Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: The expression of S100A8 in microglial cell induces the Cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE 2) pathway. BV-2 cells were transfected with S100A8 shRNA or Scramble vector. After 24 h, cells were incubated in hypoxic condition for 48 h. ( A ) The mRNA and ( B ) the protein levels of S100A8 and COX-2 were detected by real-time PCR and western blotting. ( C ) Secretion of PGE 2 level analyzed by ELISA. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, ** p < 0.01 versus control; # p < 0.05, ### p < 0.001 versus hypoxia-exposed sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, shRNA, Plasmid Preparation, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Huoxuezhitong capsule ameliorates MIA-induced osteoarthritis of rats through suppressing PI3K/ Akt/ NF-κB pathway.

doi: 10.1016/j.biopha.2020.110471

Figure Lengend Snippet: Fig. 5. HXZT presented anti-inflammatory activities in OA rats. After 5 weeks treatment, the serum PGE2 (A), TNF-α (B) and IL-1β (C) levels were examined by ELISA. Data were presented as mean ± SD. (n = 6, #p < 0.05, ##p < 0.01 compared with the Sham group; *p < 0.05, **p < 0.01 compared with the OA group).

Article Snippet: Next, the levels of PGE2 and IL-1β, TNF-α in serum were determined by ELISA according to the manufacturer's instruction (Elabscience, Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: BMC cancer

Article Title: Possible predisposition for colorectal carcinogenesis due to altered gene expressions in normal appearing mucosa from patients with colorectal neoplasia.

doi: 10.1186/s12885-019-5833-8

Figure Lengend Snippet: Fig. 1 Simplified schematic illustration of pathways for Wnt/β-catenin, ERK/MAPK and PI3K/Akt and PGE2-metabolism. A) Canonical Wnt/β-catenin signaling. The engagement of the Wnt receptor, Frizzled, leads to the inhibition of the β-catenin destruction complex, composed of APC, axin and GSK3β. β-catenin thereby avoids ubiquitination and subsequent degradation, thus allowing it to translocate to the nucleus to activate an array of regulatory genes. B) The RAS/RAF/MEK/ERK MAPK pathway. Stimulation of the receptor tyrosine kinase (RTK) or G-protein coupled receptors (GPCRs) leads to sequential activation of RAS, RAF, MEK, and ERK causing modification of substrates promoting cell survival and proliferation. C) In the PI3K/Akt pathway, activation of the RTK or GPCRs leads to sequential modification of phosphatidyl inositol residues of the phospholipid bilayer. In this process, PI3K generates PIP3. PIP3 in association with PDK1 activates Akt. Akt then modulates the activity of downstream substrates including mTOR, thus promoting proliferation and cell survival. D) PGE2-metabolism. PGE2-synthesis begins with catalytic hydrolysis of membrane phospholipids by cytoplasmic phospholipase A2 (cPLA2), thus releasing arachidonic acid (AA). By the action of the COX-1 and COX-2, AA is converted to prostaglandin H2 (PGH2). PGH2 is then converted to PGE2 by prostaglandin E synthase (PTGES). The main exporter of PGE2 is thought to be multi-drug resistance related polypeptide 4 (MRP4). Removal of PGE2 from the extracellular compartment around target cells occurs by diffusion to the blood stream and subsequent uptake and degradation in lung, liver or kidney endothelial cells or by import to colonic epithelial cells through the prostaglandin transporter (PGT) and subsequent degradation by 15-prostaglandin dehydrogenase (15-PGDH). Through autocrine and paracrine signaling, extracellular PGE2 stimulates the prostaglandin receptors EP1–4. The EPs are GPCRs with EP1 being Gαq-coupled while EP2 and EP4 are Gαs-coupled. EP3 is capable of coupling with different G-proteins including Gαi, Gαs and Gαq

Article Snippet: In order to inhibit both synthesis and catabolism of PGE2, the Ringer’s solution additionally contained 13 μM of indomethacin from Sigma-Aldrich (Seelze, Germany) and 110 μM of the 15-PGDH-inhibitor 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxy-benzeneacetic acid (Santa Cruz Biotechnology, Heidelberg, Germany, cat. no. 78028–01-0).

Techniques: Inhibition, Ubiquitin Proteomics, Activation Assay, Modification, Activity Assay, Membrane, Diffusion-based Assay

Journal: BMC cancer

Article Title: Possible predisposition for colorectal carcinogenesis due to altered gene expressions in normal appearing mucosa from patients with colorectal neoplasia.

doi: 10.1186/s12885-019-5833-8

Figure Lengend Snippet: Fig. 5 Mucosal prostaglandin E2 contents. On the left: Mucosal prostaglandin E2 concentrations. On the right: Mucosal concentration of prostaglandin E metabolites. Each mark represents one study patient. White squares = CTRL, control group (N = 14), Black dots = CRN group (N = 14)

Article Snippet: In order to inhibit both synthesis and catabolism of PGE2, the Ringer’s solution additionally contained 13 μM of indomethacin from Sigma-Aldrich (Seelze, Germany) and 110 μM of the 15-PGDH-inhibitor 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxy-benzeneacetic acid (Santa Cruz Biotechnology, Heidelberg, Germany, cat. no. 78028–01-0).

Techniques: Concentration Assay, Control

Journal: Pediatric research

Article Title: Lipocalin-2 and calprotectin as stool biomarkers for predicting necrotizing enterocolitis in premature neonates.

doi: 10.1038/s41390-021-01680-7

Figure Lengend Snippet: Fig. 1 Detection of CALPRO, LCN2, and HAPTO in the stools of NEC and non-NEC premature infants by ELISA. Stool samples analyzed were those collected over a 10-day period preceding NEC diagnosis (round symbols) and in matched non-NEC infants (square symbols). Simple linear regression for the levels of biomarkers in the stools were calculated for both NEC and non-NEC with R2 values varying between 0.02 and 0.0003 while the slopes were considered nonsignificant for all. For simplicity, only the regression line (black line, R2) for NEC samples are shown (left panels). The biomarker data were then subjected to ROC curve analyses (right panels) showing sensitivity and specificity values and cut-off evaluation (dotted lines *, left panels).

Article Snippet: Calprotectin (CALPRO) ELISA (ALPCO cat # 30-CALHU-E01), prostaglandin E2 (PGE2) ELISA (ALPCO cat # 74-PG2HU-E05), hemaglobin/haptoglobin complex (HAPTO-HEMO) ELISA (ALPCO cat# 30-HAPHU-E01), lysozyme (LYZ) ELISA (ALPCO Cat # 30-LYSHU-E01), osteoprotegerin (OPGN) ELISA (Biomedica Medizinprodukte, Cat# BI-20403, Vienna, Austria), and lipocalin2 (LCN2/NGAL) ELISA (Epitope Diagnostics Inc., Cat# KRT-853, San Diego, CA) were all provided at no cost by ALPCO Immunoassay.

Techniques: Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Response of chondrocytes to shear stress: antagonistic effects of the binding partners Toll-like receptor 4 and caveolin-1.

doi: 10.1096/fj.11-184861

Figure Lengend Snippet: Figure 3. TLR4 and caveolin-1 exert antagonistic effects on shear-induced IL-6 synthesis in human chondrocytes. T/C-28a2 chondrocytes were subjected to shear stress (20 dyn/cm2) or static conditions (0 dyn/cm2) for 2 h (A, B) or 48 h (C, D). In select experiments, T/C-28a2 chondrocytes were transfected with either a siRNA oligonucleotide sequence specific for TLR4 (A) or caveolin-1 (D) or scrambled siRNA control before their exposure to fluid shear. In separate experiments, cells were transfected with either a plasmid containing the cDNA of caveolin-1 (B) or TLR4 (C) or an empty vector (control) prior to shear exposure. TLR4 (A, C) or caveolin-1 (B, D) and IL-6 protein (top panels) and mRNA expression (bottom panels) were determined by Western blot analysis and qRT-PCR, respectively. -Actin and GAPDH served as internal controls in immunoblotting and qRT-PCR assays, respectively. Western blots are representative of 3 independent experiments, all revealing similar results. Data represent means se of 3 independent qRT-PCR experiments. *P 0.05 vs. static control; ŒP 0.05 vs. shear alone.

Article Snippet: The caveolin-1, TLR4, mPGES-1, and L-PGDS, EP2, EP3 cDNA plasmids were obtained from Origene Technologies (Rockville, MD, USA), and subcloned to the pCMV6-XL vector.

Techniques: Shear, Transfection, Sequencing, Control, Plasmid Preparation, Expressing, Western Blot, Quantitative RT-PCR

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Response of chondrocytes to shear stress: antagonistic effects of the binding partners Toll-like receptor 4 and caveolin-1.

doi: 10.1096/fj.11-184861

Figure Lengend Snippet: Figure 8. Fluid shear induces the binding of the NF-B p65 subunit to the IL-6 promoter in human chondrocytes. T/C-28a2 chondrocytes were subjected to fluid shear stress (20 dyn/cm2) or static conditions (0 dyn/cm2) for 48 h (A, B, F, G) or 2 h (C–E, H–J). In select experiments, T/C-28a2 cells were transfected with the indicated siRNAs or cDNA constructs before exposure to shear stress. In separate experiments, T/C-28a2 cells were subjected to fluid shear stress (20 dyn/cm2) for 2 h in the presence or absence of a MEK1/2 inhibitor [U0126 (10 M) or PD98095 (20 M)]. A–E) Nuclear extracts were then isolated, and NF-B-specific DNA-protein complex formation was determined by gel shift. F–J) Supershift assays using an anti-p65 Ab were carried out as outlined in Materials and Methods. Results of a competition experiment using 200-fold unlabeled NF-B oligonucleotide (cold probe) are shown. Gels are representative of 3 independent experiments, all revealing similar results.

Article Snippet: The caveolin-1, TLR4, mPGES-1, and L-PGDS, EP2, EP3 cDNA plasmids were obtained from Origene Technologies (Rockville, MD, USA), and subcloned to the pCMV6-XL vector.

Techniques: Shear, Binding Assay, Transfection, Construct, Isolation, Gel Shift

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Response of chondrocytes to shear stress: antagonistic effects of the binding partners Toll-like receptor 4 and caveolin-1.

doi: 10.1096/fj.11-184861

Figure Lengend Snippet: Figure 9. Fluid shear induces the binding of the NF-B p65 subunit to the IL-6 promoter in human chondrocytes. T/C-28a2 cells were subjected to fluid shear stress (20 dyn/cm2) or static conditions (0 dyn/cm2) for 48 h (A, B) or 2 h (C–G). Cells were transfected with the indicated siRNAs or cDNA constructs or treated with prescribed pharmacological inhibitors before exposure to shear stress. Cross-linked chromatin was immunoprecipitated using an antip65 antibody (left panel). In ChIP assays, the anti-RNA polymerase II antibody was used as a positive control, whereas the normal mouse IgG and anti-TLR4 antibodies were used as negative controls. DNA purified from both the immunoprecipitated (IP) and preimmune (input) specimens were subjected to PCR amplification using primers for the GAPDH (control) and p65 promoter genes. All experiments are representative of 3 independent experiments, all revealing similar results.

Article Snippet: The caveolin-1, TLR4, mPGES-1, and L-PGDS, EP2, EP3 cDNA plasmids were obtained from Origene Technologies (Rockville, MD, USA), and subcloned to the pCMV6-XL vector.

Techniques: Shear, Binding Assay, Transfection, Construct, Immunoprecipitation, Positive Control, Control

Journal: International Journal of Molecular Sciences

Article Title: Impact of Mechanical Load on the Expression Profile of Synovial Fibroblasts from Patients with and without Osteoarthritis

doi: 10.3390/ijms20030585

Figure Lengend Snippet: Impact of static compressive force application on the expression of proinflammatory genes. Gene expression and protein secretion of ( a ) TNF-α, ( b ) COX-2/PG-E2 and ( c ) IL-6 of N-SF and OA-SF after 48 h with or without static compressive force application. AU: arbitrary units; RT-qPCR: n = 9; ELISA: n = 6. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤0.001. Statistics: Welch-corrected ANOVA with Games-Howell post-hoc-tests.

Article Snippet: For quantification of tumour necrosis factor α (TNFα) and prostaglandin E2 (PGE2), interleukin-6 (IL-6), collagen-1 (COL-1) and fibronectin (FN-1) protein secretion into the synovial fibroblast supernatant, we used commercially available ELISA kits according to the manufacturers’ instructions (TNFα: EK0525, Boster Biological Technology; PGE2: 514010; Cayman chemical; IL-6: EK0410 Boster Biological Technology; COL-1: ab210966, Abcam; FN-1: EK0349, Boster Biological Technology).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay