Journal: Scientific Reports

Article Title: A phenotypic high-content, high-throughput screen identifies inhibitors of NLRP3 inflammasome activation

doi: 10.1038/s41598-021-94850-w

Figure Lengend Snippet: NLRP3 inflammasome inhibitor hits validation in the speck assay. ( a ) Schematic of the priming protocol. ( b ) Schematic of the activation protocol. ASC-mCherry iBMDM cells were treated with indicated concentrations of ( c ) PU H71, ( d ) MPC-3100, ( e ) momelotinib, ( f ) CEP-33779, ( g ) ACHP or ( h ) MLN120B with LPS (1 µg/ml) for 2 h followed by nigericin treatment (10 µM, 2 h) after which PFA was added (priming—blue trace) and specks counted. In a parallel experiment the compounds were added after LPS treatment just prior to nigericin (activation—red trace). The images are representative of the nuclei and speck formation for each compound at 10 µM in both priming and activation protocols. Data are presented as mean ± SEM, n = 3 independent experiments. Data in ( c – h ) was analyzed using GraphPad Prism version 7 software ( https://www.graphpad.com/scientific-software/prism/ ).

Article Snippet: MCC950 (CAS number: 256373-96-3) was purchased from Tocris; PU H71 (Cat. number: 1856) and momelotinib (CAS number: 1056634-68-4) were purchased from Axon Medchem; MPC-3100 (Cat. number: A4063) was purchased from ApexBio; MLN120B (CAS number: 783348-36-7), CEP-33779 (CAS number: 958025-66-6) and ACHP (CAS number: 406209-26-5) were purchased from MedChemExpress.

Techniques: Activation Assay, Software

Journal: Cell host & microbe

Article Title: Mitochondria-derived vesicles deliver antimicrobial reactive oxygen species to control phagosome-localized Staphylococcus aureus

doi: 10.1016/j.chom.2018.10.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: NEMO binding domain (NBD) inhibitor peptide and control peptide , Novus Biologicals , Cat# NBP2-26504.

Techniques: Mutagenesis, Virus, Transformation Assay, Binding Assay, Control, Molecular Weight, Flow Cytometry, shRNA, Reverse Transcription, Transfection, Software, Imaging

Journal: Antioxidants

Article Title: Gastro-Protective Effects of Albizia anthelmintica Leaf Extract on Indomethacin-Induced Gastric Ulcer in Wistar Rats: In Silico and In Vivo Studies

doi: 10.3390/antiox10020176

Figure Lengend Snippet: Effects of indomethacin alone and with oral pre-treatments of famotidine or A. anthelmintica (200, 100 mg/kg) on ( A ) gastric inhibitor of nuclear factor kappa-B kinase subunit beta (IKKB), ( B ) gastric nuclear factor KB (NF-κB), ( C ) gastric TNF-α, and ( D ) gastric IL-6 in rats. Statistical analyses were carried out by one-way ANOVA followed by Tukey’s post hoc test, mean ± SEM. * Significantly different from control group at p < 0.05. @ Significantly different from indomethacin alone group at p < 0.05.

Article Snippet: Primary rabbit polyclonal antibodies against inhibitor of nuclear factor kappa-B kinase subunit beta (IKK-β, catalogue number: PA2036-1) and rabbit monoclonal antibodies against β-actin (catalogue number: M01263) were purchased from Boster Biological Technology (Pleasanton, CA, USA) and diluted at 1:1000 and 1:5000, respectively.

Techniques: Control

Journal: PLoS ONE

Article Title: Regional Susceptibility to TNF-α Induction of Murine Brain Inflammation via Classical IKK/NF-κB Signalling

doi: 10.1371/journal.pone.0039049

Figure Lengend Snippet: A: p65 expression in primary cortical neurons, showing increased expression with TNF-α and decreased expression with IKK Inhibitor (F = 177.79; p<0.001; df = 2,12). B: NF-κ B expression in a control neuron, using anti-p65 antibody and FITC labelled secondary, NF-κ B expression in a TNF-α stimulated neuron and NF-κ B expression in a TNF-α stimulated neuron with NF-κ B inhibitor. C: The percentage of neurons with anti-p65 staining in the nucleus (F = 30.63; p<0.001; df = 2,14), and D: the levels of fluorescence in each sample where fluorescence is rated between 0 and 3 on the scale bar (F = 17.65; p<0.002; df = 2,57). Molecular weight markers are in kilodaltons (kDa).

Article Snippet: For inhibition studies, 1 μg/ml IKK Inhibitor (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 150 μg/ml sodium salicylate (Santa Cruz Biotechnology) were added.

Techniques: Expressing, Control, Staining, Fluorescence, Molecular Weight

Journal: PLoS ONE

Article Title: Regional Susceptibility to TNF-α Induction of Murine Brain Inflammation via Classical IKK/NF-κB Signalling

doi: 10.1371/journal.pone.0039049

Figure Lengend Snippet: Tissue inflammation in Control (Ctrl), 10 ng/ml TNF-α (TNF), TNF-α with IKK Inhibitor (IKK), and TNF-α with sodium salicylate samples (Sodium S). A–C: Measurements at 0.5, 1 and 2 hours stimulation and after TNF-α removal (3 and 4 hours). Results are presented as for , data is significant after 2 hours of stimulation (F = 22.53; p = 0.001; df = 3,96). D-F: Plot of % volumetric increase of tissue vs. normalized overall expression of NF-κB p65 from quantification of Western blot samples with a superimposed line of linear regression.

Article Snippet: For inhibition studies, 1 μg/ml IKK Inhibitor (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 150 μg/ml sodium salicylate (Santa Cruz Biotechnology) were added.

Techniques: Control, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Regional Susceptibility to TNF-α Induction of Murine Brain Inflammation via Classical IKK/NF-κB Signalling

doi: 10.1371/journal.pone.0039049

Figure Lengend Snippet: Tissue inflammation in Control (Ctrl), 10 ng/ml TNF-α (TNF), TNF-α with IKK Inhibitor (IKK), and TNF-α with sodium salicylate samples (Sodium S). A–C: Measurements at 24, 48, 72 and 96 hour periods in A: frontal lobe, B: temporal region, and C: cerebellum. Data is significant after 24 hours of stimulation. Results are presented as a percentage increase of original value (F = 816.22; p<0.001; df = 3,96). D–F: Confirmation of inflammation with hematoxylin and eosin (H&E) stain. Tissue stimulated with TNF-α 10 ng/ml in frontal lobe (D), temporal region (E) and cerebellum (F). Calibration bars are 50 µm.

Article Snippet: For inhibition studies, 1 μg/ml IKK Inhibitor (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 150 μg/ml sodium salicylate (Santa Cruz Biotechnology) were added.

Techniques: Control, Staining

Journal: Cell Death & Disease

Article Title: Mesenchymal stem cells reverse EMT process through blocking the activation of NF-κB and Hedgehog pathways in LPS-induced acute lung injury

doi: 10.1038/s41419-020-03034-3

Figure Lengend Snippet: a The cocultured system with LPS-treated MLE-12 cells and MSCs was built. b Luciferase reporter assays detected the relative luciferase activity of various signaling pathways (Notch, Wnt, Nanog, NF-κB, PI3K/AKT, Oct4, Hedgehog, MAPK/JNK, and MAPK/ERK) in LPS-treated MLE-12 cells treated with or without MSC. c The mRNA level of Ikbkb, Chuk, Ikbkg, or RelA was examined by RT-qPCR after cocultured with MSC. d Western blot analysis determined the levels of IKKβ, p-IκBα, and p-IκBβ in the whole cell lysates as well as the nuclear protein level of p65 after cocultured with MSCs. e RT-qPCR analyzed the mRNA levels of hedgehog pathway key factors (Shh, Dhh, Ihh, Ptch1, Smo, and Gli1) in control cells and cocultured cells. f The luciferase activity of hedgehog pathway was detected in cocultured cells. g Relative mRNA level of Shh was measured by RT-qPCR in LPS-treated MLE-12 cells under four different conditions (control, MSC, KINK-1, and KINK-1+MSC). h Relative luciferase activity of hedgehog pathway was measured in LPS-treated MLE-12 cells under the same four conditions. ** p < 0.01. n.s. no statistical significance.

Article Snippet: The kinase inhibitor of NF-κB-1 (KINK-1; 5 μM), IKKβ inhibitor, was procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Luciferase, Activity Assay, Protein-Protein interactions, Quantitative RT-PCR, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Mesenchymal stem cells reverse EMT process through blocking the activation of NF-κB and Hedgehog pathways in LPS-induced acute lung injury

doi: 10.1038/s41419-020-03034-3

Figure Lengend Snippet: a After treated with CHX in three different time points, the protein level of IKKβ was tested in LPS-treated MLE-12 cells with or without MSC-exosome. b The protein level of IKKβ was detected in LPS-treated MLE-12 cells treated with control or MSC-exosome or control+MG132 or MSC-exosome+MG132. c Ubiquitination assay detected the ubiquitination of IKKβ protein in LPS-treated MLE-12 cells treated with MSC-exosome. d Pull-down silver staining was applied to unveil the proteins that might interact with IKKβ. e Co-IP assay demonstrated the interaction between Usp5 and IKKβ. f RT-qPCR and western blot examined the overexpression efficiency of Usp5 and the protein level of IKKβ in response to Usp5 overexpression. g Western blot analyzed the protein level of IKKβ in LPS-treated MLE-12 cells under four situations (control, MSC-exosome, MSC-exosome+pcDNA3.1/Usp5, or MSC-exosome+pcDNA3.1/Usp5+miR-182-5p inhibitor). h Luciferase reporter assays indicated the relative luciferase activity of Usp5 promoter in LPS-treated MLE-12 cells treated with MSC-exosome. i RT-qPCR and western blot detected the mRNA level and protein level of Usp5 in LPS-treated MLE-12 cells treated with MSC-exosome or MSC/sh-Dicer-exosome. ** p < 0.01. n.s. no statistical significance.

Article Snippet: The kinase inhibitor of NF-κB-1 (KINK-1; 5 μM), IKKβ inhibitor, was procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control, Ubiquitin Proteomics, Silver Staining, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Western Blot, Over Expression, Luciferase, Activity Assay

Journal: Cell Death & Disease

Article Title: Mesenchymal stem cells reverse EMT process through blocking the activation of NF-κB and Hedgehog pathways in LPS-induced acute lung injury

doi: 10.1038/s41419-020-03034-3

Figure Lengend Snippet: a StarBase v2.0 predicted miRNAs targeted to Usp5 were subjected to RT-qPCR analysis in LPS-treated MLE-12 cells with or without MSC coculture. b RT-qPCR analyzed miR-23a-3p expression in LPS-treated MLE-12 cells treated with MSC-exosome. c RT-qPCR analyzed miR-23a-3p expression in LPS-treated MLE-12 cells transfected with miR-23a-3p mimics. d The binding sequence between miR-23a-3p and Usp5 was shown. e Luciferase reporter assay examined the luciferase activity of indicated vectors in LPS-treated MLE-12 cells and HEK-293T cells co-transfected with miR-23a-3p mimics or NC mimics. f The expression level of Usp5 was detected by RT-qPCR in LPS-treated MLE-12 cells after the transfection of miR-23a-3p mimics. g Western blot measured the protein level of IKKβ in LPS-treated MLE-12 cells treated with different groups (control, MSC-exosome, MSC-exosome+miR-23a-3p inhibitor or MSC-exosome+miR-23a-3p inhibitor+miR-182-5p inhibitor). h After CHX treatment, the half-life of IKKβ was detected in LPS-treated MLE-12 cells transfected with NC mimics, miR-23a-3p mimics or miR-23a-3p mimics+pcDNA3.1/Usp5. i The ubiquitination level of IKKβ was detected in LPS-treated MLE-12 cells transfected with NC mimics, miR-23a-3p mimics or miR-23a-3p mimics+pcDNA3.1/Usp5. j Western blot examined the protein level of IKKβ in LPS-treated MLE-12 cells under seven conditions (control, MSC-exosome, MSC-exosome+miR-23a-3p inhibitor, MSC-exosome+miR-182-5p inhibitor, control+MG132, MSC-exosome+MG132, and MSC-exosome+miR-23a-3p inhibitor+miR-182-5p inhibitor+MG132). ** p < 0.01. n.s. no statistical significance.

Article Snippet: The kinase inhibitor of NF-κB-1 (KINK-1; 5 μM), IKKβ inhibitor, was procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Binding Assay, Sequencing, Luciferase, Reporter Assay, Activity Assay, Western Blot, Control, Ubiquitin Proteomics

Journal: Cell Death & Disease

Article Title: Mesenchymal stem cells reverse EMT process through blocking the activation of NF-κB and Hedgehog pathways in LPS-induced acute lung injury

doi: 10.1038/s41419-020-03034-3

Figure Lengend Snippet: Rescue assays were carried out in LPS-treated MLE-12 cells under four different contexts (control, MSC-exosome, MSC-exosome+miR-182-5p inhibitor, and MSC-exosome+miR-182-5p inhibitor+miR-23a-3p inhibitor). a The levels of nuclear p65, IKKβ, p-IKBα, and p-IKBβ were detected in LPS-treated MLE-12 cells using western blot. b IF staining examined the nuclear translocation of p65 in LPS-treated MLE-12 cells under diverse conditions. Scale bar = 50 μm. c The protein level of p65 in nucleus or cytoplasm was examined by western blot analysis in LPS-treated MLE-12 cells with MSC-exosome, MSC-exosome+miR-182-5p inhibitor or MSC-exosome+miR-23a-3p inhibitor. d Luciferase reporter assay examined the luciferase activity of hedgehog pathway in LPS-treated MLE-12 cells. e – i RT-qPCR detected the mRNA level of E-cadherin, α-SMA, TGF-β1, Collagen type I, and Collagen type III in indicated LPS-treated MLE-12 cells. j Western blot detected the protein level of E-cadherin, α-SMA, TGF-β1, Collagen type I, and Collagen type III in indicated LPS-treated MLE-12 cells. * p < 0.05, ** p < 0.01.

Article Snippet: The kinase inhibitor of NF-κB-1 (KINK-1; 5 μM), IKKβ inhibitor, was procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control, Western Blot, Staining, Translocation Assay, Luciferase, Reporter Assay, Activity Assay, Quantitative RT-PCR

Journal: Pharmaceuticals

Article Title: Natural Product-Based Screening for Lead Compounds Targeting SARS CoV-2 M pro

doi: 10.3390/ph16050767

Figure Lengend Snippet: ( a ) Scatter diagram of inhibition rates’ distribution of 2526 compounds to M pro at 80 μM. The gradient inhibition rates of compounds ( b ) fraxetin, ( c ) vanitiolide, ( d ) (−)–gallocatechin gallate, ( e ) hematoxylin, ( f ) wedelolactone, ( g ) β,β−dimethylacrylalkannin, ( h ) hydroxytyrosol ccetate, ( i ) Cholesterol 3–Sulfate Sodium Salt, ( j ) ginkgolic acid C15:1, ( k ) melanin, and ( l ) baicalein to M pro under two conditions; error bars: mean ± S.D. of three independent replicates.

Article Snippet: The Natural Product Library for HTS (catalog number L6000, 50 μL per hole, 10 mM, TargetMol, Shanghai, China, September 2020) was purchased from Shanghai Taosu Biochemical Technology Co., Ltd., including the following: fraxetin (catalog number T2909, TargetMol), vanitiolide (catalog number T0243, TargetMol), (−)–gallocatechin gallate (catalog number T3807, TargetMol), hematoxylin (catalog number T1686, TargetMol), wedelolactone (catalog number T3384, TargetMol), β,β–dimethylacrylalkannin (catalog number T5703, TargetMol), hydroxytyrosol acetate (catalog number T8168, TargetMol), cholesteryl sodium sulfate (catalog number T5239, TargetMol), ginkgolic acid C15:1 (catalog number T6S2123, TargetMol), melanin (catalog number HY-113485, MedChemExpress, Shanghai, China), baicalein (catalog number T2858, TargetMol), sanguinarine (catalog number T2781, TargetMol), corylifol A (catalog number T4S0145, TargetMol), M pro fluorescent substrate (MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2,C223JGC240-1/PE0977, GenScript, Nanjing, China), Sensor Chip SA (10291121, GE Healthcare, Shanghai, China), NHS-PEG12-Biotin (21313, Thermo Scientific, Beijing, China), ultrafiltration centrifuge tube (UFC901096, Millipore), 96-well Microtiter™ microplate (2305-11, Thermo Scientific), black 96-well plate (655077, Greiner Bio-One, Beijing, China), Triton™ X-100 (T109026, Aladdin, Beijing, China), Eppendorf tube (23-2052LK, Crystalgen, Beijing, China).

Techniques: Inhibition

Journal: Pharmaceuticals

Article Title: Natural Product-Based Screening for Lead Compounds Targeting SARS CoV-2 M pro

doi: 10.3390/ph16050767

Figure Lengend Snippet: The denaturation profile and the curves of the first derivative of the fluorescence as a function of temperature (−dF/dT) of ( a ) M pro + baicalein, ( b ) M pro + wedelolactone, ( c ) M pro + β,β–dimethylacrylalkannin, ( d ) M pro + cholesteryl sodium sulfate and ( e ) M pro + melanin, the T m value is represented as the highest part of the curve. The K obs for ( f ) baicalein, ( g ) wedelolactone, ( h ) β,β–dimethylacrylalkannin, ( i ) cholesteryl sodium sulfate and ( j ) melanin binding to M pro were derived from plotting ΔT m against log10 of compound concentrations and fitting to linear regression models.

Article Snippet: The Natural Product Library for HTS (catalog number L6000, 50 μL per hole, 10 mM, TargetMol, Shanghai, China, September 2020) was purchased from Shanghai Taosu Biochemical Technology Co., Ltd., including the following: fraxetin (catalog number T2909, TargetMol), vanitiolide (catalog number T0243, TargetMol), (−)–gallocatechin gallate (catalog number T3807, TargetMol), hematoxylin (catalog number T1686, TargetMol), wedelolactone (catalog number T3384, TargetMol), β,β–dimethylacrylalkannin (catalog number T5703, TargetMol), hydroxytyrosol acetate (catalog number T8168, TargetMol), cholesteryl sodium sulfate (catalog number T5239, TargetMol), ginkgolic acid C15:1 (catalog number T6S2123, TargetMol), melanin (catalog number HY-113485, MedChemExpress, Shanghai, China), baicalein (catalog number T2858, TargetMol), sanguinarine (catalog number T2781, TargetMol), corylifol A (catalog number T4S0145, TargetMol), M pro fluorescent substrate (MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2,C223JGC240-1/PE0977, GenScript, Nanjing, China), Sensor Chip SA (10291121, GE Healthcare, Shanghai, China), NHS-PEG12-Biotin (21313, Thermo Scientific, Beijing, China), ultrafiltration centrifuge tube (UFC901096, Millipore), 96-well Microtiter™ microplate (2305-11, Thermo Scientific), black 96-well plate (655077, Greiner Bio-One, Beijing, China), Triton™ X-100 (T109026, Aladdin, Beijing, China), Eppendorf tube (23-2052LK, Crystalgen, Beijing, China).

Techniques: Fluorescence, Binding Assay, Derivative Assay

Journal: Pharmaceuticals

Article Title: Natural Product-Based Screening for Lead Compounds Targeting SARS CoV-2 M pro

doi: 10.3390/ph16050767

Figure Lengend Snippet: Binding pattern of compounds ( a ) baicalein, ( b ) cholesteryl sodium sulfate, ( c ) ginkgolic acid C15:1, ( d ) wedelolactone, ( e ) (−)–gallocatechin gallate, ( f ) melanin, ( g ) β,β–dimethylacrylalkannin, ( h ) hematoxylin to M pro (PDB code: 7RFS) active site. The red and blue colors on the protein surface are the surface electrostatic potential, with red representing negative charge and blue representing positive charge.

Article Snippet: The Natural Product Library for HTS (catalog number L6000, 50 μL per hole, 10 mM, TargetMol, Shanghai, China, September 2020) was purchased from Shanghai Taosu Biochemical Technology Co., Ltd., including the following: fraxetin (catalog number T2909, TargetMol), vanitiolide (catalog number T0243, TargetMol), (−)–gallocatechin gallate (catalog number T3807, TargetMol), hematoxylin (catalog number T1686, TargetMol), wedelolactone (catalog number T3384, TargetMol), β,β–dimethylacrylalkannin (catalog number T5703, TargetMol), hydroxytyrosol acetate (catalog number T8168, TargetMol), cholesteryl sodium sulfate (catalog number T5239, TargetMol), ginkgolic acid C15:1 (catalog number T6S2123, TargetMol), melanin (catalog number HY-113485, MedChemExpress, Shanghai, China), baicalein (catalog number T2858, TargetMol), sanguinarine (catalog number T2781, TargetMol), corylifol A (catalog number T4S0145, TargetMol), M pro fluorescent substrate (MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2,C223JGC240-1/PE0977, GenScript, Nanjing, China), Sensor Chip SA (10291121, GE Healthcare, Shanghai, China), NHS-PEG12-Biotin (21313, Thermo Scientific, Beijing, China), ultrafiltration centrifuge tube (UFC901096, Millipore), 96-well Microtiter™ microplate (2305-11, Thermo Scientific), black 96-well plate (655077, Greiner Bio-One, Beijing, China), Triton™ X-100 (T109026, Aladdin, Beijing, China), Eppendorf tube (23-2052LK, Crystalgen, Beijing, China).

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Natural Product-Based Screening for Lead Compounds Targeting SARS CoV-2 M pro

doi: 10.3390/ph16050767

Figure Lengend Snippet: The drug-like properties and pharmacokinetic parameters of screening compounds.

Article Snippet: The Natural Product Library for HTS (catalog number L6000, 50 μL per hole, 10 mM, TargetMol, Shanghai, China, September 2020) was purchased from Shanghai Taosu Biochemical Technology Co., Ltd., including the following: fraxetin (catalog number T2909, TargetMol), vanitiolide (catalog number T0243, TargetMol), (−)–gallocatechin gallate (catalog number T3807, TargetMol), hematoxylin (catalog number T1686, TargetMol), wedelolactone (catalog number T3384, TargetMol), β,β–dimethylacrylalkannin (catalog number T5703, TargetMol), hydroxytyrosol acetate (catalog number T8168, TargetMol), cholesteryl sodium sulfate (catalog number T5239, TargetMol), ginkgolic acid C15:1 (catalog number T6S2123, TargetMol), melanin (catalog number HY-113485, MedChemExpress, Shanghai, China), baicalein (catalog number T2858, TargetMol), sanguinarine (catalog number T2781, TargetMol), corylifol A (catalog number T4S0145, TargetMol), M pro fluorescent substrate (MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2,C223JGC240-1/PE0977, GenScript, Nanjing, China), Sensor Chip SA (10291121, GE Healthcare, Shanghai, China), NHS-PEG12-Biotin (21313, Thermo Scientific, Beijing, China), ultrafiltration centrifuge tube (UFC901096, Millipore), 96-well Microtiter™ microplate (2305-11, Thermo Scientific), black 96-well plate (655077, Greiner Bio-One, Beijing, China), Triton™ X-100 (T109026, Aladdin, Beijing, China), Eppendorf tube (23-2052LK, Crystalgen, Beijing, China).

Techniques: Molecular Weight, Solubility, Permeability