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: If not otherwise indicated, stimuli and inhibitors were used at the following concentrations: PLX-4720 (10 μM, MedChem Express, HY-51424); SB203580 (10 μM, MedChem Express, HY-10256); Doramapimod (10 μM, MedChem Express, HY-10320); BX795 (1 μM, Invivogen, Tlrl-bx7); Abemaciclib (25 nM, MedChem Express, HY-16297A); Ribociclib (40 nM, MedChem Express, HY-15777); Palbociclib (25 nM, MedChem Express, HY-50767); Fadraciclib (25 nM, MedChem Express, HY-101212); Roniciclib (25 nM, MedChem Express, HY-13914); SCH772984 (1 μM, MedChem Express, HY-50846); SP600125 (10 μM, MedChem Express, HY-12041); TAK-1 inhib HS-276 (10-20 μM, MedChem Express, HY-147141); TAO Kinase inhibitor 1 (5 μM, MedChem Express, HY-112136); Selonsertib (10 μM, MedChem Express, HY-18938); 5z-7-Oxozeaenol (5 μM, MedChem Express, HY-12686); GW806742X (1 μM, MedChem Express, HY-112292A); DN1289 (1 μM, MedChem Express, HY-152142); Gossypetin (40 μM, MedChem Express, HY-119917); BSJ-04-122 (10 μM, MedChem Express, HY-152185); GSK-872 (10 μM, MedChem Express, HY-101872); Bortezomib (1 μM, Selleck, SE-S1013-5MG); MLN9424 (1 μM, Tocris Bioscience, 6499); DT-061 (1-20 μM; MedChem Express, HY-112929); Dinophysistoxin (250 nM, Bertin Technologies); Okadaic acid (100-600 nM, Bertin Technologies, WP-10011490); Cantharidin (5-25 μM, Sigma-Aldrich, C7632-25MG); Calyculin-A (1 μM, Bertin Technologies, WP-19246); LB-100 (50 μM, Bertin Technologies, 29105); Raphin-1 (50 μM, Tocris Bioscience, 6760/10); Sephin-1 (100 μM, Tocris Bioscience, 5553/10); Anisomycin (1-5 μM, Selleck, SE-S7409-10MG); Valboro-Pro/ Talabostat (10 μM, Selleck, SE-S8455-5MG); Phosphatase Inhibitor Library (117 items) (10 μM, MedChem Express, HY-L081).

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

Journal: bioRxiv

Article Title: Anticipating on-target resistance to WRN inhibitors in microsatellite unstable cancers

doi: 10.64898/2026.01.22.700152

Figure Lengend Snippet: ( A ) Analysis of cell-line dependency on WRN and susceptibility to HRO761, stratified by MSI status; MSS: microsatellite stable; NA: non-applicable ( Table S3 ). ( B ) Clonogenic assays on a representative panel of MSI-high and MSS/MSI-low cell lines in response to HRO761 (1 μM). The X axis represents the surviving fraction compared to the DSMO control counts. ( C ) Schematic of the genome-wide synthetic lethality screen design. ( D ) Representation of NormZ values per gene generated using DrugZ, comparing DMSO-treated versus HRO761-treated RKO cell populations. ( E ) Survival curves of RKO cells transduced with sgRNAs targeting PPM1D (left) and DCLRE1C (right) using a one-vector system. Six days post-infection, cells were exposed to HRO761 for 7 days, and confluency was measured by total DAPI intensity. Mean ± s.e.m. compared to DMSO-treated control cells. N = 4; except DCLRE1C_1, N = 2. (F) ZIP synergy scores derived for the combination of HRO761 with either GSK-2830371 (WIP1i) or AZD-7648 (DNA-PKi) in RKO using the matrix datasets in Fig. S3D. Mean for N = 2 ( G ) Survival analysis of RKO cells treated with either HRO761 (WRNi) at 179 nM, GSK-2830371 (WIP1i) at 5 μM or both (combo) for 7 days. Confluency was measured with Incucyte. Relative survival is calculated with respect to a DMSO-treated control. Mean ± SD for N = 2. ( H ) Survival analysis of RKO cells treated with either HRO761 at 89 nM, AZD-7648 (DNA-PKi) at 1.78 μM or both for 7 days, as in ( G ). Mean ± SD for N = 2. ( I ) ZIP synergy scores derived for the combination of HRO761 with either GSK-2830371 (WIP1i) or AZD-7648 (DNA-PKi) in HCT116 cells. Mean for N = 2 ( J ) Survival analysis of HCT116 cells treated with either HRO761 at 70 nM, GSK-2830371 at 1.6 μM or both; HRO761 at 45 nM, AZD-7648 (DNA-PKi) at 6.45 μM or both for 7 days. Mean ± SD for N = 2.

Article Snippet: GSK-2830371 and AZD-7648 were acquired from MedChemExpress.

Techniques: Control, Genome Wide, Generated, Transduction, Plasmid Preparation, Infection, Derivative Assay

Journal: bioRxiv

Article Title: Anticipating on-target resistance to WRN inhibitors in microsatellite unstable cancers

doi: 10.64898/2026.01.22.700152

Figure Lengend Snippet: ( A ) Analysis of cell-line dependency on WRN and susceptibility to HRO761, stratified by MSI status; MSS: microsatellite stable; NA: non-applicable ( Table S3 ). ( B ) Clonogenic assays on a representative panel of MSI-high and MSS/MSI-low cell lines in response to HRO761 (1 μM). The X axis represents the surviving fraction compared to the DSMO control counts. ( C ) Schematic of the genome-wide synthetic lethality screen design. ( D ) Representation of NormZ values per gene generated using DrugZ, comparing DMSO-treated versus HRO761-treated RKO cell populations. ( E ) Survival curves of RKO cells transduced with sgRNAs targeting PPM1D (left) and DCLRE1C (right) using a one-vector system. Six days post-infection, cells were exposed to HRO761 for 7 days, and confluency was measured by total DAPI intensity. Mean ± s.e.m. compared to DMSO-treated control cells. N = 4; except DCLRE1C_1, N = 2. (F) ZIP synergy scores derived for the combination of HRO761 with either GSK-2830371 (WIP1i) or AZD-7648 (DNA-PKi) in RKO using the matrix datasets in Fig. S3D. Mean for N = 2 ( G ) Survival analysis of RKO cells treated with either HRO761 (WRNi) at 179 nM, GSK-2830371 (WIP1i) at 5 μM or both (combo) for 7 days. Confluency was measured with Incucyte. Relative survival is calculated with respect to a DMSO-treated control. Mean ± SD for N = 2. ( H ) Survival analysis of RKO cells treated with either HRO761 at 89 nM, AZD-7648 (DNA-PKi) at 1.78 μM or both for 7 days, as in ( G ). Mean ± SD for N = 2. ( I ) ZIP synergy scores derived for the combination of HRO761 with either GSK-2830371 (WIP1i) or AZD-7648 (DNA-PKi) in HCT116 cells. Mean for N = 2 ( J ) Survival analysis of HCT116 cells treated with either HRO761 at 70 nM, GSK-2830371 at 1.6 μM or both; HRO761 at 45 nM, AZD-7648 (DNA-PKi) at 6.45 μM or both for 7 days. Mean ± SD for N = 2.

Article Snippet: GSK-2830371 and AZD-7648 were acquired from MedChemExpress.

Techniques: Control, Genome Wide, Generated, Transduction, Plasmid Preparation, Infection, Derivative Assay