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: Antibodies used in this study were anti-Gasdermin D (E8G3F) Monoclonal antibody (1:1000, Cell signaling, 97558S); anti-Caspase 1 (p20) (human) mAb (Bally-1) antibody (1:500, Adipogen, AG-20B-0048); anti-DFNA5/Gasdermin E (EPR19859) Monoclonal antibody (1:1000,Abcam, ab215191); anti-Caspase 3 antibody (1:500, Cell signaling, 9662S); anti-Human IL-1β/IL-1F2 Polyclonal antibody (1:500, R&D systems, AF-201-NA); anti-Cleaved IL-1β (Asp116) Monoclonal antibody (1:750, Cell signaling, 83186S); anti-Tubulin-α antibody (1:1000, Abcam, ab4074); anti-PP1alpha Polyclonal antibody (1:1000, Invitrogen, PA5-119781); anti-PP1CB-Specific antibody (1:1000, ProteinTech, PR-55136-AP-150); anti-PP1CC antibody (1:1000, ProteinTech, PR-11082-1-AP-150); PP2A C Subunit antibody (1:500, Cell signaling, 2038S); anti-Purified NLRP1 (N-terminal) antibody (1:300, Biolegend, 679802); anti-NLRP1 (C-terminal) Polyclonal antibody (1:500,Abcam, ab36852); anti-GFP antibody (1:1000, Abcam, ab6673); anti-SNAP/CLIP-tag Monoclonal antibody (1:1000, ProteinTech, 6F9-100); anti-ZAKα Polyclonal antibody (1:1000, Bethyl Laboratories, A301-993A); anti-P38 MAPK antibody (1:1000, Cell signaling, 9212S); anti-Phospho-p38 (Thr180/Tyr182) (D3F9) Monoclonal antibody (1:1000, Cell signaling, 4511S); anti-Puromycine antibody Clone 12D10 (1:1000, Sigma-Aldrich, MABE343); anti-TAK1 antibody (1:1000, Cell signaling, 4505S); anti-Phospho-TAK1 (Ser412) antibody (1:1000, Cell signaling, 9339S); anti-Phospho-TAK1 (Thr184/187) 90C7 Monoclonal antibody (1:1000, Cell signaling, 4508S); anti-P38 alpha/MAPK14 antibody (E229) (1:1000, Abcam, ab170099); anti-P38 MAPK beta Monoclonal antibody (1:1000, Invitrogen, MA514-950); anti-P38 beta MAPK (C28C2) Monoclonal antibody (1:1000, Cell Signaling, 2339S); anti-P38 gamma/MAPK12 antibody (EPR6528N) (1:1000, Abcam, ab205926); anti-P38 gamma MAPK antibody (1:1000, Cell Signaling, 2307S); anti-Human P38 delta antibody (1:1000, R&D system, AF1519); anti-P38δ MAPK (10A8) mAb (1:1000, Cell Signaling, 2308S); anti-GAPDH antibody (1:1000, GeneTex, GTX100118); Goat anti-rabbit HRP secondary antibody (1:5000, Advansta R-05072-500); Goat anti-mouse HRP secondary antibody (1:5000, Advansta, R-05071-500); Goat anti-rat IgG H&L (HRP) (1:5000, Abcam, ab97057); Goat IgG HRP-conjugated Antibody (1:5000, Biotechne, HAF109).

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

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: Antibodies used in this study were anti-Gasdermin D (E8G3F) Monoclonal antibody (1:1000, Cell signaling, 97558S); anti-Caspase 1 (p20) (human) mAb (Bally-1) antibody (1:500, Adipogen, AG-20B-0048); anti-DFNA5/Gasdermin E (EPR19859) Monoclonal antibody (1:1000,Abcam, ab215191); anti-Caspase 3 antibody (1:500, Cell signaling, 9662S); anti-Human IL-1β/IL-1F2 Polyclonal antibody (1:500, R&D systems, AF-201-NA); anti-Cleaved IL-1β (Asp116) Monoclonal antibody (1:750, Cell signaling, 83186S); anti-Tubulin-α antibody (1:1000, Abcam, ab4074); anti-PP1alpha Polyclonal antibody (1:1000, Invitrogen, PA5-119781); anti-PP1CB-Specific antibody (1:1000, ProteinTech, PR-55136-AP-150); anti-PP1CC antibody (1:1000, ProteinTech, PR-11082-1-AP-150); PP2A C Subunit antibody (1:500, Cell signaling, 2038S); anti-Purified NLRP1 (N-terminal) antibody (1:300, Biolegend, 679802); anti-NLRP1 (C-terminal) Polyclonal antibody (1:500,Abcam, ab36852); anti-GFP antibody (1:1000, Abcam, ab6673); anti-SNAP/CLIP-tag Monoclonal antibody (1:1000, ProteinTech, 6F9-100); anti-ZAKα Polyclonal antibody (1:1000, Bethyl Laboratories, A301-993A); anti-P38 MAPK antibody (1:1000, Cell signaling, 9212S); anti-Phospho-p38 (Thr180/Tyr182) (D3F9) Monoclonal antibody (1:1000, Cell signaling, 4511S); anti-Puromycine antibody Clone 12D10 (1:1000, Sigma-Aldrich, MABE343); anti-TAK1 antibody (1:1000, Cell signaling, 4505S); anti-Phospho-TAK1 (Ser412) antibody (1:1000, Cell signaling, 9339S); anti-Phospho-TAK1 (Thr184/187) 90C7 Monoclonal antibody (1:1000, Cell signaling, 4508S); anti-P38 alpha/MAPK14 antibody (E229) (1:1000, Abcam, ab170099); anti-P38 MAPK beta Monoclonal antibody (1:1000, Invitrogen, MA514-950); anti-P38 beta MAPK (C28C2) Monoclonal antibody (1:1000, Cell Signaling, 2339S); anti-P38 gamma/MAPK12 antibody (EPR6528N) (1:1000, Abcam, ab205926); anti-P38 gamma MAPK antibody (1:1000, Cell Signaling, 2307S); anti-Human P38 delta antibody (1:1000, R&D system, AF1519); anti-P38δ MAPK (10A8) mAb (1:1000, Cell Signaling, 2308S); anti-GAPDH antibody (1:1000, GeneTex, GTX100118); Goat anti-rabbit HRP secondary antibody (1:5000, Advansta R-05072-500); Goat anti-mouse HRP secondary antibody (1:5000, Advansta, R-05071-500); Goat anti-rat IgG H&L (HRP) (1:5000, Abcam, ab97057); Goat IgG HRP-conjugated Antibody (1:5000, Biotechne, HAF109).

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

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: Antibodies used in this study were anti-Gasdermin D (E8G3F) Monoclonal antibody (1:1000, Cell signaling, 97558S); anti-Caspase 1 (p20) (human) mAb (Bally-1) antibody (1:500, Adipogen, AG-20B-0048); anti-DFNA5/Gasdermin E (EPR19859) Monoclonal antibody (1:1000,Abcam, ab215191); anti-Caspase 3 antibody (1:500, Cell signaling, 9662S); anti-Human IL-1β/IL-1F2 Polyclonal antibody (1:500, R&D systems, AF-201-NA); anti-Cleaved IL-1β (Asp116) Monoclonal antibody (1:750, Cell signaling, 83186S); anti-Tubulin-α antibody (1:1000, Abcam, ab4074); anti-PP1alpha Polyclonal antibody (1:1000, Invitrogen, PA5-119781); anti-PP1CB-Specific antibody (1:1000, ProteinTech, PR-55136-AP-150); anti-PP1CC antibody (1:1000, ProteinTech, PR-11082-1-AP-150); PP2A C Subunit antibody (1:500, Cell signaling, 2038S); anti-Purified NLRP1 (N-terminal) antibody (1:300, Biolegend, 679802); anti-NLRP1 (C-terminal) Polyclonal antibody (1:500,Abcam, ab36852); anti-GFP antibody (1:1000, Abcam, ab6673); anti-SNAP/CLIP-tag Monoclonal antibody (1:1000, ProteinTech, 6F9-100); anti-ZAKα Polyclonal antibody (1:1000, Bethyl Laboratories, A301-993A); anti-P38 MAPK antibody (1:1000, Cell signaling, 9212S); anti-Phospho-p38 (Thr180/Tyr182) (D3F9) Monoclonal antibody (1:1000, Cell signaling, 4511S); anti-Puromycine antibody Clone 12D10 (1:1000, Sigma-Aldrich, MABE343); anti-TAK1 antibody (1:1000, Cell signaling, 4505S); anti-Phospho-TAK1 (Ser412) antibody (1:1000, Cell signaling, 9339S); anti-Phospho-TAK1 (Thr184/187) 90C7 Monoclonal antibody (1:1000, Cell signaling, 4508S); anti-P38 alpha/MAPK14 antibody (E229) (1:1000, Abcam, ab170099); anti-P38 MAPK beta Monoclonal antibody (1:1000, Invitrogen, MA514-950); anti-P38 beta MAPK (C28C2) Monoclonal antibody (1:1000, Cell Signaling, 2339S); anti-P38 gamma/MAPK12 antibody (EPR6528N) (1:1000, Abcam, ab205926); anti-P38 gamma MAPK antibody (1:1000, Cell Signaling, 2307S); anti-Human P38 delta antibody (1:1000, R&D system, AF1519); anti-P38δ MAPK (10A8) mAb (1:1000, Cell Signaling, 2308S); anti-GAPDH antibody (1:1000, GeneTex, GTX100118); Goat anti-rabbit HRP secondary antibody (1:5000, Advansta R-05072-500); Goat anti-mouse HRP secondary antibody (1:5000, Advansta, R-05071-500); Goat anti-rat IgG H&L (HRP) (1:5000, Abcam, ab97057); Goat IgG HRP-conjugated Antibody (1:5000, Biotechne, HAF109).

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