Journal: Cell death & disease

Article Title: Inhibition of keratinocyte necroptosis mediated by RIPK1/RIPK3/MLKL provides a protective effect against psoriatic inflammation.

doi: 10.1038/s41419-020-2328-0

Figure Lengend Snippet: Fig. 1 Expression of necroptosis-related genes in the epidermis of human psoriasis lesions. a Samples from normal skin and psoriatic lesions were stained with RIPK1, RIPK3, MLKL, and pMLKL(S358). Representative images are shown. Scale bar represents 200 μm. b mRNA levels of RIPK1, RIPK3, and MLKL in skin tissues from the normal and psoriasis groups were analyzed by real-time quantitative PCR. c, d Protein levels of RIPK1, RIPK3, pRIPK3(S227), MLKL and pMLKL (S358) were quantified by western blotting. GAPDH was used as a loading control. Relative protein expression was normalized to that of the internal control. Results are representative of three independent experiments. Error bars (b, d) represent mean ± standard deviation (SD). *p < 0.05, **p < 0.01, and ***p < 0.001 when compared.

Article Snippet: HMGB1 (ab79823), RIPK3 (ab56164), pRIPK3(S227, ab209384), pRIPK3 (S232, ab195117), MLKL (ab194699), pMLKL (S345, ab196436) and pMLKL (S358, ab187091) (Abcam, Cambridge, UK). β-actin and GAPDH (Affinity Biosciences, USA), N-cadherin (13769-1-AP) and S100A8 (15792-1- AP) (Proteintech, Rosemont, IL, USA).

Techniques: Expressing, Staining, Real-time Polymerase Chain Reaction, Western Blot, Control, Standard Deviation

Journal: Cell death & disease

Article Title: Inhibition of keratinocyte necroptosis mediated by RIPK1/RIPK3/MLKL provides a protective effect against psoriatic inflammation.

doi: 10.1038/s41419-020-2328-0

Figure Lengend Snippet: Fig. 2 Inhibition of RIPK1 attenuated RIPK1/RIPK3- mediated necroptosis in vitro. a–h HaCaT cells were treated with TNF-α (100 ng/mL), Smac (100 nM), and z-VAD-fmk (20 μM) for 8 h (hereafter abbreviated as TSZ) to induce necroptosis and then stimulated with Nec-1s (10 μM). a Cell viability was measured by the Cell Counting Kit-8 (CCK-8) assay. b LDH release was measured by the cytotoxicity LDH assay kit. c Protein levels of RIPK1, RIPK3, pRIPK3(S227), MLKL and pMLKL (S358) were quantified by western blotting. GAPDH served as the loading control. d mRNA level of RIPK1, RIPK3, and MLKL was determined by real-time quantitative PCR in the three groups. e–h Real-time quantitative PCR was performed to analyze the mRNA level of IL-1β (e), IL-6 (f), CXCL1 (g), and IL-8 (h). i, j Knockdown of RIPK1 by shRNA in HaCaT cells was confirmed by western blotting (i) and real-time quantitative PCR (j). k, l After RIPK1-knockdown in HaCAT cells, they were treated with TSZ. Cell viability was measured by the Cell Counting Kit-8 (CCK-8) assay. LDH release was measured by the cytotoxicity LDH assay kit. Error bars represent mean ± standard deviation (SD). ns p > 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001 when compared. All the assays were repeated three times and the results were consistent.

Article Snippet: HMGB1 (ab79823), RIPK3 (ab56164), pRIPK3(S227, ab209384), pRIPK3 (S232, ab195117), MLKL (ab194699), pMLKL (S345, ab196436) and pMLKL (S358, ab187091) (Abcam, Cambridge, UK). β-actin and GAPDH (Affinity Biosciences, USA), N-cadherin (13769-1-AP) and S100A8 (15792-1- AP) (Proteintech, Rosemont, IL, USA).

Techniques: Inhibition, In Vitro, Cell Counting, CCK-8 Assay, Lactate Dehydrogenase Assay, Western Blot, Control, Real-time Polymerase Chain Reaction, Knockdown, shRNA, Standard Deviation

Journal: Cell death & disease

Article Title: Inhibition of keratinocyte necroptosis mediated by RIPK1/RIPK3/MLKL provides a protective effect against psoriatic inflammation.

doi: 10.1038/s41419-020-2328-0

Figure Lengend Snippet: Fig. 3 Nec-1s improved the morphological and histological features and the necroptosis-related protein expression in mice with IMQ- induced psoriasiform dermatitis. a Schematic diagram of the animal experiment protocol for the Control, IMQ, and IMQ + Nec-1s groups (n = 6). b Representative macroscopic views and H&E staining of cross-sectional slices of the dorsal skin of BALB/c mice on the 8th day. Scale bar represents 200 μm. c The epidermal thickness of the dorsal skin on the 8th day was measured by four randomly selected fields per section of each mouse. d Daily assessment of epidermal erythema, scales, and thickening of the dorsal skin. The PASI score was calculated by adding the scores of three independent criteria (ranging from 0 to 12). e Immunohistochemical staining for RIPK1 and MLKL in the dorsal mouse skin samples for the three groups. Representative images are shown. Scale bar represents 200 μm. f Protein levels of RIPK1, pRIPK3(S232), RIPK3, MLKL and pMLKL (S345) were quantified by western blotting. GAPDH served as the loading control. Error bars represent mean ± standard deviation (SD). *p < 0.05, **p < 0.01, and ***p < 0.001 when compared. All assays were repeated three times with consistent results.

Article Snippet: HMGB1 (ab79823), RIPK3 (ab56164), pRIPK3(S227, ab209384), pRIPK3 (S232, ab195117), MLKL (ab194699), pMLKL (S345, ab196436) and pMLKL (S358, ab187091) (Abcam, Cambridge, UK). β-actin and GAPDH (Affinity Biosciences, USA), N-cadherin (13769-1-AP) and S100A8 (15792-1- AP) (Proteintech, Rosemont, IL, USA).

Techniques: Expressing, Control, Staining, Immunohistochemical staining, Western Blot, Standard Deviation

Journal: Cell death & disease

Article Title: Inhibition of keratinocyte necroptosis mediated by RIPK1/RIPK3/MLKL provides a protective effect against psoriatic inflammation.

doi: 10.1038/s41419-020-2328-0

Figure Lengend Snippet: Fig. 6 Comparison of the inhibitory effect of Nec-1s, GSK’872 and NSA on necroptosis in vitro. a–c HaCaT cells were treated with TNF-α (100 ng/mL) and Smac (100 nM) for 8 h to induce apoptosis (hereafter abbreviated as TS). Necroptosis of HaCaT cells was induced by TNF-α (100 ng/ mL), Smac (100 nM), and z-VAD-fmk (20 μM) for 8 h (hereafter abbreviated as TSZ). The necroptosis cells were treated separately with the RIPK1 inhibitor Nec-1s (10 μM), the RIPK3-inhibitor GSK’872 (10 μM), and the MLKL-inhibitor NSA (5 μM). a Representative transmission electron microscopy (TEM) pictures of HaCaT cells treated as above. Representative images are shown. Scale bars represent 1 μm. b Protein levels of RIPK1, RIPK3, MLKL, pMLKL (S358) and HMGB1 were analyzed by western blotting. GAPDH was used as a loading control. c The mRNA level of IL-1β, IL-6, IL-8, and CXCL1 was analyzed by real-time quantitative PCR. d MLKL oligomerized in membrane faction upon necrosis induction. The cells were harvested and then separated into cytoplasmic and membrane proteins. Protein levels of MLKL and pMLKL (S358) were analyzed by western blotting. GAPDH was used as cytoplasm protein control. N-cadherin was used as membrane protein control. Results shown are representative data of three independent experiments. Error bars represent mean ± standard deviation (SD). ns p > 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001 when compared.

Article Snippet: HMGB1 (ab79823), RIPK3 (ab56164), pRIPK3(S227, ab209384), pRIPK3 (S232, ab195117), MLKL (ab194699), pMLKL (S345, ab196436) and pMLKL (S358, ab187091) (Abcam, Cambridge, UK). β-actin and GAPDH (Affinity Biosciences, USA), N-cadherin (13769-1-AP) and S100A8 (15792-1- AP) (Proteintech, Rosemont, IL, USA).

Techniques: Comparison, In Vitro, Transmission Assay, Electron Microscopy, Western Blot, Control, Real-time Polymerase Chain Reaction, Membrane, Standard Deviation

Journal: Oncotarget

Article Title: Alcohol intake aggravates adipose browning and muscle atrophy in cancer-associated cachexia

doi: 10.18632/oncotarget.22243

Figure Lengend Snippet: (A-C) Pro-caspase 1, activated caspase 1, Pro-caspase 3 and activated caspase 3 protein contents in GA muscle. (D-F) NLRP3 and IL-1β protein contents in GA muscle. ( * p < 0.05, mean ± SEM).

Article Snippet: Antibody against PYD-domains-containing protein 3 (NLRP3) (#PA1665) was purchased from Boster Biological Technology (Fremont, CA).

Techniques:

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/BCJ20230035

Figure Lengend Snippet: ( A ) Schematic of the necroptosis pathway. TNF (T) activates TNFR1, the Smac-mimetic Compound A (S) blocks cIAP activity and the pan-caspase inhibitor Q-VD-OPh (Q) blocks caspase-8 activity. This TSQ stimulus results in activation of RIPK1 and RIPK3, and subsequent phosphorylation and activation of MLKL, which causes MLKL-mediated membrane disruption and cell death. ( B ) Schematic of the constitutively activated mouse MLKL mutant, Q343A. Expression of MLKL Q343A using doxycycline causes cell death in the absence of upstream necroptotic stimuli. This enabled a cell-based phenotypic screen for small molecules that modulate necroptosis at the level or downstream of MLKL activation. The skull and crossbones image (Mycomorphbox_Deadly.png; by Sven Manguard) in ( A , B ) was used under a Creative Commons Attribution-Share Alike 4.0 license. ( C ) Schematic of the cell-based phenotypic screen. A total of 5632 compounds from the WEHI small molecule library along with 40 kinase inhibitors were screened against wild-type or Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the MLKL Q343A mutant. The ability of the small molecules to inhibit cell death was measured by CellTiter-Glo cell viability assays. ABT-869, a VEGF and PDGF receptor tyrosine kinase inhibitor, was identified as a hit. See also Supplementary Figure S1A. ( D ) Chemical structure of ABT-869 and its analogue WEHI-615. ( E ) Wild-type mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( F ) Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( G ) Wild-type mouse dermal fibroblast (MDF) cells were stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) to induce necroptosis and treated with increasing concentrations of ABT-869 or WEHI-615. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 4 independent experiments and errors bars represent SEM.

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques: Activity Assay, Activation Assay, Phospho-proteomics, Membrane, Disruption, Mutagenesis, Expressing, Staining, Flow Cytometry

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/BCJ20230035

Figure Lengend Snippet: ( A , B ) Wild-type mouse dermal fibroblast (MDF) cells were treated with increasing concentrations of ABT-869 or control compounds, RIPK3 inhibitors GSK′872 and GSK′843, DMSO alone or left untreated (UT) for 1 h then stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) ( A ) or TSZ (TNF, Smac-mimetic, z-VAD-fmk) ( B ) for 24 h to induce necroptosis. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 3 ( A ) or n = 4 ( B ) independent experiments and error bars represent SEM. ( C – F ) Human U937 cells were treated with increasing concentrations of ABT-869 or control compounds, MLKL inhibitor NSA and RIPK1 inhibitor GSK′481, DMSO alone or left untreated (UT) for 1 h then stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) for 48 h ( C ) or TSI (TNF, Smac-mimetic, IDN-6556) for 24 h ( E ) to induce necroptosis. Parallel experiments were performed to assess protection of TSQ ( D ) or TSI ( F ) induced death in the presence of the ABT-869 analogue, WEHI-615. Cell death was monitored by SPY505 (live cells) and propidium iodide (PI; dead cells) uptake using IncuCyte live cell imaging. One representative result shown from n = 4 ( C , D ) or n = 3 ( E , F ) independent experiments. See also Supplementary Figure S2A–H.

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques: Control, Staining, Flow Cytometry, Live Cell Imaging

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/BCJ20230035

Figure Lengend Snippet: ( A ) Binding affinities ( K D ) of ABT-869 and WEHI-615 for human full-length MLKL, RIPK1 kinase domain and RIPK3 kinase domain measured by competition binding assays from the DiscoverX KINOME scan platform using the Kd ELECT service. Each value is the mean of two replicates. ( B – D ) Cellular Thermal Shift Assays (CETSA) in mouse and human cells. Mlkl −/− mouse dermal fibroblast (MDF) cells expressing MLKL Q343A ( B ), wild-type MDF cells ( C ) and human U937 cells ( D ) were treated with DMSO, ABT-869, WEHI-615, RIPK1 inhibitor Nec-1s, RIPK3 inhibitor GSK′872 or human RIPK1 inhibitor GSK′481 (all 20 µM). Cells were subjected to an increasing temperature gradient focused around the melting temperature of the protein of interest. Following the separation of soluble and insoluble proteins, the remaining soluble proteins were detected by Western blot. Red asterisks denote protein standards. One representative result shown from n = 3 ( B , C ) or n = 2–3 ( D ) independent experiments. See also Supplementary Figure S3A–C.

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques: Binding Assay, Expressing, Western Blot

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/BCJ20230035

Figure Lengend Snippet: Thermal Shift Assays (TSA) with mouse and human RIPK1 and RIPK3 kinase domains. Increasing concentrations of ABT-869 or WEHI-615 were tested for their ability to alter the melting temperature ( T M ) of mouse RIPK1 (9.5 µg) ( A , B ), human RIPK1 (12 µg) ( C , D ), mouse RIPK3 (10 µg) ( E , F ) and human RIPK3 (6.5 µg) ( G , H ) compared with the positive controls Compound 2 for mouse RIPK1, GSK′481 for human RIPK1 and GSK′872 for mouse and human RIPK3 (all 30 µM). Data represent the mean of n = 3 independent experiments and error bars represent SEM. See also Supplementary Figure S4A–H.

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques:

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/BCJ20230035

Figure Lengend Snippet: ( A – H ) In vitro phosphorylation assays with mouse and human RIPK1 and RIPK3 kinase domains measured by ADP-Glo Kinase Assays. Increasing concentrations of ABT-869 or WEHI-615 were tested for their ability to inhibit the autophosphorylation (IC 50 ) of mouse RIPK1 (200 nM) ( A , B ), human RIPK1 (200 nM) ( C , D ), mouse RIPK3 (10 nM) ( E , F ) and human RIPK3 (10 nM) ( G , H ). Data represent the mean of n = 3 ( A , B , E , F ) or n = 2 ( C , D , G , H ) independent experiments and error bars represent SEM. ( I ) Cellular phosphorylation assays. Wild-type mouse dermal fibroblast (MDF) cells were treated with DMSO, ABT-869, WEHI-615, RIPK1 inhibitor Nec-1s or RIPK3 inhibitor GSK′872 for 2 h then stimulated with TSI (TNF, Smac-mimetic, IDN-6556) for 2 h to induce autophosphorylation of RIPK1 and RIPK3. Ripk1 −/− Mlkl −/− MDF cells and Ripk3 −/− MDF cells were included as controls. Phospho-RIPK1 and phospho-RIPK3 protein levels were detected from whole cell lysates by Western blot. Red asterisks denote protein standards. One representative result shown from n = 3 independent experiments. See also Supplementary Figure S5A–C.

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques: In Vitro, Phospho-proteomics, Western Blot

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/BCJ20230035

Figure Lengend Snippet:

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques: Western Blot, Produced, Phospho-proteomics, Transduction

Journal: Biochemical Journal

Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

doi: 10.1042/BCJ20230035

Figure Lengend Snippet:

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques:

Journal: bioRxiv

Article Title: Transcriptome-based lead generation, ligand- and structure-based prioritization and experimental validation of TLR5-activating molecules

doi: 10.64898/2026.02.25.707690

Figure Lengend Snippet: This figure shows all the H-bond interactions between robust CMap-generated leads and TLR5 which fall below 3 Å. The minimum 3 H-bond interactions between CMap-generated leads and TLR5 were showed by both Glide module and HADDOCK tool in each complex.

Article Snippet: TLR5 secreted in the culture supernatants were measured using Human Toll-like Receptor 5, TLR5 ELISA kit (BT LAB, #E0374Hu) according to the manufacturer’s protocol.

Techniques: Generated

Journal: bioRxiv

Article Title: Transcriptome-based lead generation, ligand- and structure-based prioritization and experimental validation of TLR5-activating molecules

doi: 10.64898/2026.02.25.707690

Figure Lengend Snippet: (A) CAL 27 cells were incubated with the CMap-generated leads: Cytarabine (200µM, 100µM, 50µM), Azacytidine (200µM, 100µM, 50µM), Penicillin (200µM, 100µM, 50µM), Streptozotocin (400µM, 200µM, 100µM), Piceatannol (400µM, 200µM, 100µM) and flagellin (5ng/ml) for 24h. (B) CAL 27 cells were incubated with the CMap-generated leads: Fenoterol (400µM, 200µM, 100µM), ABT-751 (200µM, 100µM, 50µM), Ganciclovir (200µM, 100µM, 50µM), Kinetin-riboside (100µM, 50µM, 25µM), and flagellin (5ng/ml) for 24h. Bar graph representing the relative expression levels of TLR5 with respect to the untreated control. Fold change is calculated by the signal of the treated sample divided by the signal of the control. If the fold change is >1, TLR5 expression is upregulated whereas fold change <1 is taken as downregulated. All values are expressed as mean ± SEM.

Article Snippet: TLR5 secreted in the culture supernatants were measured using Human Toll-like Receptor 5, TLR5 ELISA kit (BT LAB, #E0374Hu) according to the manufacturer’s protocol.

Techniques: Incubation, Generated, Expressing, Control