Journal: Oncology Reports

Article Title: TNF-α promotes the malignant transformation of intestinal stem cells through the NF-κB and Wnt/β-catenin signaling pathways

doi: 10.3892/or.2020.7631

Figure Lengend Snippet: Effects of inhibitors of the NF-κB and Wnt/β-catenin pathways on their activities in TNF-α-induced NCM460s cells. NCM460s cells were respectively incubated with 1, 10, 100 µM of (A) PDTC or (B) IWP-2 without or with 1 ng/ml TNF-α for 3 h. Western blotting was used to analyze the protein expression of genes related with NF-κB and Wnt/β-catenin signaling in the total cell extracts and the nuclear/cytoplasmic extracts. TNF, tumor necrosis factor; NCM460s, NCM460 spheroid; PDTC, pyrrolidine dithiocarbamate; IKKα/β, IκB kinase complex α/β; GSK, glycogen synthase kinase.

Article Snippet: The NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC; cat. no. M4005) and Wnt inhibitor IWP-2 (cat. no. M2237) were purchased from Abmole (Abmole Bioscience, Inc.).

Techniques: Incubation, Western Blot, Expressing

Journal: Marine Drugs

Article Title: FGFC1 Exhibits Anti-Cancer Activity via Inhibiting NF-κB Signaling Pathway in EGFR -Mutant NSCLC Cells

doi: 10.3390/md20010076

Figure Lengend Snippet: FGFC1 suppressed the NF-κB signaling pathway in NSCLC PC9 cells. ( A ) Volcano plot of DEGs, x -axis represents log2 transformed fold change, the y -axis represents -log10 transformed significance, red points represent up-regulated DEG, blue points represent down-regulated DEG, black points represent non-DEGs. ( B ) NF-κB-related genes were significantly suppressed by the treatment of FGFC1. PC9 cells were treated with DMSO or FGFC1 (10 µM) for 24 h, respectively. Total mRNA of different groups was prepared, heatmap of 28 DEGs that were significantly affected by FGFC1. Red indicated up-regulated genes and green indicated the down-regulated genes. Values were calculated from the regularized log transformation of fold change. The sequencing data were provided by The Beijing Genomics Institute (BGI). ( C ) Real-time PCR assays validated the inhibitory effects of the indicated FGFC1 (0, 5, 10, and 20 µM, 24 h) on NF-κB downstream genes (IL-6, ICAM-1, TNF-α). ( D ) FGFC1 remarkably suppressed the phosphorylation of the NF-κB pathway and inhibited its downstream targets. PC9 and H1299 cells were treated with the indicated concentrations of FGFC1 (0, 5, 10, and 20 µM) for 24 h. The expression of p-IKKα/β, IKKα, IKKβ, p-p65, p65, p-IκBα, IκBα, TNF-α, and IL-6 was examined by Western blotting. β-actin was used as an endogenous loading control. ( E ) Quantitative results of the protein levels in ( D ). p-IKKα/β/IKKα, p-IKKα/β/IKKβ, p-p65/p65, p-IκBα/IκBα, TNF-α/β-actin and IL-6/β-actin ratios in Western blotting. ( F ) PC9 cells were treated with FGFC1 (10 µM) and PDTC (10 µM) alone or in combination for 24 h. The expression of p-p65, p65, p-IκB, IκB, TNF-α, IL-6, CDK4, and Cyclin D1 was evaluated by Western blotting. β-actin was used as an endogenous loading control. ( G ) Quantitative results of the protein levels in ( F ). p-p65/p65, p-IκBα/IκBα, TNF-α/β-actin, IL-6/β-actin, CDK4/β-actin, Cyclin D1/β-actin, cleaved-PARP-1/β-actin, and cleaved-caspase-3/β-actin ratios in Western blotting. ( H ) FGFC1 significantly inhibited the colony formation of PC9 cells through the NF-κB signaling pathway. PC9 cells were treated with FGFC1 (10 µM) and PDTC (10 µM) for 10 days. Colonies were stained with 0.1% crystal violet and photographed. ( I ) Quantification of the cell colony numbers in ( H ). All data were represented as the means ± SD for at least three independent experiments. (* p < 0.05 and ** p < 0.01 vs. the DMSO control).

Article Snippet: Cell Counting Kit-8 (CCK8), RIPA lysis buffer, crystal violet, NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC), and Cell Cycle and Apoptosis Analysis Kit were purchased from Beyotime Biotechnology Co. (Shanghai, China).

Techniques: Transformation Assay, Sequencing, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Staining

Journal: Clinical and Experimental Immunology

Article Title: Dynamin inhibition interferes with inflammasome activation and cytokine gene expression in Streptococcus pyogenes -infected human macrophages

doi: 10.1111/cei.12425

Figure Lengend Snippet: Multiple signalling pathways are involved in group A streptococcus (GAS)-induced cytokine gene expression. Macrophages were left untreated or treated with pharmacological signalling inhibitors for 30 min prior to stimulation with live GAS [multiplicity of infection (MOI) 10]. Cells were collected at 3 h after stimulation, total cellular RNA was isolated and (a) interleukin (IL)-1β, tumour necrosis factor (TNF)-α, (b) interferon (IFN)-β, chemokine (C-X-C motif) ligand (CXCL)-10 and (c) NACHT-domain-, leucine-rich repeat- and PYD-containing protein 3 (NALP3) mRNA levels were determined by quantitative reverse transcription–polymerase chain reaction (qRT–PCR). Relative mRNA levels were normalized against β-actin and the relative level of mRNA was calculated with the ΔΔ comparative threshold (Ct) method using dimethylsulphoxide (DMSO)-treated cells as a calibrator. The used inhibitors were: PD98059 [mitogen-activated protein kinase (MEK)1 inhibitor, 10 μmol/l], SB202190 (p38 inhibitor, 10 μmol/l), SP600125 [stress-activated protein kinase/c-Jun N-terminal kinase (JNK) inhibitor, 10 μmol/l], LY294002 (PI3K inhibitor, 50 μmol/l), and pyrrolidine dithiocarbamate (PDTC) [nuclear factor (NF)-κB inhibitor, 100 μmol/l]. Results are the means (± standard deviation) of three independent experiments performed with cells of four blood donors (n = 12). *P < 0·05 between DMSO-treated and inhibitor-treated GAS-infected cells. Note the differences in scales.

Article Snippet: Signalling inhibitors PD98059 [mitogen-activated protein kinase (MEK1) inhibitor, used at 10 μmol/l] and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}} LY294002 (PI3K inhibitor, 50 μmol/l) were obtained from Calbiochem (San Diego, CA, USA) and SB202190 (p38 inhibitor, 10 μmol/l), SP600125 [stress-activated protein kinase/c-Jun N-terminal kinase (JNK) inhibitor, 10 μmol/l] and pyrrolidine dithiocarbamate [PDTC, specific nuclear factor (NF)-κB inhibitor, 100 μmol/l] were purchased from Alexis Biochemicals (Lausen, Switzerland).

Techniques: Expressing, Infection, Isolation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation

Journal: Clinical and Experimental Immunology

Article Title: Dynamin inhibition interferes with inflammasome activation and cytokine gene expression in Streptococcus pyogenes -infected human macrophages

doi: 10.1111/cei.12425

Figure Lengend Snippet: Pharmacological signalling inhibitors interfere with the processing and secretion of interleukin (IL)-1β. (a) Macrophages from three blood donors were left untreated or treated with different signalling inhibitors 30 min prior to stimulation with live group A streptococcus (GAS) [multiplicity of infection (MOI) 10] for 9 h. Cell lysates were prepared, proteins were separated on 15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting was performed with rabbit anti-human-IL-1β antibody. Staining of β-actin functions as a loading control. (b) Cell culture supernatants were collected at 9 h after GAS-stimulation and secreted IL-1β levels were determined by enzyme-linked immunosorbent assay (ELISA). The used inhibitors were: PD98059 [specific mitogen-activated protein kinase (MEK)1 inhibitor, 10 μmol/l], SB202190 (p38 inhibitor, 10 μmol/l), SP600125 [stress-activated protein kinase/janus kinase (JNK0 inhibitor, 10 μmol/l], LY294002 (PI3K inhibitor, 50 μmol/l), and pyrrolidine dithiocarbamate (PDTC) [nuclear factor (NF)-κB inhibitor, 100 μmol/l]. The experiment was carried out with cells obtained from three different blood donors, the columns represent the means and error bars indicate standard deviations of the means. *P < 0·05 between dimethylsulphoxide (DMSO)-treated and inhibitor-treated GAS-infected cells.

Article Snippet: Signalling inhibitors PD98059 [mitogen-activated protein kinase (MEK1) inhibitor, used at 10 μmol/l] and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}} LY294002 (PI3K inhibitor, 50 μmol/l) were obtained from Calbiochem (San Diego, CA, USA) and SB202190 (p38 inhibitor, 10 μmol/l), SP600125 [stress-activated protein kinase/c-Jun N-terminal kinase (JNK) inhibitor, 10 μmol/l] and pyrrolidine dithiocarbamate [PDTC, specific nuclear factor (NF)-κB inhibitor, 100 μmol/l] were purchased from Alexis Biochemicals (Lausen, Switzerland).

Techniques: Infection, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical and Translational Hepatology

Article Title: Mitochondrial GRIM19 Loss Induces Liver Fibrosis through NLRP3/IL33 Activation via Reactive Oxygen Species/NF-кB Signaling

doi: 10.14218/JCTH.2023.00562

Figure Lengend Snippet: (A–F) GRIM19 loss promotes NF-кB/p65 activation in vitro and in vivo. Total p65, p-p65, and NF-кB downstream targets IL6, TNFα, VEGF, VCAM1, and ICAM1 were analyzed by western blotting in GRIM19-deficient AML12 cells (A) and liver tissues (B). NF-кB-regulatory proteins pIKKα/β, IKKα/β, pIкBα, and IкBα were detected by western blotting in GRIM19-deficient AML12 cells and liver tissues (C). NF-кB p65 levels in nuclear or cytoplasmic extractions were analyzed by western blotting in GRIM19-deficient AML12 cells (D). p65 and p-p65 co-expression was detected by dual immunofluorescence (IF) staining in GRIM19-deficient AML12 cells (E) and liver tissues (F). (G) NF-кB inhibition reverses GRIM19 loss-driven NF-кB/p65 activation in vitro . GRIM19-deficient AML12 cells were treated with NF-кB inhibitor PDTC (0, 5, 10 µM) for 16 h. The expression of p65, p-p65, and NF-кB downstream targets were determined by western blotting. (H, I) Reactive oxygen species (ROS) scavenger abrogates GRIM19 loss-driven NF-кB/p65 activation in vitro . GRIM19-deficient AML12 cells were treated with NAC (0, 5, 10 mM) for 16 h, then p65, p-p65, and NF-кB downstream targets were detected by western blotting (H). NF-кB-regulatory proteins pIKKα/β, IKKα/β, pIкBα, and IкBα were detected by western blotting in GRIM19-deficient AML12 cells after NAC treatment (0, 5, 10 mM) for 16 h (I). β-actin was used as a loading control. DAPI was used to stain the nuclei. Mean fluorescent intensity (MFI) was used to quantify the expression of proteins in IF staining. Data are expressed as mean±SD. Scale bars: 50 µm. * p <0.05, ** p <0.01, *** p <0.001 between the indicated groups determined by unpaired student’s t-test . AML-12, alpha mouse liver 12; NC, negative control; NAC, N-acetylcysteine; PDTC, ammonium pyrrolidinedithiocarbamate; IL-6, interleukin 6; VCAM-1, vascular cell adhesion molecule 1; ICAM-1, intercellular adhesion molecular 1; VEGF, vascular endothelial growth factor; TNF, tumour necrosis factor; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The ROS scavenger N-acetylcysteine (NAC), NF-кB inhibitor PDTC, and Caspase1 inhibitor VX765 were obtained from AbMole Bioscience (Houston, TX, USA).

Techniques: Activation Assay, In Vitro, In Vivo, Western Blot, Expressing, Immunofluorescence, Staining, Inhibition, Control, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: Mitochondrial GRIM19 Loss Induces Liver Fibrosis through NLRP3/IL33 Activation via Reactive Oxygen Species/NF-кB Signaling

doi: 10.14218/JCTH.2023.00562

Figure Lengend Snippet: (A) GRIM19 loss increases IL1β and IL33 levels. IL1β and IL33 cytokines were detected by flow cytometry in GRIM19-deficient AML12 cells. (B) GRIM19 loss triggers NLRP3 inflammasome activation in vitro . NLRP3 inflammasome complex, as well as IL1β and IL33, were detected by western blotting in GRIM19-deficient AML12 cells. (C) ROS inhibition attenuates GRIM19 loss-induced NLRP3 inflammasome activation in vitro . GRIM19-deficient AML12 cells were treated with NAC (0, 5, 10 mM) for 16 h. NLRP3 inflammasome, IL1β and IL33 were detected by western blotting. (D) NF-кB blockage decreases GRIM19 loss-triggered NLRP3 inflammasome activation in vitro . GRIM19-deficient AML12 cells were treated with NF-кB inhibitor PDTC (0, 5, 10 µM) for 16 h. NLRP3 inflammasome, IL1β, and IL33 levels were detected by western blotting. (E) NLRP3 inhibition reduces GRIM19 loss-induced IL1β and IL33 expression in vitro . GRIM19-deficient AMl12 cells were treated with NLRP3 inhibitor MCC950 (0, 2.5, 5 µM) for 16 h. NLRP3 inflammasome, IL1β, and IL33 were detected by western blotting. (F) Caspase1 repression attenuates GRIM19 loss-triggered IL1β and IL33 expression in vitro . GRIM19-deficient AML12 cells were treated with Caspase1 inhibitor VX765 (0, 20, 40 µM) for 24h. Caspase1, IL1β, and IL33 expressions were determined by western blotting. β-actin was used as a loading control. Data are presented as mean±SD of three independent experiments. Representative images are shown. IL, interleukin; NAC, N-acetylcysteine; PDTC, ammonium pyrrolidine dithiocarbamate; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; NC, negative control.

Article Snippet: The ROS scavenger N-acetylcysteine (NAC), NF-кB inhibitor PDTC, and Caspase1 inhibitor VX765 were obtained from AbMole Bioscience (Houston, TX, USA).

Techniques: Flow Cytometry, Activation Assay, In Vitro, Western Blot, Inhibition, Expressing, Control, Negative Control

Journal: Regenerative Biomaterials

Article Title: Calcium silicate enhances immunosuppressive function of MSCs to indirectly modulate the polarization of macrophages

doi: 10.1093/rb/rbab056

Figure Lengend Snippet: Effects of CS ionic products on the expression of immunosuppressive factors in HBMSCs. ( A ) Gene expression of COX-2, TSG-6, PTGES2 and HGF in HBMSCs cultured with CS ionic products (MSCM CS 1/64 and MSCM CS 1/128) or without CS ionic products (MSCM CS0). ( B , C ) Immunofluorescence staining and western blot analysis results of COX-2 in HBMSCs cultured with (MSCM CS 1/64 and MSCM CS 1/128) or without CS ionic products (MSCM CS0). ( D ) Effects of CS ionic products on the gene expression of immunosuppressive factors in HBMSCs pre-treated with or without NF-κB inhibitor. * P < 0.05 and ** P < 0.01

Article Snippet: For inhibiting NF-κB pathway in HBMSCs, 1 mM NF-κB inhibitor, pyrrolidinedithiocarbamic acid ammonium salt (PDTC, Beyotime, China) was added to half of the MSCM CS 0 and MSCM CS 1/64 groups while another half of groups was kept as original.

Techniques: Expressing, Gene Expression, Cell Culture, Immunofluorescence, Staining, Western Blot