or not to EXOA (10 ng/ml) or its inactive mutant EXOA H426A for 3 h. Immunoblots show lysates from one experiment performed at least three times. (E) Immunoblotting characterization of genetic invalidation of P38α and P38β in A549 NLRP1+/ASC-GFP cells using CRISPR-Cas9. Immunoblots show lysates from one experiment performed at least three times. (F) Fluorescence microscopy of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/ ZAKα - reporter cell lines expressing hACE2 infected for 24 h with various SARS-CoV-2 MOI. ASC-GFP (green) pictures were taken in the dish after viral infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. Source data are available for this figure: .
    Figure Legend Snippet: Multiple EEF2-targeting toxins activate the hNLRP1 inflammasome in a ZAKα-dependent manner. (A) Schematic mechanism of EXOA and related toxin-mediated translation inhibition. tRNA, transfer RNA; E, exit; P, peptidyl; A, aminoacyl. (B) Determination of ribosome inactivation in A549 NLRP1+/ASC-GFP and A549 NLRP1−/ASC-GFP reporter cell lines exposed to EXOA (10 ng/ml) for 2 and 6 h by measuring ribosome polysome accumulation and puromycin incorporation. Images are representative of one experiment performed at least three times. (C) Immunoblotting of ADP-ribosylated proteins, EEF2, and Tubulin in A549 NLRP1+/ASC-GFP cell lysates treated or not with VbP (15 µM) or EXOA (10 ng/ml) in the presence of Nicotinamide adenine dinucleotide-Biotin (NAD-Biot). Immunoblots show lysates from one experiment performed at least three times. (D) Immunoblotting of NLRP1, Tubulin, and phosphorylated P38 and JNK in A549 NLRP1+ and A549 NLRP1− reporter cell lines exposed or not to EXOA (10 ng/ml) or its inactive mutant EXOA H426A for 3 h. Immunoblots show lysates from one experiment performed at least three times. (E) Immunoblotting characterization of genetic invalidation of P38α and P38β in A549 NLRP1+/ASC-GFP cells using CRISPR-Cas9. Immunoblots show lysates from one experiment performed at least three times. (F) Fluorescence microscopy of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/ ZAKα - reporter cell lines expressing hACE2 infected for 24 h with various SARS-CoV-2 MOI. ASC-GFP (green) pictures were taken in the dish after viral infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. Source data are available for this figure: .

    Techniques Used: Inhibition, Western Blot, TNKS1 Histone Ribosylation Assay, Mutagenesis, CRISPR, Fluorescence, Microscopy, Expressing, Infection

    EEF2 inactivation drives ZAKα and P38 MAPK activation and subsequent NLRP1 inflammasome nucleation. (A) Immunoblotting of P38, JNK, ZAKα, NLRP1, Tubulin, and phosphorylated P38 and JNK in A549 NLRP1+ and A549 NLRP1+/ZAKα- reporter cell lines exposed or not to EXOA (10 ng/ml) for 3 h. Immunoblots show lysates from one experiment performed at least three times. (B) Cell lysis (LDH release), florescence microscopy, and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/ZAKα- reporter cell lines exposed to EXOA (10 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positives for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. (C) Cell lysis (LDH release), fluorescence microscopy, and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/P38α/β- reporter cell lines exposed to EXOA (10 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. Graphs show one experiment performed in triplicate at least three times. (D) Western blot examination of NLRP1 using an anti-NLRP1 N-terminal antibody (aa 1–323) in A549 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for 112 TST 114 / 112 AAA 114 or 178 TST 180 / 178 AAA 180 after 4 h exposure to EXOA (10 ng/ml) or VbP (15 µM). Images shown are from one experiment and are representative of n = 3 independent experiments. (E) Cell lysis (LDH release), fluorescence microscopy, and associated quantifications of ASC-GFP specks in A549 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for 112 TST 114 / 112 AAA 114 or 178 TST 180 / 178 AAA 180 after 10 h exposure to EXOA (10 ng/ml) or VbP (15 µM). ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times. Source data are available for this figure: .
    Figure Legend Snippet: EEF2 inactivation drives ZAKα and P38 MAPK activation and subsequent NLRP1 inflammasome nucleation. (A) Immunoblotting of P38, JNK, ZAKα, NLRP1, Tubulin, and phosphorylated P38 and JNK in A549 NLRP1+ and A549 NLRP1+/ZAKα- reporter cell lines exposed or not to EXOA (10 ng/ml) for 3 h. Immunoblots show lysates from one experiment performed at least three times. (B) Cell lysis (LDH release), florescence microscopy, and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/ZAKα- reporter cell lines exposed to EXOA (10 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positives for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. (C) Cell lysis (LDH release), fluorescence microscopy, and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/P38α/β- reporter cell lines exposed to EXOA (10 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. Graphs show one experiment performed in triplicate at least three times. (D) Western blot examination of NLRP1 using an anti-NLRP1 N-terminal antibody (aa 1–323) in A549 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for 112 TST 114 / 112 AAA 114 or 178 TST 180 / 178 AAA 180 after 4 h exposure to EXOA (10 ng/ml) or VbP (15 µM). Images shown are from one experiment and are representative of n = 3 independent experiments. (E) Cell lysis (LDH release), fluorescence microscopy, and associated quantifications of ASC-GFP specks in A549 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for 112 TST 114 / 112 AAA 114 or 178 TST 180 / 178 AAA 180 after 10 h exposure to EXOA (10 ng/ml) or VbP (15 µM). ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times. Source data are available for this figure: .

    Techniques Used: Activation Assay, Western Blot, Lysis, Microscopy, Fluorescence, Plasmid Preparation, Construct

    List of reagents used in the study
    Figure Legend Snippet: List of reagents used in the study

    Techniques Used: Protease Inhibitor, Staining, Marker, CyQUANT Assay, Mutagenesis, Membrane, Recombinant



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    93
    InvivoGen a549 asc gfp ace2
    P. aeruginosa triggers human NLRP1 inflammasome activation in corneal and nasal epithelial cells. (A) Cell lysis (LDH) and IL-1β/IL-18 release evaluation in pHCECs and pHNECs upon P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. When specified, the pan Caspase inhibitor (Z-VAD, 20 µM), Caspase-1 inhibitor (Z-YVAD, 20 µM), Caspase-3/7 inhibitor (Z-DEVD, 20 µM), and Caspase-8 inhibitor (Z-IETD, 20 µM) were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. (B) Immunoblotting examination of NLRP1, NLRP3, and Tubulin in resting, PAO1-exposed as in A or LPS-primed pHCECs and pHNECs or in pHCECs and pHNECs genetically invalidated for NLRP1 using CRISPR-Cas9. PMA (100 µg/ml)- or LPS (100 ng/ml)-primed THP1 monocytic cell line was used as a positive control for NLRP3 expression. Immunoblots show lysates from one experiment performed three times. (C) Florescence microscopy and associated quantifications of <t>ASC-GFP</t> specks in <t>A549</t> NLRP1+/ASC-GFP and A549 NLRP1−/ASC-GFP reporter cell lines exposed to P. aeruginosa (PAO1, 1.10 5 bacteria) for 24 h. ASC-GFP (green) pictures were taken in the dish after the infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (D) Immunoblotting characterization of genetic invalidation of NLRP1 in pHCECs and pHNECs population using CRISPR-Cas9 and microscopy visualization of plasma membrane permeabilization (PI incorporation, orange) in pHCECs co-cultured with PAO1 (1.10 5 bacteria) for 24 h. (E) sgRNA CD8 (SgCD8) was used as control and served as WT cells during subsequent experiments described in E. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 20 µm. Cell lysis (LDH), IL-18 release, and CFU evaluation in WT (SgCD8, D) or NLRP1 -deficient pHCECs and pHNECs, upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. For CFU analysis 1 × 10 4 (MOI 1) or 1 × 10 5 (MOI 10) bacteria were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. Source data are available for this figure: .
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    P. aeruginosa triggers human NLRP1 inflammasome activation in corneal and nasal epithelial cells. (A) Cell lysis (LDH) and IL-1β/IL-18 release evaluation in pHCECs and pHNECs upon P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. When specified, the pan Caspase inhibitor (Z-VAD, 20 µM), Caspase-1 inhibitor (Z-YVAD, 20 µM), Caspase-3/7 inhibitor (Z-DEVD, 20 µM), and Caspase-8 inhibitor (Z-IETD, 20 µM) were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. (B) Immunoblotting examination of NLRP1, NLRP3, and Tubulin in resting, PAO1-exposed as in A or LPS-primed pHCECs and pHNECs or in pHCECs and pHNECs genetically invalidated for NLRP1 using CRISPR-Cas9. PMA (100 µg/ml)- or LPS (100 ng/ml)-primed THP1 monocytic cell line was used as a positive control for NLRP3 expression. Immunoblots show lysates from one experiment performed three times. (C) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1−/ASC-GFP reporter cell lines exposed to P. aeruginosa (PAO1, 1.10 5 bacteria) for 24 h. ASC-GFP (green) pictures were taken in the dish after the infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (D) Immunoblotting characterization of genetic invalidation of NLRP1 in pHCECs and pHNECs population using CRISPR-Cas9 and microscopy visualization of plasma membrane permeabilization (PI incorporation, orange) in pHCECs co-cultured with PAO1 (1.10 5 bacteria) for 24 h. (E) sgRNA CD8 (SgCD8) was used as control and served as WT cells during subsequent experiments described in E. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 20 µm. Cell lysis (LDH), IL-18 release, and CFU evaluation in WT (SgCD8, D) or NLRP1 -deficient pHCECs and pHNECs, upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. For CFU analysis 1 × 10 4 (MOI 1) or 1 × 10 5 (MOI 10) bacteria were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. Source data are available for this figure: .

    Journal: The Journal of Experimental Medicine

    Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

    doi: 10.1084/jem.20230104

    Figure Lengend Snippet: P. aeruginosa triggers human NLRP1 inflammasome activation in corneal and nasal epithelial cells. (A) Cell lysis (LDH) and IL-1β/IL-18 release evaluation in pHCECs and pHNECs upon P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. When specified, the pan Caspase inhibitor (Z-VAD, 20 µM), Caspase-1 inhibitor (Z-YVAD, 20 µM), Caspase-3/7 inhibitor (Z-DEVD, 20 µM), and Caspase-8 inhibitor (Z-IETD, 20 µM) were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. (B) Immunoblotting examination of NLRP1, NLRP3, and Tubulin in resting, PAO1-exposed as in A or LPS-primed pHCECs and pHNECs or in pHCECs and pHNECs genetically invalidated for NLRP1 using CRISPR-Cas9. PMA (100 µg/ml)- or LPS (100 ng/ml)-primed THP1 monocytic cell line was used as a positive control for NLRP3 expression. Immunoblots show lysates from one experiment performed three times. (C) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1−/ASC-GFP reporter cell lines exposed to P. aeruginosa (PAO1, 1.10 5 bacteria) for 24 h. ASC-GFP (green) pictures were taken in the dish after the infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (D) Immunoblotting characterization of genetic invalidation of NLRP1 in pHCECs and pHNECs population using CRISPR-Cas9 and microscopy visualization of plasma membrane permeabilization (PI incorporation, orange) in pHCECs co-cultured with PAO1 (1.10 5 bacteria) for 24 h. (E) sgRNA CD8 (SgCD8) was used as control and served as WT cells during subsequent experiments described in E. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 20 µm. Cell lysis (LDH), IL-18 release, and CFU evaluation in WT (SgCD8, D) or NLRP1 -deficient pHCECs and pHNECs, upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. For CFU analysis 1 × 10 4 (MOI 1) or 1 × 10 5 (MOI 10) bacteria were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. Source data are available for this figure: .

    Article Snippet: A549 ASC GFP ACE2 , a549-ascov2 , Invivogen.

    Techniques: Activation Assay, Lysis, Bacteria, Co-Culture Assay, Western Blot, CRISPR, Positive Control, Expressing, Microscopy, Infection, Membrane, Cell Culture, Control

    P. aeruginosa –activated hNLRP1 inflammasome requires proteasome activity. (A) Fluorescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to 1 × 10 5 P. aeruginosa clinical isolates from patients with infected lung (strains 3039 1533 and 2348 4390) or with infected cornea (strain 0236 1921) for 24 h. ASC-GFP (green) pictures were taken in the dish after infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from each experiment were analyzed. Values are expressed as mean ± SEM. One-way ANOVA. (B) Schematic drawing of P. aeruginosa co-culture experiments performed with human corneal epithelial cells. (C) Immunoblotting of NLRP1, Gasdermin-D, and Tubulin in pHCECs upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h in presence/absence of proteasome inhibitor bortezomib. Immunoblots show lysates from one experiment performed at least three times. (D) Cell lysis (LDH) and IL-1B release evaluation in pHCECs and pHNECs, upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h in presence/absence of proteasome inhibitor bortezomib. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM from one experiment (in triplicate) performed at least three times. Source data are available for this figure: .

    Journal: The Journal of Experimental Medicine

    Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

    doi: 10.1084/jem.20230104

    Figure Lengend Snippet: P. aeruginosa –activated hNLRP1 inflammasome requires proteasome activity. (A) Fluorescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to 1 × 10 5 P. aeruginosa clinical isolates from patients with infected lung (strains 3039 1533 and 2348 4390) or with infected cornea (strain 0236 1921) for 24 h. ASC-GFP (green) pictures were taken in the dish after infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from each experiment were analyzed. Values are expressed as mean ± SEM. One-way ANOVA. (B) Schematic drawing of P. aeruginosa co-culture experiments performed with human corneal epithelial cells. (C) Immunoblotting of NLRP1, Gasdermin-D, and Tubulin in pHCECs upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h in presence/absence of proteasome inhibitor bortezomib. Immunoblots show lysates from one experiment performed at least three times. (D) Cell lysis (LDH) and IL-1B release evaluation in pHCECs and pHNECs, upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h in presence/absence of proteasome inhibitor bortezomib. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM from one experiment (in triplicate) performed at least three times. Source data are available for this figure: .

    Article Snippet: A549 ASC GFP ACE2 , a549-ascov2 , Invivogen.

    Techniques: Activity Assay, Fluorescence, Microscopy, Infection, Co-Culture Assay, Western Blot, Bacteria, Lysis

    P. aeruginosa EEF2-inactivating EXOA promotes NLRP1 inflammasome response. (A) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to 1 × 10 5 P. aeruginosa (PAO1) and associated isogenic mutants for various secretion systems (PAO1 ΔT3SS , PAO1 ΔT2SS , PAO1 ΔT1SS ) for 24 h. ASC-GFP (green) pictures were taken in the dish after infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (B) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to 1 × 10 5 P. aeruginosa (PAO1) and associated isogenic mutants for various T2SS virulence effectors (PAO1 ΔPLCN , PAO1 ΔPLCH , PAO1 ΔLASB , and PAO1 ΔEXOA ) for 24 h. ASC-GFP (green) pictures were taken in the dish after infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nulcei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (C) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to EXOA (10 ng/ml) or its catalytically dead mutant EXOA H426A (500 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (D) Schematic mechanism of P. aeruginosa EXOA and related toxins at mediating EEF2 ribosylation and inactivation and subsequent ribosome inactivation. (E) Immunoblotting characterization of genetic invalidation of DPH1 in A549 NLRP1+/ASC-GFP cells using CRISPR-Cas9. The red arrow shows the selected KO cells for subsequent experiments. (F) Fluorescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/ DPH1− reporter cell lines exposed to VbP (15 µM), EXOA (10 ng/ml), cholix toxin (CT, 10 ng/ml), and diphtheria toxin (DT, 20 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (G) Plasma membrane permeabilization determination over time using PI incorporation in WT or NLRP1 -deficient pHCECs exposed to VbP (15 µM), EXOA (10 ng/ml) or EXOA H426A (10 ng/ml) for indicated times. ***P ≤ 0.001, T test. Values are expressed as mean ± SEM from one experiment (in triplicate) performed at least three times. Source data are available for this figure: .

    Journal: The Journal of Experimental Medicine

    Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

    doi: 10.1084/jem.20230104

    Figure Lengend Snippet: P. aeruginosa EEF2-inactivating EXOA promotes NLRP1 inflammasome response. (A) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to 1 × 10 5 P. aeruginosa (PAO1) and associated isogenic mutants for various secretion systems (PAO1 ΔT3SS , PAO1 ΔT2SS , PAO1 ΔT1SS ) for 24 h. ASC-GFP (green) pictures were taken in the dish after infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (B) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to 1 × 10 5 P. aeruginosa (PAO1) and associated isogenic mutants for various T2SS virulence effectors (PAO1 ΔPLCN , PAO1 ΔPLCH , PAO1 ΔLASB , and PAO1 ΔEXOA ) for 24 h. ASC-GFP (green) pictures were taken in the dish after infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nulcei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (C) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP reporter cell lines exposed to EXOA (10 ng/ml) or its catalytically dead mutant EXOA H426A (500 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (D) Schematic mechanism of P. aeruginosa EXOA and related toxins at mediating EEF2 ribosylation and inactivation and subsequent ribosome inactivation. (E) Immunoblotting characterization of genetic invalidation of DPH1 in A549 NLRP1+/ASC-GFP cells using CRISPR-Cas9. The red arrow shows the selected KO cells for subsequent experiments. (F) Fluorescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/ DPH1− reporter cell lines exposed to VbP (15 µM), EXOA (10 ng/ml), cholix toxin (CT, 10 ng/ml), and diphtheria toxin (DT, 20 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (G) Plasma membrane permeabilization determination over time using PI incorporation in WT or NLRP1 -deficient pHCECs exposed to VbP (15 µM), EXOA (10 ng/ml) or EXOA H426A (10 ng/ml) for indicated times. ***P ≤ 0.001, T test. Values are expressed as mean ± SEM from one experiment (in triplicate) performed at least three times. Source data are available for this figure: .

    Article Snippet: A549 ASC GFP ACE2 , a549-ascov2 , Invivogen.

    Techniques: Microscopy, Infection, Mutagenesis, Western Blot, CRISPR, Fluorescence, Membrane

    Multiple EEF2-targeting toxins activate the hNLRP1 inflammasome in a ZAKα-dependent manner. (A) Schematic mechanism of EXOA and related toxin-mediated translation inhibition. tRNA, transfer RNA; E, exit; P, peptidyl; A, aminoacyl. (B) Determination of ribosome inactivation in A549 NLRP1+/ASC-GFP and A549 NLRP1−/ASC-GFP reporter cell lines exposed to EXOA (10 ng/ml) for 2 and 6 h by measuring ribosome polysome accumulation and puromycin incorporation. Images are representative of one experiment performed at least three times. (C) Immunoblotting of ADP-ribosylated proteins, EEF2, and Tubulin in A549 NLRP1+/ASC-GFP cell lysates treated or not with VbP (15 µM) or EXOA (10 ng/ml) in the presence of Nicotinamide adenine dinucleotide-Biotin (NAD-Biot). Immunoblots show lysates from one experiment performed at least three times. (D) Immunoblotting of NLRP1, Tubulin, and phosphorylated P38 and JNK in A549 NLRP1+ and A549 NLRP1− reporter cell lines exposed or not to EXOA (10 ng/ml) or its inactive mutant EXOA H426A for 3 h. Immunoblots show lysates from one experiment performed at least three times. (E) Immunoblotting characterization of genetic invalidation of P38α and P38β in A549 NLRP1+/ASC-GFP cells using CRISPR-Cas9. Immunoblots show lysates from one experiment performed at least three times. (F) Fluorescence microscopy of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/ ZAKα - reporter cell lines expressing hACE2 infected for 24 h with various SARS-CoV-2 MOI. ASC-GFP (green) pictures were taken in the dish after viral infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. Source data are available for this figure: .

    Journal: The Journal of Experimental Medicine

    Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

    doi: 10.1084/jem.20230104

    Figure Lengend Snippet: Multiple EEF2-targeting toxins activate the hNLRP1 inflammasome in a ZAKα-dependent manner. (A) Schematic mechanism of EXOA and related toxin-mediated translation inhibition. tRNA, transfer RNA; E, exit; P, peptidyl; A, aminoacyl. (B) Determination of ribosome inactivation in A549 NLRP1+/ASC-GFP and A549 NLRP1−/ASC-GFP reporter cell lines exposed to EXOA (10 ng/ml) for 2 and 6 h by measuring ribosome polysome accumulation and puromycin incorporation. Images are representative of one experiment performed at least three times. (C) Immunoblotting of ADP-ribosylated proteins, EEF2, and Tubulin in A549 NLRP1+/ASC-GFP cell lysates treated or not with VbP (15 µM) or EXOA (10 ng/ml) in the presence of Nicotinamide adenine dinucleotide-Biotin (NAD-Biot). Immunoblots show lysates from one experiment performed at least three times. (D) Immunoblotting of NLRP1, Tubulin, and phosphorylated P38 and JNK in A549 NLRP1+ and A549 NLRP1− reporter cell lines exposed or not to EXOA (10 ng/ml) or its inactive mutant EXOA H426A for 3 h. Immunoblots show lysates from one experiment performed at least three times. (E) Immunoblotting characterization of genetic invalidation of P38α and P38β in A549 NLRP1+/ASC-GFP cells using CRISPR-Cas9. Immunoblots show lysates from one experiment performed at least three times. (F) Fluorescence microscopy of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/ ZAKα - reporter cell lines expressing hACE2 infected for 24 h with various SARS-CoV-2 MOI. ASC-GFP (green) pictures were taken in the dish after viral infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. Source data are available for this figure: .

    Article Snippet: A549 ASC GFP ACE2 , a549-ascov2 , Invivogen.

    Techniques: Inhibition, Western Blot, TNKS1 Histone Ribosylation Assay, Mutagenesis, CRISPR, Fluorescence, Microscopy, Expressing, Infection

    EEF2 inactivation drives ZAKα and P38 MAPK activation and subsequent NLRP1 inflammasome nucleation. (A) Immunoblotting of P38, JNK, ZAKα, NLRP1, Tubulin, and phosphorylated P38 and JNK in A549 NLRP1+ and A549 NLRP1+/ZAKα- reporter cell lines exposed or not to EXOA (10 ng/ml) for 3 h. Immunoblots show lysates from one experiment performed at least three times. (B) Cell lysis (LDH release), florescence microscopy, and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/ZAKα- reporter cell lines exposed to EXOA (10 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positives for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. (C) Cell lysis (LDH release), fluorescence microscopy, and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/P38α/β- reporter cell lines exposed to EXOA (10 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. Graphs show one experiment performed in triplicate at least three times. (D) Western blot examination of NLRP1 using an anti-NLRP1 N-terminal antibody (aa 1–323) in A549 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for 112 TST 114 / 112 AAA 114 or 178 TST 180 / 178 AAA 180 after 4 h exposure to EXOA (10 ng/ml) or VbP (15 µM). Images shown are from one experiment and are representative of n = 3 independent experiments. (E) Cell lysis (LDH release), fluorescence microscopy, and associated quantifications of ASC-GFP specks in A549 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for 112 TST 114 / 112 AAA 114 or 178 TST 180 / 178 AAA 180 after 10 h exposure to EXOA (10 ng/ml) or VbP (15 µM). ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times. Source data are available for this figure: .

    Journal: The Journal of Experimental Medicine

    Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

    doi: 10.1084/jem.20230104

    Figure Lengend Snippet: EEF2 inactivation drives ZAKα and P38 MAPK activation and subsequent NLRP1 inflammasome nucleation. (A) Immunoblotting of P38, JNK, ZAKα, NLRP1, Tubulin, and phosphorylated P38 and JNK in A549 NLRP1+ and A549 NLRP1+/ZAKα- reporter cell lines exposed or not to EXOA (10 ng/ml) for 3 h. Immunoblots show lysates from one experiment performed at least three times. (B) Cell lysis (LDH release), florescence microscopy, and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/ZAKα- reporter cell lines exposed to EXOA (10 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positives for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. (C) Cell lysis (LDH release), fluorescence microscopy, and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1+/ASC-GFP/P38α/β- reporter cell lines exposed to EXOA (10 ng/ml) for 10 h. ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. Graphs show one experiment performed in triplicate at least three times. (D) Western blot examination of NLRP1 using an anti-NLRP1 N-terminal antibody (aa 1–323) in A549 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for 112 TST 114 / 112 AAA 114 or 178 TST 180 / 178 AAA 180 after 4 h exposure to EXOA (10 ng/ml) or VbP (15 µM). Images shown are from one experiment and are representative of n = 3 independent experiments. (E) Cell lysis (LDH release), fluorescence microscopy, and associated quantifications of ASC-GFP specks in A549 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for 112 TST 114 / 112 AAA 114 or 178 TST 180 / 178 AAA 180 after 10 h exposure to EXOA (10 ng/ml) or VbP (15 µM). ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times. Source data are available for this figure: .

    Article Snippet: A549 ASC GFP ACE2 , a549-ascov2 , Invivogen.

    Techniques: Activation Assay, Western Blot, Lysis, Microscopy, Fluorescence, Plasmid Preparation, Construct

    List of reagents used in the study

    Journal: The Journal of Experimental Medicine

    Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

    doi: 10.1084/jem.20230104

    Figure Lengend Snippet: List of reagents used in the study

    Article Snippet: A549 ASC GFP ACE2 , a549-ascov2 , Invivogen.

    Techniques: Protease Inhibitor, Staining, Marker, CyQUANT Assay, Mutagenesis, Membrane, Recombinant