gsk872  (MedChemExpress)

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: Inflammation

Article Title: ZBP1 Mediates Renal Tubular Injury in Diabetic Nephropathy Through RIPK3-mediated Necroptosis

doi: 10.1007/s10753-025-02362-w

Figure Lengend Snippet: ZBP1 expression in renal tissue is associated with tubular necroptosis in patients with DN. (a) The IHC of RIPK3 and MLKL in each group. Scale bars = 50 μm. (b) Immunofluorescence double staining of p-MLKL and AQP1. Scale bars = 50 μm. (c) We investigated transmission electron micrographs from NC group ( n = 5), DN1 group ( n = 3), and DN2 group ( n = 7). The images depicted in the figure show electron micrographs of DN2 group patients. TEM observed thickened glomerular basement membranes, some renal tubular atrophy, and swollen proximal tubular epithelial cells in the DN group (red puncta). Abbreviations: IHC, immunohistochemistry; NC, normal control; DN1, diabetic nephropathy patients with interstitial fibrosis and tubular atrophy score < 2; DN2, diabetic nephropathy patients with interstitial fibrosis and tubular atrophy score ≥ 2; AQP, aquaporin. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Additionally, MTECs were treated with the RIPK3 inhibitor GSK’872 (MCE, HY-101872) for 24 h for further study.

Techniques: Expressing, Immunofluorescence, Double Staining, Transmission Assay, Immunohistochemistry, Control

Journal: Inflammation

Article Title: ZBP1 Mediates Renal Tubular Injury in Diabetic Nephropathy Through RIPK3-mediated Necroptosis

doi: 10.1007/s10753-025-02362-w

Figure Lengend Snippet: ZBP1 knockdown inhibits RIPK3-mediated necroptosis in db/db mice. (a) IHC assay for RIPK3 and MLKL protein expression on tubules in db/db mice. Scale bars = 50 μm. (b) IF double staining for p-MLKL and AQP1 in the db/db kidney. Scale bars = 50 μm. (c) RIPK3, p-RIPK3, MLKL and p-MLKL levels were assessed by Western blot. (d) TEM showed swollen endoplasmic reticulum and mitochondria (red puncta) in the proximal renal tubular epithelial cells, as well as loss of mitochondrial cristae in db/db mice compared to NC group, whereas the ZBP1 knockdown group exhibited alleviated lesions. Abbreviations: IHC, immunohistochemistry; EV, empty vector; KD, knockdown; IF, immunofluorescence; TEM, transmission electron microscopy. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Additionally, MTECs were treated with the RIPK3 inhibitor GSK’872 (MCE, HY-101872) for 24 h for further study.

Techniques: Knockdown, Expressing, Double Staining, Western Blot, Immunohistochemistry, Plasmid Preparation, Immunofluorescence, Transmission Assay, Electron Microscopy

Journal: Inflammation

Article Title: ZBP1 Mediates Renal Tubular Injury in Diabetic Nephropathy Through RIPK3-mediated Necroptosis

doi: 10.1007/s10753-025-02362-w

Figure Lengend Snippet: ZBP1 knockdown reduces HG-induced MTECs damage and necroptosi s in vitro. (a and b) The knockdown effect of ZBP1 was detected by real-time PCR and Western blot. (c) Western blot analysis of KIM1 in MTECs. (d) IF of KIM1 in MTECs. ZBP1 knockdown reduced KIM-1 expression in HG-induced MTECs. Scale bars = 50 μm. (e) Quantitative data and Western blotting of RIPK3, p-RIPK3, MLKL and p-MLKL. (f) Immunofluorescence analysis of p-MLKL in MTECs. Scale bars = 50 μm. (g) TEM revealed that HG increased MTEC volume, induced cell membrane rupture (blue puncta), cytoplasmic translucency (yellow puncta), organelle swelling (red puncta) and cytoplasmic content leakage. These pathological changes were attenuated in the ZBP1 knockdown group. Abbreviations: EV, empty vector; KD, knockdown; IHC, immunohistochemistry; HG, high glucose; IF, immunofluorescence; TEM, transmission electron microscopy. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Additionally, MTECs were treated with the RIPK3 inhibitor GSK’872 (MCE, HY-101872) for 24 h for further study.

Techniques: Knockdown, In Vitro, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Membrane, Plasmid Preparation, Immunohistochemistry, Transmission Assay, Electron Microscopy

Journal: Inflammation

Article Title: ZBP1 Mediates Renal Tubular Injury in Diabetic Nephropathy Through RIPK3-mediated Necroptosis

doi: 10.1007/s10753-025-02362-w

Figure Lengend Snippet: ZBP1 knockdown attenuates AGEs-induced MTECs damage and necroptosis in vitro. (a and b) Western blot and real-time PCR of KIM-1 in MTECs. (c) Quantitative data and Western blotting of RIPK3, p-RIPK3, MLKL and p-MLKL. (d) Immunofluorescence analysis of p-MLKL in MTECs. Scale bars = 25 μm. (e) Real-time PCR analysis mRNA levels of IL-1β and TNF-α. (f) Quantitative data and Western blot analysis of p-p65 in MTECs stimulated with AGEs. (g) Western blot showed that inhibition of ZBP1 expression could inhibit the expression of α-SMA and COL-I. Abbreviations: AGEs, advanced glycation end-products; EV, empty vector; KD, knockdown. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Additionally, MTECs were treated with the RIPK3 inhibitor GSK’872 (MCE, HY-101872) for 24 h for further study.

Techniques: Knockdown, In Vitro, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Inhibition, Expressing, Plasmid Preparation

Journal: Inflammation

Article Title: ZBP1 Mediates Renal Tubular Injury in Diabetic Nephropathy Through RIPK3-mediated Necroptosis

doi: 10.1007/s10753-025-02362-w

Figure Lengend Snippet: ZBP1 interacts with RIPK3 in HG-induced MTECs. ( a ) Western blot showed the protein levels of RIPK3, p-RIPK3, MLKL and p-MLKL. ( b ) Immunofluorescence images showed that ZBP1 knockdown and GSK’872 reduced the phosphorylation and membrane translocation of MLKL in HG-induced MTECs. ( c ) Western blot revealed that overexpressing RIPK3 negated the protective effects of ZBP1 knockdown on necroptosis. ( d - f ) Co-IP, SPR and molecular docking of ZBP1 and RIPK3. ( e ) Molecular docking of ZBP1 and RIPK3. Docking scale = 6.88 Kcal/mol. Abbreviations: EV, empty vector; KD, knockdown; HG, high glucose; IF, immunofluorescence; IP, immunoprecipitation; SPR, surface plasmon resonance. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Additionally, MTECs were treated with the RIPK3 inhibitor GSK’872 (MCE, HY-101872) for 24 h for further study.

Techniques: Western Blot, Immunofluorescence, Knockdown, Phospho-proteomics, Membrane, Translocation Assay, Co-Immunoprecipitation Assay, Plasmid Preparation, Immunoprecipitation, SPR Assay