Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: 27-Hydroxycholesterol accelerates cellular senescence in human lung resident cells.

doi: 10.1152/ajplung.00351.2015

Figure Lengend Snippet: Fig. 8. Effects of 27-OHC on prostaglandin (PG) synthesis and effects of cyclooxygenase (COX) inhibitors on 27-OHC-enhanced cellular senes- cence. HFL-1 cells were exposed to various concentrations of 27-OHC for 2 wk. COX-1 and COX-2 expression was evaluated by Western blotting (A and B). PGE2 and PGD2 release was quantified by enzyme-linked immunoassay (ELISA) (C and D). Cells were treated with 27-OHC in the presence or absence of indo- methacin, a nonselective COX inhibitor; cele- coxib, a selective COX-2 inhibitor; and PF- 04418948, a selective prostaglandin E2 receptor 2 (EP2) antagonist. Cells were harvested and assayed for 3-NT formation (E) and SA--gal activity (F and G). Relative intensity was calcu- lated by dividing each protein band intensity by the -actin band intensity. Values are means SE (n 4). *P 0.05 and **P 0.01 vs. control group. †P 0.05 and ††P 0.01 vs. 27-OHC-treated group.

Article Snippet: Commercially available reagents were obtained as follows: mouse monoclonal anti-p53 antibody, mouse monoclonal anti-p16 antibody, mouse monoclonal anti-cyclooygenase-1 (COX-1) antibody, mouse monoclonal anti-COX-2 antibody, mouse monoclonal anti- -actin antibody, and mouse monoclonal anti-lamin A/C antibody were from Santa Cruz Biotechnology (Dallas, TX); rabbit polyclonal anti-p21 antibody, rabbit polyclonal anti-phosphorylated (p)-retinoblastoma (pRB) antibody and rabbit polyclonal anti-p-p53 antibody were from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antisterol 27-hydroxylase antibody and 27-OHC were from Avanti Polar Lipids (Alabaster, AL); mouse anti-CD68 antibody was from BioLegend (San Diego, CA); fibronectin enzyme-linked immunosorbent assay (ELISA) kit, goat fluorescein isothiocyanate polyclonal secondary antibody, and rabbit Dylight 650 polyclonal secondary antibody were from Abcam (Cambridge, UK); indomethacin, a nonselective COX inhibitor, and celecoxib, a selective 27-OHC-2 inhibitor, were from Sigma (St. Louis, MO); manganese (III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a peroxynitrite scavenger, was from Calbiochem (La Jolla, CA); diaminofluorescein-2, a fluorescent indicator, was from Sekisui Medical (Tokyo, Japan); PF-04418948, a selective prostaglandin E2 receptor 2 (EP2) antagonist and EIA prostaglandin kit were from Cayman Chemical (San Diego, CA); Dulbecco’s Modified Eagle’s Medium (DMEM), fetal calf serum (FCS), and antibiotic-antimycotic were purchased from Invitrogen Life Technologies (Grand Island, NY).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Control

Journal: Immunology

Article Title: Prostaglandin E 2 directly inhibits the conversion of inducible regulatory T cells through EP2 and EP4 receptors via antagonizing TGF‐β signalling

doi: 10.1111/imm.13417

Figure Lengend Snippet: EP2 and EP4 receptors mediate PGE 2 suppression of iTreg cell differentiation in vitro . (a,b) Percentages of Foxp3 + T cells in EP2 +/+ (a) or EP2 −/− (b) CD4 + CD25 − naïve T cells cultured with IL‐2 and TGF‐β1 with dm‐PGE 2 or selective agonists for each EP1‐4 receptor for 3 days. (c,d) Percentages of Foxp3 + T cells in EP4 +/+ (c) or EP4 −/− (d) CD4 + CD25 − naïve T cells cultured with IL‐2 and TGF‐β1 with dm‐PGE 2 or selective agonists for each EP1‐4 receptor for 3 days. (e) Percentages of Foxp3 + T cells in wild‐type C57BL/6 CD4 + CD25 − naïve T cells cultured with IL‐2 and TGF‐β1 in the absence or presence of PGE 2 , EP2 antagonist or EP4 antagonist or both EP2 and EP4 antagonists for 3 days. (f) Percentages of Foxp3 + T cells in wild‐type C57BL/6 CD4 + CD25 − naïve T cells cultured with IL‐2 and TGF‐β1 with db‐cAMP or IBMX for 3 days. (g) Percentages of Foxp3 + T cells in wild‐type C57BL/6 CD4 + CD25 − naïve T cells cultured with IL‐2 and TGF‐β1 with PGE 2 , a PKA inhibitor (H‐89) or a PI3K inhibitor (LY‐294002) for 3 days. All experiments were performed in triplicates and repeated at least twice independently. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ns, not significant

Article Snippet: Selective antagonists against EP2 (PF‐04418948) and EP4 (L‐161,982) were purchased from Cayman.

Techniques: Cell Differentiation, In Vitro, Cell Culture

Journal: Oncotarget

Article Title: Inhibition of EP2/EP4 signaling abrogates IGF-1R-mediated cancer cell growth: Involvement of protein kinase C-θ activation

doi:

Figure Lengend Snippet: A, The levels of COX-1, COX-2, EP1-EP4, IR, IGF-1R, IGF-2R, EGFR, ErbB4, NRDc, and β-actin mRNA and IGF-1R protein were measured in MiaPaCa-2, BxPC-3, PANC-1, and Capan-1 human pancreatic cancer cell lines. B, CM (serum-free for 48 h) from these cell lines were also subjected to PGE 2 enzyme immunoassays. Columns , mean values ( n = 4) based on two independent experiments; bars , SD. C, Pancreatic cancer cell lines were treated with 0.5 and 5 μM AH6809/GW627368X for 48 h and the cell viabilities were measured using the MTT assay. The A 550 values for untreated cells were assigned as 100% and the relative percentages for treated cells are shown. Columns , mean percentages (n = 6); bars , SD.

Article Snippet: The EP2-selective antagonist AH6809 and EP4-selective antagonist GW627368X were purchased from Cayman Chemical (Ann Arbor, MI).

Techniques: Enzyme Immunoassay, MTT Assay

Journal: Oncotarget

Article Title: Inhibition of EP2/EP4 signaling abrogates IGF-1R-mediated cancer cell growth: Involvement of protein kinase C-θ activation

doi:

Figure Lengend Snippet: A, Cells were stimulated with HB-EGF (50 ng/mL), IGF-1 (20 ng/mL), or IGF-2 (50 ng/mL) for 48 h in the absence or presence of AH6809/GW627368X (5 μM each) pretreatment for 3 h and cell growth was measured using the MTT assay. The A 550 values for untreated cells were assigned as 100% and the relative percentages for the treated cells are shown. Columns , mean percentages ( n = 6); bars , SD. B, BxPC-3 cells were stimulated with HB-EGF (50 ng/mL), IGF-1 (20 ng/mL) and IGF-2 (50 ng/mL) for 20 min in the absence or presence of AH6809/GW627368X (5 μM each) pretreatment for 3 h. The levels of phosphorylated MEK, total MEK, phosphorylated ERK, and total ERK were determined by immunoblotting. The relative levels of phospho-MEK and -ERK were calculated using ImageJ software.

Article Snippet: The EP2-selective antagonist AH6809 and EP4-selective antagonist GW627368X were purchased from Cayman Chemical (Ann Arbor, MI).

Techniques: MTT Assay, Western Blot, Software

Journal: Oncotarget

Article Title: Inhibition of EP2/EP4 signaling abrogates IGF-1R-mediated cancer cell growth: Involvement of protein kinase C-θ activation

doi:

Figure Lengend Snippet: A, BxPC-3 cells were stimulated with IGF-1 (20 ng/mL) for 20 min in the absence or presence of AH6809/GW627368X (5 μM each) pretreatment for 3 h. The levels of phosphorylated PKC-θ and total PKC-θ were determined by immunoblotting. B, BxPC-3 cells were treated with AH6809/GW627368X (5 μM each) for 1 and 3 h. The levels of phosphorylated PKC-θ and total PKC-θ were determined by immunoblot analysis. C, A pseudosubstrate of PKC-θ (10 μM, Millipore) was added with AH6809/GW627368X (5 μM each) pretreatment and the cell growth and phosphorylation of MEK and ERK were determined by MTT assays and immunoblotting, respectively. The A 550 values for untreated cells were assigned as 100% and the relative percentages for treated cells are shown. The relative levels of phospho-MEK and -ERK were calculated using ImageJ software. PS , pseudosubstrate; Columns , mean percentages ( n = 6); bars , SD.

Article Snippet: The EP2-selective antagonist AH6809 and EP4-selective antagonist GW627368X were purchased from Cayman Chemical (Ann Arbor, MI).

Techniques: Western Blot, Phospho-proteomics, Software

Journal: Oncotarget

Article Title: Inhibition of EP2/EP4 signaling abrogates IGF-1R-mediated cancer cell growth: Involvement of protein kinase C-θ activation

doi:

Figure Lengend Snippet: A, Knockdown using PKC-θ siRNA was performed in BxPC-3 cells and confirmed by immunoblotting. Cells transfected with negative control siRNA and PKC-θ siRNA were stimulated with IGF-1 for 48 h or 20 min in the absence or presence of AH6809/GW627368X pretreatment for 3 h, and then tested by growth stimulation assays and immunoblotting. The A 550 values for untreated cells were assigned as 100% and the relative percentages for treated cells are shown. The relative levels of phospho-MEK and -ERK were calculated using ImageJ software. Columns , mean percentages (n = 6); bars , SD. B, The expression levels of PKC-α, PKC-β, PKC-γ, PKC-δ, PKC-ε, PKC-η, PKC-θ, PKC-ζ, PKC-ι, and β-actin mRNA were determined in BxPC-3 cells transfected with negative control siRNA and PKC-θ siRNA.

Article Snippet: The EP2-selective antagonist AH6809 and EP4-selective antagonist GW627368X were purchased from Cayman Chemical (Ann Arbor, MI).

Techniques: Knockdown, Western Blot, Transfection, Negative Control, Software, Expressing

Journal: Oncotarget

Article Title: Inhibition of EP2/EP4 signaling abrogates IGF-1R-mediated cancer cell growth: Involvement of protein kinase C-θ activation

doi:

Figure Lengend Snippet: A, BxPC-3 cells were stimulated with IGF-1 (20 ng/mL) for 20 min in the absence or presence of AH6809/GW627368X (5 μM each) pretreatment for 3 h. The levels of phosphorylated PDK1, total PDK1, phosphorylated AMPKα, total AMPKα, and MAP4K3 were determined by immunoblotting. B, Knockdown was performed with specific siRNAs for MAP4K3 and PDK1 in BxPC-3 cells and confirmed by immunoblotting. C, Cells transfected with negative control siRNA, MAP4K3 siRNA (#0140), and PDK1 siRNA (#4485) were stimulated with IGF-1 (20 ng/mL) for 48 h in the absence or presence of AH6809/GW627368X (5 μM each) pretreatment for 3 h. Cell growth was measured by the MTT assay. The A 550 values for untreated cells were assigned as 100% and the relative percentages for treated cells are shown. Columns , mean percentages ( n = 6); bars , SD. D, Cells transfected with negative control siRNA, MAP4K3 siRNA (#0140), and PDK1 siRNA (#4485) were stimulated with IGF-1 (20 ng/mL) for 20 min in the absence or presence of AH6809/GW627368X (5 μM each) pretreatment for 3 h. The levels of phosphorylated PKC-θ, total PKC-θ, phosphorylated MEK, total MEK, phosphorylated ERK, and total ERK were determined by immunoblotting. The relative levels of phospho-PKC-θ, -MEK and -ERK were calculated using ImageJ software.

Article Snippet: The EP2-selective antagonist AH6809 and EP4-selective antagonist GW627368X were purchased from Cayman Chemical (Ann Arbor, MI).

Techniques: Western Blot, Knockdown, Transfection, Negative Control, MTT Assay, Software

Journal: Oncotarget

Article Title: Inhibition of EP2/EP4 signaling abrogates IGF-1R-mediated cancer cell growth: Involvement of protein kinase C-θ activation

doi:

Figure Lengend Snippet: A, The body weights of mice were measured (left) at 0, 7, 14, 21, 28, and 35 days after injection. Bars , SD. Macroscopic tumors from control and AH6809/GW627368X-treated mice (center). The average tumor weights were calculated for each group (right). Columns , mean; bars , SD. B, H&E staining and immunohistochemical staining of IGF-1 and Ki-67 in tumor lesions. The percentage of Ki-67-positive cells was calculated. Columns , mean; bars , SD. C, Proteins from tumors were subjected to immunoblotting and the relative levels of phospho-PKC-θ, -MEK, and -ERK were calculated using ImageJ software. Asterisk , targeted band; Columns , mean; bars , SD.

Article Snippet: The EP2-selective antagonist AH6809 and EP4-selective antagonist GW627368X were purchased from Cayman Chemical (Ann Arbor, MI).

Techniques: Injection, Control, Staining, Immunohistochemical staining, Western Blot, Software