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: Simplified schematic illustration of pathways for Wnt/β-catenin, ERK/MAPK and PI3K/Akt and PGE 2 -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) PGE 2 -metabolism. PGE 2 -synthesis begins with catalytic hydrolysis of membrane phospholipids by cytoplasmic phospholipase A2 (cPLA 2 ), thus releasing arachidonic acid (AA). By the action of the COX-1 and COX-2, AA is converted to prostaglandin H 2 (PGH 2 ). PGH 2 is then converted to PGE 2 by prostaglandin E synthase (PTGES). The main exporter of PGE 2 is thought to be multi-drug resistance related polypeptide 4 (MRP4). Removal of PGE 2 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 PGE 2 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: The following antibodies were tested: Mouse anti-EP3 antibody (5F5), sc-57,105 (Santa Cruz Biotechnology), Rabbit anti-EP3 antibody, 101,760 (Cayman Chemicals, Ann Arbor, MI, USA), Rabbit anti-EP3 antibody, APR-065 (Alomone Labs, Jerusalem, Israel), Rabbit anti-EP3 antibody (PTGER3), NBP1–84835 (Bio-techne, Abingdon, United Kingdom).

Techniques: Inhibition, Activation Assay, Modification, Activity Assay, 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: Quantitative real-time PCR primer sequences

Article Snippet: The following antibodies were tested: Mouse anti-EP3 antibody (5F5), sc-57,105 (Santa Cruz Biotechnology), Rabbit anti-EP3 antibody, 101,760 (Cayman Chemicals, Ann Arbor, MI, USA), Rabbit anti-EP3 antibody, APR-065 (Alomone Labs, Jerusalem, Israel), Rabbit anti-EP3 antibody (PTGER3), NBP1–84835 (Bio-techne, Abingdon, United Kingdom).

Techniques: Real-time Polymerase Chain Reaction

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: Difference in mRNA expression in CRN versus controls

Article Snippet: The following antibodies were tested: Mouse anti-EP3 antibody (5F5), sc-57,105 (Santa Cruz Biotechnology), Rabbit anti-EP3 antibody, 101,760 (Cayman Chemicals, Ann Arbor, MI, USA), Rabbit anti-EP3 antibody, APR-065 (Alomone Labs, Jerusalem, Israel), Rabbit anti-EP3 antibody (PTGER3), NBP1–84835 (Bio-techne, Abingdon, United Kingdom).

Techniques: Expressing

Journal: Chemical Biology & Drug Design

Article Title: Investigation on the antipyretic mechanism of Chaiqin Qingning capsule for the treatment of fever based on network pharmacology, molecular docking, and in vitro experimental validation

doi: 10.1111/cbdd.14451

Figure Lengend Snippet: FIGURE 7 Western blot detection of CQQNC on the expression of COX-1, COX-2, cAMP, EP3, and mPGES1. Statistical significance: ∆∆∆∆p < .0001, ∆∆p < .01, and ∆p < .05 represents IL-1β group versus control group. ****p < .0001, **p < .01, and *p < .05 represent CQQNC group versus IL-1β group (n = 3).

Article Snippet: AntiCOX- 1 (batch number: 00100314), COX- 2 (batch number: 10027896), EP3 (batch number: 00071265), mouse and rabbit GAPDH (batch number: 10021642 and 0013796) antibody were purchased from Proteintech.

Techniques: Western Blot, Expressing, Control

Journal: Pharmacology Research & Perspectives

Article Title: Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1 α in HCA-7 human colon cancer cells

doi: 10.1002/prp2.83

Figure Lengend Snippet: EP4 receptors were down-regulated in a cellular density-dependent manner in HCA-7 cells. Cells were cultured at low (L; 3 × 10 4 cells/each 6 well), middle (M; 1 × 10 5 cells/each 6 well), and high (H; 3 × 10 5 cells/each 6 well) densities. Each cellular density was then subjected to immunoblot analysis with an antibody against EP4 (A) or EGFR (B), quantitative PCR (C), the EP4 promoter-luciferase assay (D), PGE 2 -stimulated cAMP assay (E), or EP2 or EP3 promoter-luciferase assay (F), as described in the Materials and Methods. (A, B) The blots were stripped and re-probed with an antibody against β -tubulin. The bar graphs represent the ratio of COX-2 to β -tubulin as assessed with pooled densitometric data (mean ± SD) from more than three independent experiments. Data are normalized to the ratio of COX-2 to β -tubulin at the low cellular density as 100%. (C) The RNA of HCA-7 cells at each cellular density was isolated and used for quantitative real-time PCR with primers specific for either human EP4 or GAPDH, as described in the Materials and Methods. Data were analyzed by comparative methods relative to the expression of GAPDH and were normalized to the results obtained at the low cellular density as 100%. Data are the mean ± SD from three independent experiments. (D, F) Each cellular density was transiently transfected with human EP4 receptor promoter- (D), human EP2 promoter-, or EP3 promoter- (F) responsive luciferase reporter genes and control pRL-CMV or pGL3-CMV plasmids, and luciferase activity was determined as described in the Materials and Methods. Data were analyzed by comparative methods relative to the results of control luciferase activity levels, and were normalized to the results obtained at the low cellular density as 100%. Data are the mean ± SD from three independent experiments. (E) Each cellular density was treated with the indicated concentrations of PGE 2 for 60 min. cAMP formation was determined as described in the Materials and Methods. Data are the means ± SEM of three independent experiments, with each being performed in duplicate. Data were normalized to the vehicle-treated control at the low cellular density as 100%. * P , † P < 0.05, ANOVA, significantly different from the vehicle-stimulated HCA-7 cells at the low cellular density. ‡ P < 0.05, ANOVA, significantly different from PGE 2 -stimulated HCA-7 cells at the low cellular density (the analyzed results were shown for 1 μ mol/L PGE 2 only).

Article Snippet: Cells were transiently transfected with firefly pGL3/EP4R-Promoter-Luc, the hypoxia response element (HRE) luciferase reporter plasmid (pGL3/HRE-Luc27) (Ji et al. ), or Renilla human EP2 or EP3 receptor promoter luciferase reporter plasmids (S713225, S722267; SwitchGear Genomics, Menlo Park, CA), and either with human HIF-1 α siRNA, renilla luciferase control plasmids, pRL-CMV (Promega), or the firefly luciferase control plasmid; pGL3-CMV (CMV promoter of pRL-CMV was replaced with the pGL3 vector) using GeneJuice (Novagen/Merck, Darmstadt, Germany) reagent.

Techniques: Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Luciferase, cAMP Assay, Isolation, Expressing, Transfection, Control, Activity Assay

Journal: Pharmacology Research & Perspectives

Article Title: Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1 α in HCA-7 human colon cancer cells

doi: 10.1002/prp2.83

Figure Lengend Snippet: HIF-1 α was cellular density- and CoCl 2 -dependently up-regulated, and EP4 receptors were down-regulated by CoCl 2 in HCA-7 cells. Cells were cultured at low (L; 3 × 10 4 cells/each 6 well), middle (M; 1 × 10 5 cells/each 6 well), and high (H; 3 × 10 5 cells/each 6 well) densities. Each cellular density was then subjected to immunoblot analysis with antibody against HIF-1 α (A, C) or the EP4 receptor (D), or the HIF-responsive HRE luciferase assay (B), as described in the Materials and Methods. A, The blots were stripped and re-probed with an antibody against β -tubulin. The bar graphs represent the ratio of HIF-1 α to β -tubulin as assessed with pooled densitometric data (mean ± SD) from more than three independent experiments. Data were normalized to the ratio of HIF-1 α to β -tubulin at the low cellular density as 100%. (B) Each cellular density was transiently transfected with the HRE luciferase reporter genes and control pRL-CMV plasmid, and luciferase activity was determined as described in the Materials and Methods. Data were analyzed by comparative methods relative to the results of control renilla luciferase activity levels, and were normalized to the results obtained at the low cellular density as 100%. Data are the mean ± SD from three independent experiments. (C, D) Each cellular density was treated with 100 μ mol/L CoCl 2 for 16 h, then subjected to immunoblot analysis with an antibody against HIF-1 α (C) or the EP4 receptor (D), as described in the Materials and Methods. The blots were stripped and re-probed with an antibody against β -tubulin. The bar graphs represent the ratio of HIF-1 α (C) or EP4 receptors (D) to β -tubulin as assessed with pooled densitometric data (mean ± SD) from more than three independent experiments. Data are normalized to the ratio of HIF-1 α (C) or EP4 receptors (D) to β -tubulin at the low cellular density as 100%. * P < 0.05, ANOVA, significantly different from the vehicle treatment. † P < 0.05, ANOVA, significantly different from the low cellular density control.

Article Snippet: Cells were transiently transfected with firefly pGL3/EP4R-Promoter-Luc, the hypoxia response element (HRE) luciferase reporter plasmid (pGL3/HRE-Luc27) (Ji et al. ), or Renilla human EP2 or EP3 receptor promoter luciferase reporter plasmids (S713225, S722267; SwitchGear Genomics, Menlo Park, CA), and either with human HIF-1 α siRNA, renilla luciferase control plasmids, pRL-CMV (Promega), or the firefly luciferase control plasmid; pGL3-CMV (CMV promoter of pRL-CMV was replaced with the pGL3 vector) using GeneJuice (Novagen/Merck, Darmstadt, Germany) reagent.

Techniques: Cell Culture, Western Blot, Luciferase, Transfection, Control, Plasmid Preparation, Activity Assay

Journal: Pharmacology Research & Perspectives

Article Title: Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1 α in HCA-7 human colon cancer cells

doi: 10.1002/prp2.83

Figure Lengend Snippet: Effects of HIF-1 α siRNA on HIF-1 α protein expression, HIF-responsive luciferase activity, EP4 receptor expression, and EP4 receptor promoter-responsive luciferase activity in HCA-7 cells cultured at different cellular densities. Cells were cultured at low (L; 3 × 10 4 cells/each 6 well), middle (M; 1 × 10 5 cells/each 6 well), and high (H; 3 × 10 5 cells/each 6 well) densities. Each cellular density was transfected with HIF-1 α siRNA for 48 h, and cells were then subjected to immunoblot analysis with antibody against HIF-1 α (A) or EP4 receptors (C), or the luciferase assay for HIF-responsive HRE (B) and EP4 receptor promoter-responsive (D) activities, as described in the Materials and Methods. (A, C) The blots were stripped and re-probed with an antibody against β -tubulin. The bar graphs represent the ratio of HIF-1 α (A) or EP4 receptors (C) to β -tubulin as assessed with pooled densitometric data (mean ± SD) from more than three independent experiments. Data were normalized to the ratio of HIF-1 α (A) or EP4 receptors (C) to β -tubulin at the low cellular density as 100%. (B, D) Each cellular density was transiently transfected with HIF-responsive HRE luciferase reporter genes (B) or EP4 receptor promoter luciferase reporter genes (D) and the control pRL-CMV plasmid 20 h after siRNA transfection, and luciferase activity was determined, as described in the Materials and Methods. Data were analyzed by comparative methods relative to the results of control renilla luciferase activity levels, and were normalized to the result obtained at the low cellular density as 100%. Data are the mean ± SD from three independent experiments. * P , ‡ P < 0.05, ANOVA, significantly different from control transfection at the low cellular density. † P , ¶ P < 0.05, ANOVA, significantly different from each density control that was not transfected with siRNA.

Article Snippet: Cells were transiently transfected with firefly pGL3/EP4R-Promoter-Luc, the hypoxia response element (HRE) luciferase reporter plasmid (pGL3/HRE-Luc27) (Ji et al. ), or Renilla human EP2 or EP3 receptor promoter luciferase reporter plasmids (S713225, S722267; SwitchGear Genomics, Menlo Park, CA), and either with human HIF-1 α siRNA, renilla luciferase control plasmids, pRL-CMV (Promega), or the firefly luciferase control plasmid; pGL3-CMV (CMV promoter of pRL-CMV was replaced with the pGL3 vector) using GeneJuice (Novagen/Merck, Darmstadt, Germany) reagent.

Techniques: Expressing, Luciferase, Activity Assay, Cell Culture, Transfection, Western Blot, Control, Plasmid Preparation