Journal: Communications Biology

Article Title: Repurposing of Ibrutinib and Quizartinib as potent inhibitors of necroptosis

doi: 10.1038/s42003-023-05353-5

Figure Lengend Snippet: a , b Stable L929 cell line expressing RIPK1-HBD ( a ) or RIPK3-HBD ( b ) were pretreated with individual necroptosis inhibitor for 1 h following treatment with 4-OHT and z-VAD. The concentration of individual compound is 15 μM. NEC-1 (20 μM) and GSK’872 (10 μM) was chose as positive control. The red line indicated the value of cell viability of DMSO control. For compounds tested, n = 4 biologically independent samples. For NEC-1, GSK’872 and DMSO, n = 16 biologically independent samples. c Summary of classification of necroptosis inhibitors. Data shown are representative of three independent experiments. Means ± SD.

Article Snippet: Pan-caspase inhibitor z-VAD, Necrostatin-1, GSK’872, Smac mimetic (SM-164), Ibrutinib, Quizartinib, BIIB068, RN486, Gilteritinib, HM43239, FLT3-IN-3, propidium iodide were purchased from TargetMol.

Techniques: Expressing, Concentration Assay, Positive Control, Control

Journal: Communications Biology

Article Title: Repurposing of Ibrutinib and Quizartinib as potent inhibitors of necroptosis

doi: 10.1038/s42003-023-05353-5

Figure Lengend Snippet: a HT-29, Hela-RIPK3, and NIH3T3-RIPK3 cells were pretreated with NEC-1 (20 μM), GSK’872 (10 μM), Ibrutinib (15 μM) or vehicle for 1 h following treatment with TSZ. Cell viabilities were determined using CCK8 method. For HT-29 and Hela-RIPK3 cells, n = 3 biologically independent samples. For NIH3T3-RIPK3 cells, n = 4 biologically independent samples. b , c HT-29 cells ( b ) or Hela-RIPK3 cells ( c ) were pretreated Ibrutinib (15 μM) or vehicle for 1 h following treatment with TSZ for indicated time. Then cells were harvested and analyzed with the indicated antibodies. d Mouse RIPK3-flag reconstituted RIPK3-KO L929 cells were pretreated Ibrutinib (15 μM) or vehicle for 1 h following treatment with TSZ for indicated time. Then cells were harvested and immunoprecipitated with M2 (anti-flag) antibody. The total cell lysates (TCL) and the immunoprecipitates were immunoblotted with the indicated antibodies. e L929 cells were pretreated Ibrutinib (15 μM) or vehicle for 1 h following treatment with TSZ for indicated time. Then the cells were lysed with Triton X-100 lysis buffer. The insoluble fractions were collected and analyzed with the indicated antibodies. Data shown are representative of three independent experiments. Means ± SD. * p < 0.05, # p < 0.01.

Article Snippet: Pan-caspase inhibitor z-VAD, Necrostatin-1, GSK’872, Smac mimetic (SM-164), Ibrutinib, Quizartinib, BIIB068, RN486, Gilteritinib, HM43239, FLT3-IN-3, propidium iodide were purchased from TargetMol.

Techniques: Immunoprecipitation, Lysis

Journal: Communications Biology

Article Title: Repurposing of Ibrutinib and Quizartinib as potent inhibitors of necroptosis

doi: 10.1038/s42003-023-05353-5

Figure Lengend Snippet: a Plasmid expressing HA-mRIPK3 or Vector was transfected into 293T cells. Then the cells were treated with indicated concentrations of Ibrutinib, GSK’872 (10 μM), NEC-1 (20 μM) for 20 h. The cells were harvested and analyzed with indicated antibodies. b Hela-RIPK3 or HT-29 was treated with Ibrutinib (15 μM) for the indicated times. Then the cells were harvested and analyzed with indicated antibodies. c In vitro ADP-Glo kinase assay using recombinant hRIPK3 protein. Recombinant hRIPK3 was incubated with DMSO, GSK’872 (10 μM) or Ibrutinib (10 μM). For DMSO and Ibrutinib, n = 4 biologically independent samples. For GSK’872, n = 5 biologically independent samples. d Molecular Docking simulation result of Ibrutinib and RIPK3 using RIPK3 structure (PDB code: 7MX3) . e L929 cells were treated with Ibrutinib (15 μM) before or after challenging with TZ. Then cell viabilities were determined using CCK8 method. n = 3 biologically independent samples. f L929 cells were treated with Ibrutinib (15 μM) before or after challenging with TZ. Then cells were harvested and analyzed with indicated antibodies. Data shown are representative of three independent experiments. Means ± SD. # p < 0.01.

Article Snippet: Pan-caspase inhibitor z-VAD, Necrostatin-1, GSK’872, Smac mimetic (SM-164), Ibrutinib, Quizartinib, BIIB068, RN486, Gilteritinib, HM43239, FLT3-IN-3, propidium iodide were purchased from TargetMol.

Techniques: Plasmid Preparation, Expressing, Transfection, In Vitro, Kinase Assay, Recombinant, Incubation

Journal: Communications Biology

Article Title: Repurposing of Ibrutinib and Quizartinib as potent inhibitors of necroptosis

doi: 10.1038/s42003-023-05353-5

Figure Lengend Snippet: a HT-29 cells were pretreated with vehicle, GSK’872 (10 μM), Quizartinib (10 μM) for 1 h following treatment with TSZ. Then the cell viabilities were determined using CCK8 method. n = 3 biologically independent samples. b HT-29 cells were pretreated with vehicle or Quizartinib (10 μM) for 1 h following treatment with TSZ for the indicated times. Then the cells were harvested and analyzed with the indicated antibodies. c Mouse RIPK3-flag reconstituted RIPK3-KO L929 cells were pretreated Quizartinib (2 μM) or vehicle for 1 h following treatment with TSZ for indicated time. Then cells were harvested and immunoprecipitated with M2 (anti-flag) antibody. The total cell lysates (TCL) and the immunoprecipitates were immunoblotted with the indicated antibodies. d L929 cells were pretreated Quizartinib (2 μM) or vehicle for 1 h following treatment with TSZ for indicated time. Then the cells were lysed with Triton X-100 lysis buffer. The insoluble fractions were collected and analyzed with the indicated antibodies. e L929 cells were pretreated vehicle or indicated compounds for 1 h following treatment with TSZ. Then the cell viabilities were determined using CCK8 method. n = 3 biologically independent samples. f Plasmid expressing HA-RIPK1 or Vector was transfected into 293T cells. Then the cells were treatment with indicated concentrations of Quizartinib, NEC-1 (10 μM) or vehicle for 20 h. The cells were harvested and analyzed with indicated antibodies. Data shown are representative of three independent experiments. Means ± SDs. # p < 0.01.

Article Snippet: Pan-caspase inhibitor z-VAD, Necrostatin-1, GSK’872, Smac mimetic (SM-164), Ibrutinib, Quizartinib, BIIB068, RN486, Gilteritinib, HM43239, FLT3-IN-3, propidium iodide were purchased from TargetMol.

Techniques: Immunoprecipitation, Lysis, Plasmid Preparation, Expressing, Transfection

Journal: bioRxiv

Article Title: A necroptotic-to-apoptotic signaling axis underlies inflammatory bowel disease

doi: 10.1101/2024.11.13.623307

Figure Lengend Snippet: (A) The intestinal organoid differentiation procedure. Representative micrographs of stem and colonocyte-enriched organoids are shown. Scale bars are 100 µm. (B) Expression of stem cell ( LGR5 ), goblet cell ( MUC2 ) and colonocyte ( ALPI , FABP1 ) markers was assessed by quantitative polymerase chain reaction (qPCR) in organoids from four donors and normalized to the housekeeping gene HPRT , with the group with the highest expression set to 1. (C, D) Organoids were treated with IFNγ (50 ng/mL) and/or TNF (50 ng/mL). (C) Percentage death was measured by Cytotox Red-stained organoid area relative to the total organoid area (n = 5 donors, mean ± SEM of 5 independent experiments) using IncuCyte live cell imaging (see ). (D) Representative micrographs after 21 hours of treatment; scale bars are 550 µm. (E) Heatmaps showing Log 2 fold change of selected genes in intestinal biopsies from patients with UC or CD, relative to non-IBD patients from published data (top) and from for organoids treated with IFNγ and/or TNF for 3.5 hours relative to untreated organoids in this work (bottom). The selected genes, their left-to-right ordering, and the annotated clusters are the same as in . (F) Heatmaps show differential gene expression between untreated organoids and the corresponding organoids treated with IFNγ and/or TNF for 3.5 hours. All entries with an adjusted p<0.05 by empirical Bayes moderated t-statistic and Benjamini–Hochberg multiple test correction. (G) Immunoblots of organoids treated with IFNγ and/or TNF as indicated (representative of 3 independent experiments). (H) Stem cell organoids were pretreated for 0.5 h with vehicle (DMSO, n=7), RIPK1 inhibitor (necrostatin-1s, 10 μM, n=2), RIPK3 inhibitor (GSK872, 10 μM, n=2), Caspase-1 inhibitor (VX-765, 40 μM, n=3), NLRP3 inhibitor (MCC950, 10 μM, n=2) or the pan-caspase inhibitor (Q-VD-OPh, 40 μM, n=6) alone, or with Q-VD-OPh/GSK872 (n=3) and Q-VD-OPh/ GSK872/ MCC950 (n=3), followed by IFNγ (50 ng/mL) and TNF (50 ng/mL) treatment. Cell death was assessed after 0, 6, 12, 18, 24 hours (n = 5 of different donors, mean ± SEM, pooled from 7 independent experiments). (I) Colonocyte organoids were pretreated for 0.5 h with DMSO (n=6), GSK872 (n=3), VX-765 (n=3), MCC950 (n=3) or Q-VD-OPh (n=6) alone, or with Q-VD-OPh/ GSK872 (n=3), Q-VD-OPh/GSK872/ MCC950 (n=3) with the same concentration indicated in (H), followed by IFNγ and TNF treatment. Cell death was assessed after 0, 6, 12, 18, 24 hours (n = 5 of different donors, mean ± SEM, pooled from 6 independent experiments).

Article Snippet: XELJANZ (tofacitinib; 20 μM, Pfizer), necrostatin-1s (10 μM, nec1s, Merck, 504297), GSK872 (10 μM, GSK872, SynKinase, SYN-5481), VX-765 (40 μM, Selleck, S2228), MCC950 (10 μM, kindly provided by A. Roberson and M. Cooper, University of Queensland, Australia), Q-VD-OPh (40 μM, QVD, MedChemExpress, HY-12305), Human Fas Ligand/TNFSF6 Antibody (10 μg/mL, R&D, MAB126), Human TRAIL/TNFSF10 Antibody (10 μg/mL, R&D, MAB375), ABT-737 (1 mM, Active Biochem; A-6044), cycloheximide (10 mg/mL, Sigma; C7698), TNF (50 ng/mL), Smac mimetic (1 mM, Compound A, TetraLogic Pharmaceuticals), Q-VD-OPh (20 μM), ABT-199 (1 mM, Active Biochem; A-1231), S63845 (10 mM, Active Biochem; A-6044) and A-1331852 (BCL-XL inhibitor; AbbVie, provided by Guillaume Lessene, WEHI) stimulations were performed as described in the relevant figure legends.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Staining, Live Cell Imaging, Gene Expression, Western Blot, Concentration Assay

Journal: bioRxiv

Article Title: A necroptotic-to-apoptotic signaling axis underlies inflammatory bowel disease

doi: 10.1101/2024.11.13.623307

Figure Lengend Snippet: (A) MDA-MB-231 cells were treated with IFNγ (50 ng/mL), TRAIL neutralizing antibody (TRAIL Ab, 10 μg/mL) and TRAIL (50 ng/mL) for 48 hours. Cell death was assessed by PI exclusion, measured by Incucyte® imaging (n = 2 independent experiments, mean ± SEM). (B) Cell death of HepG2 cells treated with TRAIL/TNFSF10 neutralizing antibody (FASL Ab,10 μg/mL) then Actinomycin D (Act D, 0.5 μg/mL) and FAS ligand (FasL, 100 ng/mL) was assessed by PI exclusion using IncuCyte imaging (n=1). (C) Cell death in colonocyte organoids pre-treated with vehicle (DMSO), TRAIL Ab (10 μg/mL), or FASL Ab (10 μg/mL) then IFNγ and TNF (n=3 different donors, mean ± SEM, pooled from 3 independent experiments). (D, E) Organoids were pre-treated with Q-VD-OPh (QVD, 10 μM), GSK’872 (10 μM), necrostatin-1s (Nec1s, 10 μM) or vehicle (DMSO) followed by stimulation with ABT-737 (1 μM) and cycloheximide (CHX, 10 mg/mL), or the MCL-1 inhibitor S63856 (5 μM) to activate mitochondrial apoptosis. Alternatively, intestinal organoids were stimulated with TNF(50 ng/mL), Smac-mimetic Compound A (1 μM) and QVD-OPh to induce necroptosis (TSQ). Cell death was assessed using Cytox Red uptake by IncuCyte live cell imaging (n=3 different donors, mean ± SEM, pooled from 3 independent experiments). (F) Mouse bone marrow-derived macrophages were primed with LPS (100 ng/mL) and treated with Nigericin (20 μM) ±pretreatment with VX-765 (20 μM) or MCC950 (10 μM) (n=3 different mice, mean ± SEM, pooled from 2 independent experiments. ( G) Caspase-3 activity was determined by DEVDase activity assay in organoids (n=3 different donors, mean ± SEM, pooled from 3 independent experiments). (H) Representative immunofluorescence images of wildtype control and BCL2 overexpressing intestinal stem cell organoids. Scale bar, 100 µm. (I) IncuCyte imaging was used to quantify cell death following induction of exogenous BCL2 by doxycycline (dox) in stem cell or colonocyte organoids and subsequent IFNγ and TNF stimulation. (n=2 donors, mean ± SEM, pooled from 2 independent experiments). (J) Heatmap of pro-survival and pro-death DEGs (see ) comparing IFNγ/TNF treated stem cell organoids versus colonocyte organoids, or untreated stem cell organoids versus colonocyte organoids. All entries with an adjusted p<0.05 by empirical Bayes moderated t-statistic and Benjamini–Hochberg multiple test correction. (K) Single-cell IBD patient sequencing data (inflamed and non-inflamed mucosa from 3 non-IBD controls and 3 UC patients; ) was re-analyzed to define pro-survival and pro-death genes enrichment scores. (L) PUMA levels of non-inflamed, inflamed and marginal IBD tissues and non-IBD controls were analyzed by immunoblot and quantified by ImageJ (mean ± SEM); data represented separately as upregulated (n=4) and downregulated (n=7) trends. Protein levels are represented as the integrated band density and normalized to TNF/Compound A treated HT29 cells. PUMA levels of HT29 cells and biopsies are analyzed by immunoblot and quantified by ImageJ (mean ± SEM); Each dot represents one biopsy, data represented separately as upregulated (n=5, left panel) and downregulated (n=7, right panel) trends. Expression was represented as the integrated band density and normalized to TNF/Compound A treated HT29 cells then adjusted for differences in GAPDH.

Article Snippet: XELJANZ (tofacitinib; 20 μM, Pfizer), necrostatin-1s (10 μM, nec1s, Merck, 504297), GSK872 (10 μM, GSK872, SynKinase, SYN-5481), VX-765 (40 μM, Selleck, S2228), MCC950 (10 μM, kindly provided by A. Roberson and M. Cooper, University of Queensland, Australia), Q-VD-OPh (40 μM, QVD, MedChemExpress, HY-12305), Human Fas Ligand/TNFSF6 Antibody (10 μg/mL, R&D, MAB126), Human TRAIL/TNFSF10 Antibody (10 μg/mL, R&D, MAB375), ABT-737 (1 mM, Active Biochem; A-6044), cycloheximide (10 mg/mL, Sigma; C7698), TNF (50 ng/mL), Smac mimetic (1 mM, Compound A, TetraLogic Pharmaceuticals), Q-VD-OPh (20 μM), ABT-199 (1 mM, Active Biochem; A-1231), S63845 (10 mM, Active Biochem; A-6044) and A-1331852 (BCL-XL inhibitor; AbbVie, provided by Guillaume Lessene, WEHI) stimulations were performed as described in the relevant figure legends.

Techniques: Imaging, Live Cell Imaging, Derivative Assay, Activity Assay, Immunofluorescence, Control, Sequencing, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21 WAF1/CIP1 proteasomal degradation in senescent cells

doi: 10.1038/s41419-024-06755-x

Figure Lengend Snippet: A – C Flow cytometrical determination of the mitochondrial membrane potential (MMP) of proliferating and senescent (day 7 after γIR) HCT116 ( A ), A549 ( B ) and MCF-7 ( C ) cells after 24 h treatment with PX-866 ( A : 10 µM; B , C : 30 µM) or Navitoclax (10 µM). D – F Proliferating and senescent (day 7 after γIR) HCT116 ( D ), A549 ( E ) and MCF-7 ( F ) cells were exposed to PX-866 ( D , E : 10 µM; F: 30 µM) together with the apoptosis inhibitor q-VD-OPh (in yellow), the necroptosis inhibitors Nec-1s and GSK’872, or the ferroptosis inhibitors Fer-1 and CPX (all 10 µM). After 24 h, LDH activity in the cell culture supernatant was determined as a marker for cell death. Only a co-treatment with q-VD-Ph inhibited the PX-866-mediated increased death of senescent cells. Significance indicators above the bars are comparing the sample of senescent cells of the corresponding inhibitor with or without PX-866 treatment. The exception is the sample of PX-866 and q-VD-OPh treated senescent cells which is not significantly altered by PX-866 treatment. This sample is significantly different to a treatment with PX-866 without q-VD-OPh (indicated by brackets). G , H Measurement of caspase-3-like (DEVDase) activity in cellular extracts of proliferating and senescent (day 7 after γIR) HCT116 ( G ) and A549 ( H ) cells after an additional treatment with PX-866 ( G : 10 µM; H : 30 µM) or Navitoclax (10 µM). Data shown are the mean ± SD of at least three independent experiments. Individual data points are represented in the bar charts by small black squares. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The necroptosis inhibitors Nec-1s (Necrostatin-2 racemate) and GSK’872, the ferroptosis inhibitors Fer-1 (Ferrostatin-1) and CPX (ciclopirox olamine), and the fluorogenic caspase-3 substrate DEVD-AMC (N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin) were also obtained from Biomol.

Techniques: Membrane, Activity Assay, Cell Culture, Marker

Journal: Cell

Article Title: RIPK3 restricts viral pathogenesis via cell death-independent neuroinflammation

doi: 10.1016/j.cell.2017.03.011

Figure Lengend Snippet: Key Resource Table

Article Snippet: PRR ligands and chemical agents The following PRR ligands and chemical inhibitors were used in cell culture experiments: Poly(I:C) (1 μg/ml, Millipore), LPS-EB Ultrapure (1 μg/ml, Invivogen), CL264 (1 μg/ml, Invivogen), zVAD (50 μM, SM Biochemicals), necrostatin-1 (30 μM, Sigma), GSK 963 (100nM, GlaxoSmithKline), GSK 843 (100nM, GlaxoSmithKline) ( Mandal et al., 2014 ), GSK 872 (100nM, GlaxoSmithKline) and QVD (20 μM, SM Biochemicals), AP1 (100 nM, ClonTech, sold as “BB Homodimerizer”).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software