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human primary keratinocytes  (PromoCell)


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    PromoCell human primary keratinocytes
    A, B. Screening methodology, associated quantifications of ASC-GFP specks in HEK293T cells individually expressing or not NLRP1 and IL-1β release in <t>NTERT-keratinocytes</t> (WT and NLRP1KO) exposed to 1µM of compounds from the phosphatase screening library for 8 h. ASC-GFP (green) pictures were taken with an EVOS7000 after adding Hoechst (nuclei staining) directly in medium. The percentage of ASC complex was performed by determining the ratios between cells positive for ASC speckles and the total of cell nuclei (Hoechst) by automatic fluorescence microscopy. At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of GSDMD and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM) and Cantharidin (Canth. 5µM). Immunoblots show combined lysates+supernatants from one experiment performed at least three times. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Immunoblotting of PP1 and PP2A catalytic subunits (PP1Cα or PP1Cγ and PP2Acα or PP2Acβ) and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes 24 hours after CRISPR-Cas9 (RNP)-mediated PP1/PP2A catalytic subunit invalidation. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times.
    Human Primary Keratinocytes, supplied by PromoCell, used in various techniques. Bioz Stars score: 97/100, based on 507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human primary keratinocytes/product/PromoCell
    Average 97 stars, based on 507 article reviews
    human primary keratinocytes - by Bioz Stars, 2026-02
    97/100 stars

    Images

    1) Product Images from "A TAK1-Driven NLRP1 Inflammasome Pathway Revealed by Phosphatase-Targeting Environmental Toxins"

    Article Title: A TAK1-Driven NLRP1 Inflammasome Pathway Revealed by Phosphatase-Targeting Environmental Toxins

    Journal: bioRxiv

    doi: 10.64898/2026.01.23.701233

    A, B. Screening methodology, associated quantifications of ASC-GFP specks in HEK293T cells individually expressing or not NLRP1 and IL-1β release in NTERT-keratinocytes (WT and NLRP1KO) exposed to 1µM of compounds from the phosphatase screening library for 8 h. ASC-GFP (green) pictures were taken with an EVOS7000 after adding Hoechst (nuclei staining) directly in medium. The percentage of ASC complex was performed by determining the ratios between cells positive for ASC speckles and the total of cell nuclei (Hoechst) by automatic fluorescence microscopy. At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of GSDMD and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM) and Cantharidin (Canth. 5µM). Immunoblots show combined lysates+supernatants from one experiment performed at least three times. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Immunoblotting of PP1 and PP2A catalytic subunits (PP1Cα or PP1Cγ and PP2Acα or PP2Acβ) and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes 24 hours after CRISPR-Cas9 (RNP)-mediated PP1/PP2A catalytic subunit invalidation. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times.
    Figure Legend Snippet: A, B. Screening methodology, associated quantifications of ASC-GFP specks in HEK293T cells individually expressing or not NLRP1 and IL-1β release in NTERT-keratinocytes (WT and NLRP1KO) exposed to 1µM of compounds from the phosphatase screening library for 8 h. ASC-GFP (green) pictures were taken with an EVOS7000 after adding Hoechst (nuclei staining) directly in medium. The percentage of ASC complex was performed by determining the ratios between cells positive for ASC speckles and the total of cell nuclei (Hoechst) by automatic fluorescence microscopy. At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of GSDMD and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM) and Cantharidin (Canth. 5µM). Immunoblots show combined lysates+supernatants from one experiment performed at least three times. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Immunoblotting of PP1 and PP2A catalytic subunits (PP1Cα or PP1Cγ and PP2Acα or PP2Acβ) and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes 24 hours after CRISPR-Cas9 (RNP)-mediated PP1/PP2A catalytic subunit invalidation. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times.

    Techniques Used: Expressing, Staining, Fluorescence, Microscopy, Western Blot, CRISPR

    A. Fluorescence microscopy of ASC-GFP specks in WT HEK293T ASC-GFP/NLRP1 reporter cells exposed to selected phosphatase inhibitory compounds for 8 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). B. Fluorescence microscopy of ASC-GFP specks in multiple HEK293T ASC-GFP reporter cells expressing NLRP1, NLRP10, PYRIN or NLRP3 inflammasome-forming sensors and exposed to selected Dinophysistoxin (100nM) compounds for 8 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). C. Cytokine analysis 8 h after exposure of WT and NLRP1KO TERT keratinocytes to Dinophysistoxin (100nM), Okadaic acid (250nM) and Cantharidin (5µM). Representative experiment of three independent replicates. D. Determination of IL-1β release in WT NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM) in presence/absence of bortezomib (1µM). ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times.
    Figure Legend Snippet: A. Fluorescence microscopy of ASC-GFP specks in WT HEK293T ASC-GFP/NLRP1 reporter cells exposed to selected phosphatase inhibitory compounds for 8 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). B. Fluorescence microscopy of ASC-GFP specks in multiple HEK293T ASC-GFP reporter cells expressing NLRP1, NLRP10, PYRIN or NLRP3 inflammasome-forming sensors and exposed to selected Dinophysistoxin (100nM) compounds for 8 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). C. Cytokine analysis 8 h after exposure of WT and NLRP1KO TERT keratinocytes to Dinophysistoxin (100nM), Okadaic acid (250nM) and Cantharidin (5µM). Representative experiment of three independent replicates. D. Determination of IL-1β release in WT NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM) in presence/absence of bortezomib (1µM). ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times.

    Techniques Used: Fluorescence, Microscopy, Staining, Expressing

    A. Schematic representation of the mechanism of ZAKα/P38 stress kinases activation upon induction of Ribotoxic Stress Response (RSR). B. Phosphotag blotting of phosphorylated NLRP1 disordered Region (DR) in HEK293T expressing the NLRP1 DR construct (aa 86-275-GFP (described in A )) and exposed to all PP1/PP2A-targeting compounds identified in /B ) or to the known RSR inducer Anisomycin (1 µg/mL) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. C. Phosphotag blotting of phosphorylated full length NLRP1 in primary human keratinocytes exposed Dinophysis toxin (100 nM) for various time. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. D. Phosphotag blotting of phosphorylated ZAKα and NLRP1 disordered Region (DR) in WT or ZAK KO NTERT NLRP1 KO + 86-275-SNAP keratinocytes exposed to Dinophysis toxin (100nM), Cantharidin (5%M) or Val-boro-Pro (VbP, 10µM) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Plasma membrane permeabilization (SYTOX Green incorporation, 16 h) and IL-1β release evaluation (10 h) in WT, ZAKα or NLRP1 KO NTERT keratinocytes after exposure to Dinophysis toxin (100nM), Cantharidin (5%M), Okadaic acid (250nM) or Anisomycin (1µM). ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. F. Immunoblotting and clonal selection (clone 3, red), fluorescence microscopy and associated quantifications of ASC-GFP specks in WT or ZAKα KO HEK293T ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or Anisomycin (1µM) for 5 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA.
    Figure Legend Snippet: A. Schematic representation of the mechanism of ZAKα/P38 stress kinases activation upon induction of Ribotoxic Stress Response (RSR). B. Phosphotag blotting of phosphorylated NLRP1 disordered Region (DR) in HEK293T expressing the NLRP1 DR construct (aa 86-275-GFP (described in A )) and exposed to all PP1/PP2A-targeting compounds identified in /B ) or to the known RSR inducer Anisomycin (1 µg/mL) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. C. Phosphotag blotting of phosphorylated full length NLRP1 in primary human keratinocytes exposed Dinophysis toxin (100 nM) for various time. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. D. Phosphotag blotting of phosphorylated ZAKα and NLRP1 disordered Region (DR) in WT or ZAK KO NTERT NLRP1 KO + 86-275-SNAP keratinocytes exposed to Dinophysis toxin (100nM), Cantharidin (5%M) or Val-boro-Pro (VbP, 10µM) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Plasma membrane permeabilization (SYTOX Green incorporation, 16 h) and IL-1β release evaluation (10 h) in WT, ZAKα or NLRP1 KO NTERT keratinocytes after exposure to Dinophysis toxin (100nM), Cantharidin (5%M), Okadaic acid (250nM) or Anisomycin (1µM). ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. F. Immunoblotting and clonal selection (clone 3, red), fluorescence microscopy and associated quantifications of ASC-GFP specks in WT or ZAKα KO HEK293T ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or Anisomycin (1µM) for 5 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA.

    Techniques Used: Activation Assay, Expressing, Construct, Western Blot, Clinical Proteomics, Membrane, Selection, Fluorescence, Microscopy, Staining

    A. Pamgene analysis of activated Serine/threonine kinases in primary human keratinocytes exposed to Dinophysis toxin (100nM) for 2 hours and subsequent determination of IL-1β release in WT NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) or Anisomycin (1µM) in presence/absence of inhibitors of identified kinases in. For all kinases, inhibitors were used at 10µM. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least two times. B. Phosphotag blotting of phosphorylated P38 kinase isoforms in NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour in presence/absence of the Pan P38 inhibitor Doramapimod (10µM). Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. C. Immunoblotting of P38, ZAKα, NLRP1 and phosphorylated P38 kinases in WT, ZAKα KO or NLRP1 KO NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. D. Immunoblotting characterization of the P38 isoform genetic knockdown (CRISPR-Cas9) and of the subsequent IL-1β release in WT, P38δ KO, P38α/β dKO, or P38α/β/δ TKO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) or Anisomycin (1µM) for 8 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. E. Western blot showing NLRP1 (anti-NLRP1 N-terminal antibody (aa 1–323)) and associated fluorescence microscopy/quantifications of ASC-GFP specks in HEK293 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for important 38 phosphorylation sites (S107A, TST112-114AAA and TST178-180AAA) after 10 h of exposure to Dinophysis toxin (100nM), Okadaic acid (250nM), Cantharidin (5µM) or Val-boro-Pro (VbP, 10µM). ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of three 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 ten fields from three independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times.
    Figure Legend Snippet: A. Pamgene analysis of activated Serine/threonine kinases in primary human keratinocytes exposed to Dinophysis toxin (100nM) for 2 hours and subsequent determination of IL-1β release in WT NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) or Anisomycin (1µM) in presence/absence of inhibitors of identified kinases in. For all kinases, inhibitors were used at 10µM. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least two times. B. Phosphotag blotting of phosphorylated P38 kinase isoforms in NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour in presence/absence of the Pan P38 inhibitor Doramapimod (10µM). Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. C. Immunoblotting of P38, ZAKα, NLRP1 and phosphorylated P38 kinases in WT, ZAKα KO or NLRP1 KO NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. D. Immunoblotting characterization of the P38 isoform genetic knockdown (CRISPR-Cas9) and of the subsequent IL-1β release in WT, P38δ KO, P38α/β dKO, or P38α/β/δ TKO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) or Anisomycin (1µM) for 8 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. E. Western blot showing NLRP1 (anti-NLRP1 N-terminal antibody (aa 1–323)) and associated fluorescence microscopy/quantifications of ASC-GFP specks in HEK293 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for important 38 phosphorylation sites (S107A, TST112-114AAA and TST178-180AAA) after 10 h of exposure to Dinophysis toxin (100nM), Okadaic acid (250nM), Cantharidin (5µM) or Val-boro-Pro (VbP, 10µM). ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of three 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 ten fields from three independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times.

    Techniques Used: Western Blot, Knockdown, CRISPR, Fluorescence, Microscopy, Plasmid Preparation, Construct, Phospho-proteomics

    A. Quantifications of ASC-GFP specks in HEK293 ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or not for 6 h in presence or absence of various MAPK inhibitors (10µM). TAK1 inhibitor; HS-276, ZAKα inhibitor; PLX4720, TAOK inhibitor; CP-43, MLKL inhibitor; necro sulfonamide, ASK1 inhibitor; GS-444217, DLK/LZK inhibitor; DN-1289, RIPK3 inhibitor; GSK-872. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three 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 ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. B. Determination of the IL-1β release in WT, P38α/β/δ TKO, TAK1 KO and NLRP1 KO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM), Anisomycin (1µM) and VbP (10µM) for 10 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of P38, TAK1, cleaved GSDMD and IL-1β and of the subsequent IL-1β release in WT, P38α/β/δ TKO, TAK1 KO, P38α/β/δ TKO/TAK1 KO and NLRP1 KO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM), Anisomycin (1µM) and VbP (10µM) for 10 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Phosphotag blotting of phosphorylated P38, TAK1 and NLRP1-DR-SNAP in WT, P38α/β/δ TKO, TAK1 KO or P38α/β/δ TKO/TAK1 KO NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Phosphotag immunoblotting of phosphorylated recombinant NLRP1 full-length protein or immunoprecipitated GFP-tagged NLRP1-DR incubated with recombinant TAK1-TAB1 fusion or P38α kinases for 60 minutes in presence/absence of lambda phosphatase. Immunoblots show proteins from one experiment performed at least three times. F. Fluorescence microscopy quantifications of ASC-GFP specks in WT, P38α/β/δ TKO or in P38α/β/δ TKO/TAK1 KO HEK293 ASC-GFP/NLRP1 reporter cells after 6 h of exposure to Dinophysis toxin (100nM) or Val-boro-Pro (VbP, 10µM). ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least ten fields from three independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times.
    Figure Legend Snippet: A. Quantifications of ASC-GFP specks in HEK293 ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or not for 6 h in presence or absence of various MAPK inhibitors (10µM). TAK1 inhibitor; HS-276, ZAKα inhibitor; PLX4720, TAOK inhibitor; CP-43, MLKL inhibitor; necro sulfonamide, ASK1 inhibitor; GS-444217, DLK/LZK inhibitor; DN-1289, RIPK3 inhibitor; GSK-872. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three 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 ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. B. Determination of the IL-1β release in WT, P38α/β/δ TKO, TAK1 KO and NLRP1 KO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM), Anisomycin (1µM) and VbP (10µM) for 10 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of P38, TAK1, cleaved GSDMD and IL-1β and of the subsequent IL-1β release in WT, P38α/β/δ TKO, TAK1 KO, P38α/β/δ TKO/TAK1 KO and NLRP1 KO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM), Anisomycin (1µM) and VbP (10µM) for 10 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Phosphotag blotting of phosphorylated P38, TAK1 and NLRP1-DR-SNAP in WT, P38α/β/δ TKO, TAK1 KO or P38α/β/δ TKO/TAK1 KO NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Phosphotag immunoblotting of phosphorylated recombinant NLRP1 full-length protein or immunoprecipitated GFP-tagged NLRP1-DR incubated with recombinant TAK1-TAB1 fusion or P38α kinases for 60 minutes in presence/absence of lambda phosphatase. Immunoblots show proteins from one experiment performed at least three times. F. Fluorescence microscopy quantifications of ASC-GFP specks in WT, P38α/β/δ TKO or in P38α/β/δ TKO/TAK1 KO HEK293 ASC-GFP/NLRP1 reporter cells after 6 h of exposure to Dinophysis toxin (100nM) or Val-boro-Pro (VbP, 10µM). ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least ten fields from three independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times.

    Techniques Used: Staining, Western Blot, Recombinant, Immunoprecipitation, Incubation, Fluorescence, Microscopy



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    ATCC primary human epidermal kcs
    A, B. Screening methodology, associated quantifications of ASC-GFP specks in HEK293T cells individually expressing or not NLRP1 and IL-1β release in <t>NTERT-keratinocytes</t> (WT and NLRP1KO) exposed to 1µM of compounds from the phosphatase screening library for 8 h. ASC-GFP (green) pictures were taken with an EVOS7000 after adding Hoechst (nuclei staining) directly in medium. The percentage of ASC complex was performed by determining the ratios between cells positive for ASC speckles and the total of cell nuclei (Hoechst) by automatic fluorescence microscopy. At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of GSDMD and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM) and Cantharidin (Canth. 5µM). Immunoblots show combined lysates+supernatants from one experiment performed at least three times. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Immunoblotting of PP1 and PP2A catalytic subunits (PP1Cα or PP1Cγ and PP2Acα or PP2Acβ) and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes 24 hours after CRISPR-Cas9 (RNP)-mediated PP1/PP2A catalytic subunit invalidation. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times.
    Primary Human Epidermal Kcs, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human oral keratinocytes hoks  (ATCC)
    96
    ATCC human oral keratinocytes hoks
    A, B. Screening methodology, associated quantifications of ASC-GFP specks in HEK293T cells individually expressing or not NLRP1 and IL-1β release in <t>NTERT-keratinocytes</t> (WT and NLRP1KO) exposed to 1µM of compounds from the phosphatase screening library for 8 h. ASC-GFP (green) pictures were taken with an EVOS7000 after adding Hoechst (nuclei staining) directly in medium. The percentage of ASC complex was performed by determining the ratios between cells positive for ASC speckles and the total of cell nuclei (Hoechst) by automatic fluorescence microscopy. At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of GSDMD and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM) and Cantharidin (Canth. 5µM). Immunoblots show combined lysates+supernatants from one experiment performed at least three times. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Immunoblotting of PP1 and PP2A catalytic subunits (PP1Cα or PP1Cγ and PP2Acα or PP2Acβ) and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes 24 hours after CRISPR-Cas9 (RNP)-mediated PP1/PP2A catalytic subunit invalidation. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times.
    Human Oral Keratinocytes Hoks, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nheks  (ATCC)
    99
    ATCC nheks
    A, B. Screening methodology, associated quantifications of ASC-GFP specks in HEK293T cells individually expressing or not NLRP1 and IL-1β release in <t>NTERT-keratinocytes</t> (WT and NLRP1KO) exposed to 1µM of compounds from the phosphatase screening library for 8 h. ASC-GFP (green) pictures were taken with an EVOS7000 after adding Hoechst (nuclei staining) directly in medium. The percentage of ASC complex was performed by determining the ratios between cells positive for ASC speckles and the total of cell nuclei (Hoechst) by automatic fluorescence microscopy. At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of GSDMD and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM) and Cantharidin (Canth. 5µM). Immunoblots show combined lysates+supernatants from one experiment performed at least three times. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Immunoblotting of PP1 and PP2A catalytic subunits (PP1Cα or PP1Cγ and PP2Acα or PP2Acβ) and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes 24 hours after CRISPR-Cas9 (RNP)-mediated PP1/PP2A catalytic subunit invalidation. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times.
    Nheks, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    adult nhpek  (ATCC)
    99
    ATCC adult nhpek
    A, B. Screening methodology, associated quantifications of ASC-GFP specks in HEK293T cells individually expressing or not NLRP1 and IL-1β release in <t>NTERT-keratinocytes</t> (WT and NLRP1KO) exposed to 1µM of compounds from the phosphatase screening library for 8 h. ASC-GFP (green) pictures were taken with an EVOS7000 after adding Hoechst (nuclei staining) directly in medium. The percentage of ASC complex was performed by determining the ratios between cells positive for ASC speckles and the total of cell nuclei (Hoechst) by automatic fluorescence microscopy. At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of GSDMD and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM) and Cantharidin (Canth. 5µM). Immunoblots show combined lysates+supernatants from one experiment performed at least three times. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Immunoblotting of PP1 and PP2A catalytic subunits (PP1Cα or PP1Cγ and PP2Acα or PP2Acβ) and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes 24 hours after CRISPR-Cas9 (RNP)-mediated PP1/PP2A catalytic subunit invalidation. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times.
    Adult Nhpek, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hacat cells  (ATCC)
    99
    ATCC hacat cells
    A, B. Screening methodology, associated quantifications of ASC-GFP specks in HEK293T cells individually expressing or not NLRP1 and IL-1β release in <t>NTERT-keratinocytes</t> (WT and NLRP1KO) exposed to 1µM of compounds from the phosphatase screening library for 8 h. ASC-GFP (green) pictures were taken with an EVOS7000 after adding Hoechst (nuclei staining) directly in medium. The percentage of ASC complex was performed by determining the ratios between cells positive for ASC speckles and the total of cell nuclei (Hoechst) by automatic fluorescence microscopy. At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of GSDMD and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM) and Cantharidin (Canth. 5µM). Immunoblots show combined lysates+supernatants from one experiment performed at least three times. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Immunoblotting of PP1 and PP2A catalytic subunits (PP1Cα or PP1Cγ and PP2Acα or PP2Acβ) and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes 24 hours after CRISPR-Cas9 (RNP)-mediated PP1/PP2A catalytic subunit invalidation. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times.
    Hacat Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pcs 200 011 hek293t atcc  (ATCC)
    99
    ATCC pcs 200 011 hek293t atcc
    A, B. Screening methodology, associated quantifications of ASC-GFP specks in HEK293T cells individually expressing or not NLRP1 and IL-1β release in <t>NTERT-keratinocytes</t> (WT and NLRP1KO) exposed to 1µM of compounds from the phosphatase screening library for 8 h. ASC-GFP (green) pictures were taken with an EVOS7000 after adding Hoechst (nuclei staining) directly in medium. The percentage of ASC complex was performed by determining the ratios between cells positive for ASC speckles and the total of cell nuclei (Hoechst) by automatic fluorescence microscopy. At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of GSDMD and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM) and Cantharidin (Canth. 5µM). Immunoblots show combined lysates+supernatants from one experiment performed at least three times. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Immunoblotting of PP1 and PP2A catalytic subunits (PP1Cα or PP1Cγ and PP2Acα or PP2Acβ) and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes 24 hours after CRISPR-Cas9 (RNP)-mediated PP1/PP2A catalytic subunit invalidation. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times.
    Pcs 200 011 Hek293t Atcc, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A, B. Screening methodology, associated quantifications of ASC-GFP specks in HEK293T cells individually expressing or not NLRP1 and IL-1β release in NTERT-keratinocytes (WT and NLRP1KO) exposed to 1µM of compounds from the phosphatase screening library for 8 h. ASC-GFP (green) pictures were taken with an EVOS7000 after adding Hoechst (nuclei staining) directly in medium. The percentage of ASC complex was performed by determining the ratios between cells positive for ASC speckles and the total of cell nuclei (Hoechst) by automatic fluorescence microscopy. At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of GSDMD and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM) and Cantharidin (Canth. 5µM). Immunoblots show combined lysates+supernatants from one experiment performed at least three times. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Immunoblotting of PP1 and PP2A catalytic subunits (PP1Cα or PP1Cγ and PP2Acα or PP2Acβ) and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes 24 hours after CRISPR-Cas9 (RNP)-mediated PP1/PP2A catalytic subunit invalidation. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times.

    Journal: bioRxiv

    Article Title: A TAK1-Driven NLRP1 Inflammasome Pathway Revealed by Phosphatase-Targeting Environmental Toxins

    doi: 10.64898/2026.01.23.701233

    Figure Lengend Snippet: A, B. Screening methodology, associated quantifications of ASC-GFP specks in HEK293T cells individually expressing or not NLRP1 and IL-1β release in NTERT-keratinocytes (WT and NLRP1KO) exposed to 1µM of compounds from the phosphatase screening library for 8 h. ASC-GFP (green) pictures were taken with an EVOS7000 after adding Hoechst (nuclei staining) directly in medium. The percentage of ASC complex was performed by determining the ratios between cells positive for ASC speckles and the total of cell nuclei (Hoechst) by automatic fluorescence microscopy. At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of GSDMD and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM) and Cantharidin (Canth. 5µM). Immunoblots show combined lysates+supernatants from one experiment performed at least three times. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Immunoblotting of PP1 and PP2A catalytic subunits (PP1Cα or PP1Cγ and PP2Acα or PP2Acβ) and analysis of the subsequent IL-1β release in WT and NLRP1-deficient NTERT-keratinocytes 24 hours after CRISPR-Cas9 (RNP)-mediated PP1/PP2A catalytic subunit invalidation. For cytokine release, ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times.

    Article Snippet: Human primary keratinocytes (pHEKs, PromoCell, C-12007) were cultivated in Keratinocyte Growth Medium 2 (PromoCell, C-20011).

    Techniques: Expressing, Staining, Fluorescence, Microscopy, Western Blot, CRISPR

    A. Fluorescence microscopy of ASC-GFP specks in WT HEK293T ASC-GFP/NLRP1 reporter cells exposed to selected phosphatase inhibitory compounds for 8 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). B. Fluorescence microscopy of ASC-GFP specks in multiple HEK293T ASC-GFP reporter cells expressing NLRP1, NLRP10, PYRIN or NLRP3 inflammasome-forming sensors and exposed to selected Dinophysistoxin (100nM) compounds for 8 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). C. Cytokine analysis 8 h after exposure of WT and NLRP1KO TERT keratinocytes to Dinophysistoxin (100nM), Okadaic acid (250nM) and Cantharidin (5µM). Representative experiment of three independent replicates. D. Determination of IL-1β release in WT NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM) in presence/absence of bortezomib (1µM). ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times.

    Journal: bioRxiv

    Article Title: A TAK1-Driven NLRP1 Inflammasome Pathway Revealed by Phosphatase-Targeting Environmental Toxins

    doi: 10.64898/2026.01.23.701233

    Figure Lengend Snippet: A. Fluorescence microscopy of ASC-GFP specks in WT HEK293T ASC-GFP/NLRP1 reporter cells exposed to selected phosphatase inhibitory compounds for 8 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). B. Fluorescence microscopy of ASC-GFP specks in multiple HEK293T ASC-GFP reporter cells expressing NLRP1, NLRP10, PYRIN or NLRP3 inflammasome-forming sensors and exposed to selected Dinophysistoxin (100nM) compounds for 8 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). C. Cytokine analysis 8 h after exposure of WT and NLRP1KO TERT keratinocytes to Dinophysistoxin (100nM), Okadaic acid (250nM) and Cantharidin (5µM). Representative experiment of three independent replicates. D. Determination of IL-1β release in WT NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM) in presence/absence of bortezomib (1µM). ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times.

    Article Snippet: Human primary keratinocytes (pHEKs, PromoCell, C-12007) were cultivated in Keratinocyte Growth Medium 2 (PromoCell, C-20011).

    Techniques: Fluorescence, Microscopy, Staining, Expressing

    A. Schematic representation of the mechanism of ZAKα/P38 stress kinases activation upon induction of Ribotoxic Stress Response (RSR). B. Phosphotag blotting of phosphorylated NLRP1 disordered Region (DR) in HEK293T expressing the NLRP1 DR construct (aa 86-275-GFP (described in A )) and exposed to all PP1/PP2A-targeting compounds identified in /B ) or to the known RSR inducer Anisomycin (1 µg/mL) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. C. Phosphotag blotting of phosphorylated full length NLRP1 in primary human keratinocytes exposed Dinophysis toxin (100 nM) for various time. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. D. Phosphotag blotting of phosphorylated ZAKα and NLRP1 disordered Region (DR) in WT or ZAK KO NTERT NLRP1 KO + 86-275-SNAP keratinocytes exposed to Dinophysis toxin (100nM), Cantharidin (5%M) or Val-boro-Pro (VbP, 10µM) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Plasma membrane permeabilization (SYTOX Green incorporation, 16 h) and IL-1β release evaluation (10 h) in WT, ZAKα or NLRP1 KO NTERT keratinocytes after exposure to Dinophysis toxin (100nM), Cantharidin (5%M), Okadaic acid (250nM) or Anisomycin (1µM). ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. F. Immunoblotting and clonal selection (clone 3, red), fluorescence microscopy and associated quantifications of ASC-GFP specks in WT or ZAKα KO HEK293T ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or Anisomycin (1µM) for 5 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA.

    Journal: bioRxiv

    Article Title: A TAK1-Driven NLRP1 Inflammasome Pathway Revealed by Phosphatase-Targeting Environmental Toxins

    doi: 10.64898/2026.01.23.701233

    Figure Lengend Snippet: A. Schematic representation of the mechanism of ZAKα/P38 stress kinases activation upon induction of Ribotoxic Stress Response (RSR). B. Phosphotag blotting of phosphorylated NLRP1 disordered Region (DR) in HEK293T expressing the NLRP1 DR construct (aa 86-275-GFP (described in A )) and exposed to all PP1/PP2A-targeting compounds identified in /B ) or to the known RSR inducer Anisomycin (1 µg/mL) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. C. Phosphotag blotting of phosphorylated full length NLRP1 in primary human keratinocytes exposed Dinophysis toxin (100 nM) for various time. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least two times. D. Phosphotag blotting of phosphorylated ZAKα and NLRP1 disordered Region (DR) in WT or ZAK KO NTERT NLRP1 KO + 86-275-SNAP keratinocytes exposed to Dinophysis toxin (100nM), Cantharidin (5%M) or Val-boro-Pro (VbP, 10µM) for an hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Plasma membrane permeabilization (SYTOX Green incorporation, 16 h) and IL-1β release evaluation (10 h) in WT, ZAKα or NLRP1 KO NTERT keratinocytes after exposure to Dinophysis toxin (100nM), Cantharidin (5%M), Okadaic acid (250nM) or Anisomycin (1µM). ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. F. Immunoblotting and clonal selection (clone 3, red), fluorescence microscopy and associated quantifications of ASC-GFP specks in WT or ZAKα KO HEK293T ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or Anisomycin (1µM) for 5 hours. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three independent experiments; scale bars, 50 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA.

    Article Snippet: Human primary keratinocytes (pHEKs, PromoCell, C-12007) were cultivated in Keratinocyte Growth Medium 2 (PromoCell, C-20011).

    Techniques: Activation Assay, Expressing, Construct, Western Blot, Clinical Proteomics, Membrane, Selection, Fluorescence, Microscopy, Staining

    A. Pamgene analysis of activated Serine/threonine kinases in primary human keratinocytes exposed to Dinophysis toxin (100nM) for 2 hours and subsequent determination of IL-1β release in WT NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) or Anisomycin (1µM) in presence/absence of inhibitors of identified kinases in. For all kinases, inhibitors were used at 10µM. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least two times. B. Phosphotag blotting of phosphorylated P38 kinase isoforms in NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour in presence/absence of the Pan P38 inhibitor Doramapimod (10µM). Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. C. Immunoblotting of P38, ZAKα, NLRP1 and phosphorylated P38 kinases in WT, ZAKα KO or NLRP1 KO NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. D. Immunoblotting characterization of the P38 isoform genetic knockdown (CRISPR-Cas9) and of the subsequent IL-1β release in WT, P38δ KO, P38α/β dKO, or P38α/β/δ TKO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) or Anisomycin (1µM) for 8 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. E. Western blot showing NLRP1 (anti-NLRP1 N-terminal antibody (aa 1–323)) and associated fluorescence microscopy/quantifications of ASC-GFP specks in HEK293 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for important 38 phosphorylation sites (S107A, TST112-114AAA and TST178-180AAA) after 10 h of exposure to Dinophysis toxin (100nM), Okadaic acid (250nM), Cantharidin (5µM) or Val-boro-Pro (VbP, 10µM). ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of three 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 ten fields from three independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times.

    Journal: bioRxiv

    Article Title: A TAK1-Driven NLRP1 Inflammasome Pathway Revealed by Phosphatase-Targeting Environmental Toxins

    doi: 10.64898/2026.01.23.701233

    Figure Lengend Snippet: A. Pamgene analysis of activated Serine/threonine kinases in primary human keratinocytes exposed to Dinophysis toxin (100nM) for 2 hours and subsequent determination of IL-1β release in WT NTERT-keratinocytes after 8 h exposure to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) or Anisomycin (1µM) in presence/absence of inhibitors of identified kinases in. For all kinases, inhibitors were used at 10µM. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least two times. B. Phosphotag blotting of phosphorylated P38 kinase isoforms in NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour in presence/absence of the Pan P38 inhibitor Doramapimod (10µM). Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. C. Immunoblotting of P38, ZAKα, NLRP1 and phosphorylated P38 kinases in WT, ZAKα KO or NLRP1 KO NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. D. Immunoblotting characterization of the P38 isoform genetic knockdown (CRISPR-Cas9) and of the subsequent IL-1β release in WT, P38δ KO, P38α/β dKO, or P38α/β/δ TKO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) or Anisomycin (1µM) for 8 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. E. Western blot showing NLRP1 (anti-NLRP1 N-terminal antibody (aa 1–323)) and associated fluorescence microscopy/quantifications of ASC-GFP specks in HEK293 ASC-GFP reporter cells reconstituted with hNLRP1 or hNLRP1 plasmid constructs mutated for important 38 phosphorylation sites (S107A, TST112-114AAA and TST178-180AAA) after 10 h of exposure to Dinophysis toxin (100nM), Okadaic acid (250nM), Cantharidin (5µM) or Val-boro-Pro (VbP, 10µM). ASC-GFP (green) pictures were taken in the dish after toxin exposure. Images shown are from one experiment and are representative of three 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 ten fields from three independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times.

    Article Snippet: Human primary keratinocytes (pHEKs, PromoCell, C-12007) were cultivated in Keratinocyte Growth Medium 2 (PromoCell, C-20011).

    Techniques: Western Blot, Knockdown, CRISPR, Fluorescence, Microscopy, Plasmid Preparation, Construct, Phospho-proteomics

    A. Quantifications of ASC-GFP specks in HEK293 ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or not for 6 h in presence or absence of various MAPK inhibitors (10µM). TAK1 inhibitor; HS-276, ZAKα inhibitor; PLX4720, TAOK inhibitor; CP-43, MLKL inhibitor; necro sulfonamide, ASK1 inhibitor; GS-444217, DLK/LZK inhibitor; DN-1289, RIPK3 inhibitor; GSK-872. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three 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 ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. B. Determination of the IL-1β release in WT, P38α/β/δ TKO, TAK1 KO and NLRP1 KO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM), Anisomycin (1µM) and VbP (10µM) for 10 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of P38, TAK1, cleaved GSDMD and IL-1β and of the subsequent IL-1β release in WT, P38α/β/δ TKO, TAK1 KO, P38α/β/δ TKO/TAK1 KO and NLRP1 KO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM), Anisomycin (1µM) and VbP (10µM) for 10 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Phosphotag blotting of phosphorylated P38, TAK1 and NLRP1-DR-SNAP in WT, P38α/β/δ TKO, TAK1 KO or P38α/β/δ TKO/TAK1 KO NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Phosphotag immunoblotting of phosphorylated recombinant NLRP1 full-length protein or immunoprecipitated GFP-tagged NLRP1-DR incubated with recombinant TAK1-TAB1 fusion or P38α kinases for 60 minutes in presence/absence of lambda phosphatase. Immunoblots show proteins from one experiment performed at least three times. F. Fluorescence microscopy quantifications of ASC-GFP specks in WT, P38α/β/δ TKO or in P38α/β/δ TKO/TAK1 KO HEK293 ASC-GFP/NLRP1 reporter cells after 6 h of exposure to Dinophysis toxin (100nM) or Val-boro-Pro (VbP, 10µM). ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least ten fields from three independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times.

    Journal: bioRxiv

    Article Title: A TAK1-Driven NLRP1 Inflammasome Pathway Revealed by Phosphatase-Targeting Environmental Toxins

    doi: 10.64898/2026.01.23.701233

    Figure Lengend Snippet: A. Quantifications of ASC-GFP specks in HEK293 ASC-GFP/NLRP1 reporter cells exposed to Dinophysis toxin (100nM) or not for 6 h in presence or absence of various MAPK inhibitors (10µM). TAK1 inhibitor; HS-276, ZAKα inhibitor; PLX4720, TAOK inhibitor; CP-43, MLKL inhibitor; necro sulfonamide, ASK1 inhibitor; GS-444217, DLK/LZK inhibitor; DN-1289, RIPK3 inhibitor; GSK-872. ASC-GFP (green) pictures were directly taken in dish after adding Hoechst (nuclei staining). Images shown are from one experiment and are representative of three 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 ten fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, one-way ANOVA. B. Determination of the IL-1β release in WT, P38α/β/δ TKO, TAK1 KO and NLRP1 KO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM), Anisomycin (1µM) and VbP (10µM) for 10 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. C. Immunoblotting of P38, TAK1, cleaved GSDMD and IL-1β and of the subsequent IL-1β release in WT, P38α/β/δ TKO, TAK1 KO, P38α/β/δ TKO/TAK1 KO and NLRP1 KO NTERT keratinocytes exposed or not to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM), Anisomycin (1µM) and VbP (10µM) for 10 hours. ***P ≤ 0.0001, one-way ANOVA. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. D. Phosphotag blotting of phosphorylated P38, TAK1 and NLRP1-DR-SNAP in WT, P38α/β/δ TKO, TAK1 KO or P38α/β/δ TKO/TAK1 KO NTERT keratinocytes exposed to Dinophysis toxin (Dino. toxin, 100nM), Okadaic acid (Ok. Acid, 250nM), Cantharidin (Canth. 5µM) for 1 hour. Tubulin-α was used as internal protein loading controls. Immunoblots show lysates from one experiment performed at least three times. E. Phosphotag immunoblotting of phosphorylated recombinant NLRP1 full-length protein or immunoprecipitated GFP-tagged NLRP1-DR incubated with recombinant TAK1-TAB1 fusion or P38α kinases for 60 minutes in presence/absence of lambda phosphatase. Immunoblots show proteins from one experiment performed at least three times. F. Fluorescence microscopy quantifications of ASC-GFP specks in WT, P38α/β/δ TKO or in P38α/β/δ TKO/TAK1 KO HEK293 ASC-GFP/NLRP1 reporter cells after 6 h of exposure to Dinophysis toxin (100nM) or Val-boro-Pro (VbP, 10µM). ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles (green, GFP) on the total nuclei (Hoechst). At least ten fields from three independent experiments were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.0001, two-way ANOVA with multiple comparisons. Graphs show one experiment performed in triplicate at least three times.

    Article Snippet: Human primary keratinocytes (pHEKs, PromoCell, C-12007) were cultivated in Keratinocyte Growth Medium 2 (PromoCell, C-20011).

    Techniques: Staining, Western Blot, Recombinant, Immunoprecipitation, Incubation, Fluorescence, Microscopy