ep4  (Santa Cruz Biotechnology)

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Receptor 4 (EP4) Affects Trophoblast Functions via Activating the cAMP-PKA-pCREB Signaling Pathway at the Maternal-Fetal Interface in Unexplained Recurrent Miscarriage

doi: 10.3390/ijms22179134

Figure Lengend Snippet: Double immunofluorescence analyses of EP4 and HLAG shown in the decidua of uRM patients and healthy controls. EP4 is co-expressed with HLAG in the cytoplasm of extravillous trophoblast cells ( A – F ). The percentage of double staining cells were counted in HLAG + EP4 + /HLAG + cells over nine slides. The expression of EP4 in EVTs was decreased in uRM patients compared to healthy controls ( G ). HLAG is specific marker for extravillous trophoblast cells (EVTs). The picture is shown in its original magnification of 40×. * p < 0.05.

Article Snippet: Cells were cultured in 6-well or 96-well saturations in RPMI 1640 solution with 10% FBS, growth averaging 60% confluence, and EP4 and controlling siRNAs were retrieved at a concentration of 20 nM through OriGene setup (CAT: SR321501, Rehovot, HaMerkaz, Israel), transferred utilizing Lipofectamine 3000 connected at a final concentration of 50 nM with producer protocols (Invitrogen, Waltham, MA, USA).

Techniques: Immunofluorescence, Double Staining, Expressing, Marker

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Receptor 4 (EP4) Affects Trophoblast Functions via Activating the cAMP-PKA-pCREB Signaling Pathway at the Maternal-Fetal Interface in Unexplained Recurrent Miscarriage

doi: 10.3390/ijms22179134

Figure Lengend Snippet: Immunohistochemical analysis of pCREB and CREB expression in placentae of uRM patients and healthy controls from the first trimester were assessed via IRS score. Expression of pCREB and CREB was identified in the nuclear of cells in the syncytium ( A , B , G , H ) and in decidual ( C , D , I , J ) of first-trimester placentas in both healthy control and uRM. The expression of pCREB is decreased in both the syncytium and decidual cells ( E , F ) were measured via IRS score. EP4 is significantly positively correlated with pCREB in both the syncytium and decidua ( M , N ). Confocal microscopy imaging showed that EP4 (red) (P, E) co-expresses and co-localizes with p-CREB (green) ( Q , V ) in extravillous trophoblasts (EVTs) (O,T) of uRM patients ( S ) and health controls ( X ). HLAG is specific marker for EVTs. The picture was shown in its original magnification of 40×.

Article Snippet: Cells were cultured in 6-well or 96-well saturations in RPMI 1640 solution with 10% FBS, growth averaging 60% confluence, and EP4 and controlling siRNAs were retrieved at a concentration of 20 nM through OriGene setup (CAT: SR321501, Rehovot, HaMerkaz, Israel), transferred utilizing Lipofectamine 3000 connected at a final concentration of 50 nM with producer protocols (Invitrogen, Waltham, MA, USA).

Techniques: Immunohistochemical staining, Expressing, Control, Confocal Microscopy, Imaging, Marker

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Receptor 4 (EP4) Affects Trophoblast Functions via Activating the cAMP-PKA-pCREB Signaling Pathway at the Maternal-Fetal Interface in Unexplained Recurrent Miscarriage

doi: 10.3390/ijms22179134

Figure Lengend Snippet: EP4 knockdown inhibits proliferation and induced apoptosis in vitro. ( A ). The expression protein EP4 is higher in HTR-8/SVneo and JEG-3 cells than BEWO. ( B ). The mRNA expression of EP4 in HTR-8/SVneo after knockdown with siRNA was detected by RT-PCR. ( C ) The mRNA mRNA of EP4 in JEG-3 after knockdown with siRNA was detected by RT-PCR. ( D ) MTT assays indicated that downregulated EP4 decreased the viability of HTR-8/SVneo compared to the negative control. ( E ) The MTT assay indicated that downregulated EP4 decreased the viability of JEG-3 compared to the negative control. ( F ) BrdU assay suggested that the proliferation of HTR-8/SVneo is inhibited by EP4 knockdown compared to the negative control. ( G ) BrdU assay suggested that the proliferation of JGE-3 is inhibited by EP4 knockdown compared to negative control. ( H ) The apoptosis test showed that the apoptosis of HTR-8/SVneo with EP4 siRNA is improved compared to the negative control and the effect was reversed by 8-Bromo-cAMP. ( I ) The apoptosis test showed that the apoptosis of JGE-3 with EP4 siRNA is reduced compared to the negative control. * p < 0.05.

Article Snippet: Cells were cultured in 6-well or 96-well saturations in RPMI 1640 solution with 10% FBS, growth averaging 60% confluence, and EP4 and controlling siRNAs were retrieved at a concentration of 20 nM through OriGene setup (CAT: SR321501, Rehovot, HaMerkaz, Israel), transferred utilizing Lipofectamine 3000 connected at a final concentration of 50 nM with producer protocols (Invitrogen, Waltham, MA, USA).

Techniques: Knockdown, In Vitro, Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, MTT Assay, BrdU Staining

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Receptor 4 (EP4) Affects Trophoblast Functions via Activating the cAMP-PKA-pCREB Signaling Pathway at the Maternal-Fetal Interface in Unexplained Recurrent Miscarriage

doi: 10.3390/ijms22179134

Figure Lengend Snippet: The expression levels of β-hCG, progesterone, and IL-6 in the supernatants of HTR-8/SVneo and JEG-3 cells after incubation with PGE2, TCS 2510, L-161,982 and siRNA. ( A ) The secretion of β-hCG was increased by 1 μM PGE2, 1 μM and 10 μM TCS 2510 in THR-8/SVneo. ( B ) The secretion of β-hCG was increased by 10 μM PGE2 and inhibited by 0.1 μM L-161,982 in JEG-3. ( C ) The production of progesterone in JEG-3 was increased by 10 μM PGE2, 1 μM and 10 μM TCS 2510. ( D ) β-hCG levels in the supernatants of HTR-8/SVneo and JEG-3 cells were dropped after slicing EP4 compared to negative control. ( E ) Progesterone levels were decreased in the supernatants of JEG-3 cells after slicing EP4 and EP4 added 8-Bromo-cAMP compared to negative controls. ( F ) IL-6 levels were attenuated in the supernatants of HTR-8/SVneo and JEG-3 cells after knockdown of EP4 compared to negative controls. * p < 0.05.

Article Snippet: Cells were cultured in 6-well or 96-well saturations in RPMI 1640 solution with 10% FBS, growth averaging 60% confluence, and EP4 and controlling siRNAs were retrieved at a concentration of 20 nM through OriGene setup (CAT: SR321501, Rehovot, HaMerkaz, Israel), transferred utilizing Lipofectamine 3000 connected at a final concentration of 50 nM with producer protocols (Invitrogen, Waltham, MA, USA).

Techniques: Expressing, Incubation, Negative Control, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Receptor 4 (EP4) Affects Trophoblast Functions via Activating the cAMP-PKA-pCREB Signaling Pathway at the Maternal-Fetal Interface in Unexplained Recurrent Miscarriage

doi: 10.3390/ijms22179134

Figure Lengend Snippet: The expression levels of cAMP, EP4, PKA and p-CREB were changed by EP4 knockdown. ( A ) The secretion of cAMP was increased by 10 μM PGE2 in HTR-8/SVneo.( B ) The secretion of cAMP was increased by PGE2, TCS 2510, PGE2 + TCS 2510 and inhibited by PG2 + L-161,982 in HTR-8/SVneo. ( C ) The production of progesterone in JEG-3 was increased by 1, 10 μM PGE2. ( D ) cAMP levels in the cell lysis of JEG-3 was increased by PGE2, TCS 2510, PGE2 + TCS 2510. ( E ) Western blot analysis showed EP4, PKA, p-CREB, CREB in HTR-8/SVneo and JEG-3 cells after silencing the EP4 gene compared with the negative control. ( F ) The density value of EP4 in HTR-8/SVneo and JEG-3 cells after knockdown of EP4 compared to negative control. ( G ) The density value of PKA in HTR-8/SVneo and JEG-3 cells after knockdown of EP4 compared to negative control. ( H ) The density value of PKA in HTR-8/SVneo and JEG-3 cells after knockdown of EP4 compared to negative control. * p < 0.05; ** p < 0.01.

Article Snippet: Cells were cultured in 6-well or 96-well saturations in RPMI 1640 solution with 10% FBS, growth averaging 60% confluence, and EP4 and controlling siRNAs were retrieved at a concentration of 20 nM through OriGene setup (CAT: SR321501, Rehovot, HaMerkaz, Israel), transferred utilizing Lipofectamine 3000 connected at a final concentration of 50 nM with producer protocols (Invitrogen, Waltham, MA, USA).

Techniques: Expressing, Knockdown, Lysis, Western Blot, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Receptor 4 (EP4) Affects Trophoblast Functions via Activating the cAMP-PKA-pCREB Signaling Pathway at the Maternal-Fetal Interface in Unexplained Recurrent Miscarriage

doi: 10.3390/ijms22179134

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Cells were cultured in 6-well or 96-well saturations in RPMI 1640 solution with 10% FBS, growth averaging 60% confluence, and EP4 and controlling siRNAs were retrieved at a concentration of 20 nM through OriGene setup (CAT: SR321501, Rehovot, HaMerkaz, Israel), transferred utilizing Lipofectamine 3000 connected at a final concentration of 50 nM with producer protocols (Invitrogen, Waltham, MA, USA).

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Receptor 4 (EP4) Affects Trophoblast Functions via Activating the cAMP-PKA-pCREB Signaling Pathway at the Maternal-Fetal Interface in Unexplained Recurrent Miscarriage

doi: 10.3390/ijms22179134

Figure Lengend Snippet: The possible role of EP4 in trophoblasts of unexplained recurrent miscarriage (uRM). Inhibiting the EP4 signaling pathway reduces the activity of the cAMP-PKA-pCREB signaling pathway, which can eventually lead to depressing the production of progesterone, β-hCG and IL-6. Downregulated of EP4 decreased proliferation and increased apoptosis in trophoblasts. These abnormal changes in trophoblasts may contribute to recurrent miscarriages.

Article Snippet: Cells were cultured in 6-well or 96-well saturations in RPMI 1640 solution with 10% FBS, growth averaging 60% confluence, and EP4 and controlling siRNAs were retrieved at a concentration of 20 nM through OriGene setup (CAT: SR321501, Rehovot, HaMerkaz, Israel), transferred utilizing Lipofectamine 3000 connected at a final concentration of 50 nM with producer protocols (Invitrogen, Waltham, MA, USA).

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: Prostaglandin E2 breaks down pericyte–endothelial cell interaction via EP1 and EP4-dependent downregulation of pericyte N-cadherin, connexin-43, and R-Ras

doi: 10.1038/s41598-020-68019-w

Figure Lengend Snippet: Primers for quantitative RT-PCR.

Article Snippet: For gene silencing, cells were transfected at approximately 60% confluency in complete medium with 30 nM siRNA targeting EP1 *, EP4 ** or PTGES * (*Dharmacon, Lafayette, CO; **ThermoFisher;) using Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific) or Dharmafect (Dharmacon).

Techniques:

Journal: Scientific Reports

Article Title: Prostaglandin E2 breaks down pericyte–endothelial cell interaction via EP1 and EP4-dependent downregulation of pericyte N-cadherin, connexin-43, and R-Ras

doi: 10.1038/s41598-020-68019-w

Figure Lengend Snippet: Inhibition of EP1, EP2 and EP4 restores pericyte–EC interaction. ( a ) RT-qPCR analysis of EP receptor expression profile in HBVPs. The different primer sets were validated for an efficiency of 2 ± 5%. EP3 expression was undetectable. ( b ) The effect of PGE2 on EP1, EP2 and EP4 expression in HBVPs was assessed by RT-qPCR 72 h post-treatment. The mRNA expression level of each EP was normalized to that of cyclophilin A using the delta-delta Ct method. Fold change relative to the control is shown. ( c ) HBVPs were treated with a cocktail of EP1 (ONO-8713), EP2 (PF-04418948) and EP4 (ONO-AE3-208) inhibitors (1 μM each) in DMSO or DMSO vehicle alone, then 5 h later with 100 nM PGE2 in DMSO or DMSO alone. Cell morphology was observed 72 h later. Scale bar 50 μm. ( d ) HBVPs (green) subjected to the same treatments as described in c were co-cultured on Matrigel with HUVECs (red) for 18 h. Scale bar 50 μm. The branching index (number of junctions/mm 2 ), average vessel length and number of vessels were measured using Angiotool and ImageJ. One-way ANOVA test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; ns, not significant.

Article Snippet: For gene silencing, cells were transfected at approximately 60% confluency in complete medium with 30 nM siRNA targeting EP1 *, EP4 ** or PTGES * (*Dharmacon, Lafayette, CO; **ThermoFisher;) using Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific) or Dharmafect (Dharmacon).

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Control, Cell Culture

Journal: Scientific Reports

Article Title: Prostaglandin E2 breaks down pericyte–endothelial cell interaction via EP1 and EP4-dependent downregulation of pericyte N-cadherin, connexin-43, and R-Ras

doi: 10.1038/s41598-020-68019-w

Figure Lengend Snippet: PGE2 transcriptionally represses RRAS expression in an EP4-dependent manner. ( a ) HBVPs were treated with PGE2 (10 or 100 nM) or control DMSO for 48 h, and RRAS expression was analyzed by quantitative RT-PCR at 48 h post-treatment. The mRNA extracts were pooled from three different cell culture dishes, and the RT-PCR analyses were repeated in two independent experiments and in triplicates. The RRAS mRNA level was normalized to that of cyclophilin A using the delta-delta Ct method. Fold change in RRAS expression is shown relative to the control. ( b ) In parallel, HBVPs were stably transduced with lentiviral particles delivering a -1907/ + 1 RRAS promoter-reporter construct. Luciferase assay was conducted to determine the promoter activity 48-h post-treatment of HBVPs with the same compounds. Luciferase assay results are shown as means ± SEM of three independent experiments performed in tri- or quadruplicates. ( c ) The specificity of RRAS promoter activity in response to PGE2 was assessed by transient transfection of 293 T cells with a wild-type (WT) RRAS promoter-reporter construct or a mutated construct (Mut) lacking cyclic AMP-responsive elements (CRE). Renilla luciferase activity was used as an internal control. The results are representative of two transfections performed in triplicates. One-way ANOVA test, **p < 0.01; ****p < 0.0001; ns: not significant. ( d ) Time-course western blot analysis of R-Ras expression in HBVPs treated with PGE2 for 12, 24 or 48 h. ( e ) HBVPs were incubated for 5 h with inhibitors of different EP receptors (EP1: ONO-8713; EP2: PF-04418948; EP3: ONO-AE5-599; EP4: ONO-AE3-208) prior to PGE2 treatment. 72 h later, R-Ras protein expression was analyzed by western blot. ( f ) EP4 expression was silenced in HBVPs using siRNA. Cells were treated with PGE2 24 h after transfection, and protein extracts were collected 48 h later for western blot. GAPDH was used as a loading control.

Article Snippet: For gene silencing, cells were transfected at approximately 60% confluency in complete medium with 30 nM siRNA targeting EP1 *, EP4 ** or PTGES * (*Dharmacon, Lafayette, CO; **ThermoFisher;) using Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific) or Dharmafect (Dharmacon).

Techniques: Expressing, Control, Quantitative RT-PCR, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transduction, Construct, Luciferase, Activity Assay, Transfection, Western Blot, Incubation

Journal: Scientific Reports

Article Title: Prostaglandin E2 breaks down pericyte–endothelial cell interaction via EP1 and EP4-dependent downregulation of pericyte N-cadherin, connexin-43, and R-Ras

doi: 10.1038/s41598-020-68019-w

Figure Lengend Snippet: PGE2 downregulates N-cadherin via EP4 and Cx43 via EP1/Ca 2+ /calpain signaling. ( a ) HBVPs were treated with inhibitors of different EP receptors for 5 h prior to PGE2 treatment. N-cadherin and Cx43 expression was analyzed 72 h later by western blot. ( b , c ) EP1 or EP4 was silenced in HBVPs. Cells were treated with PGE2 24 h after siRNA transfection, and protein extracts were collected 48 h later for western blot analysis. d. The changes in intracellular calcium levels induced by PGE2 were monitored in HBVPs by Fluo4-calcium assay. One-way ANOVA test, ***p < 0.001; ns: not significant. ( e ) HBVPs were incubated 4 h with a μ-calpain inhibitor before PGE2 or DMSO treatment. N-cadherin and Cx43 expression was analyzed 72 h later by western blot. ( f ) A proposed model of Cx43 downregulation by PGE2/EP1/Ca 2+ /calpain signaling. ( g ) In order to verify that PGE2-mediated decrease of Cx43 is not a result of epitope loss following cleavage by calpain, Cx43 expression was analyzed in HBVPs following PGE2 treatment using an anti-Cx43 antibody targeting a N-terminal epitope. h. HBVPs were treated with various inhibitors of proteins suspected to be involved in PGE2-induced N-cadherin degradation. Pepstatin A was used as an inhibitor for γ-secretase and Gö6976 for PKC. The cells were treated with each inhibitor 4 h prior PGE2 treatment, and protein extracts were collected after a total of 72 h.

Article Snippet: For gene silencing, cells were transfected at approximately 60% confluency in complete medium with 30 nM siRNA targeting EP1 *, EP4 ** or PTGES * (*Dharmacon, Lafayette, CO; **ThermoFisher;) using Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific) or Dharmafect (Dharmacon).

Techniques: Expressing, Western Blot, Transfection, Calcium Assay, Incubation

Journal: Scientific Reports

Article Title: Prostaglandin E2 breaks down pericyte–endothelial cell interaction via EP1 and EP4-dependent downregulation of pericyte N-cadherin, connexin-43, and R-Ras

doi: 10.1038/s41598-020-68019-w

Figure Lengend Snippet: Silencing mPGES-1 prevents colon cancer cells from disrupting pericytes. HT29 human colon cancer cells were transfected with control- or PTGES (mPGES-1 gene)-targeting siRNA for 48 h. ( a ) Cell lysates were examined by RT-qPCR to analyze mPGES-1 expression in HT29 cells. The PTGES mRNA level was normalized to cyclophilin A. Fold change relative to the control is shown. ( b ) ELISA was performed to measure the concentration of PGE2 in the conditioned media. Student t -test, ****p < 0.0001. ( c ) HBVPs were incubated either with fresh culture medium as a control condition (–) or with conditioned medium (CM) from control (siCtrl) or PTGES -silenced HT29 cells. Western blot was performed to analyze N-cadherin, Cx43 and R-Ras expression 48 h later. ( d ) HBVPs were incubated with DMSO or PGE2 (100 nM) for 72 h, or treated with conditioned medium from control- or PTGES -silenced HT29 cells for 48 h. The cells were detached, and the adhesion of these cells to new culture plate was determined by crystal violet staining and absorbance at 595 nm. The data shown is a representative of three independent experiments performed in quadruplicates. One-way ANOVA test, **p < 0.01; ****p < 0.0001. ( e , f ) HBVPs (green) were incubated either with fresh culture medium as a control, or with conditioned medium from control- or PTGES -silenced HT29 cells for 48 h prior to coculturing with HUVECs (red) on culture plates. Cells were stained for N-cadherin ( e ) or Cx43 ( f ) 18 h later. Scale bar 50 μm.

Article Snippet: For gene silencing, cells were transfected at approximately 60% confluency in complete medium with 30 nM siRNA targeting EP1 *, EP4 ** or PTGES * (*Dharmacon, Lafayette, CO; **ThermoFisher;) using Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific) or Dharmafect (Dharmacon).

Techniques: Transfection, Control, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Incubation, Western Blot, Staining

Journal: Scientific Reports

Article Title: Prostaglandin E2 breaks down pericyte–endothelial cell interaction via EP1 and EP4-dependent downregulation of pericyte N-cadherin, connexin-43, and R-Ras

doi: 10.1038/s41598-020-68019-w

Figure Lengend Snippet: Exposure to PGE2 disrupts pericytes and damages vessel wall integrity. In normal condition, pericytes are closely interacting and communicating with ECs via adherens junctions (N-cadherin), gap junctions (Cx43), and cell adhesion to basement membrane extracellular matrix (yellow). The exposure to PGE2-high environment in pathological conditions, such as colon cancer, disrupts normal pericyte function, which in turn causes disruption of the EC lining of blood vessel wall. The use of EP1 and EP4 inhibitors can restore the vascular damages induced by PGE2.

Article Snippet: For gene silencing, cells were transfected at approximately 60% confluency in complete medium with 30 nM siRNA targeting EP1 *, EP4 ** or PTGES * (*Dharmacon, Lafayette, CO; **ThermoFisher;) using Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific) or Dharmafect (Dharmacon).

Techniques: Membrane, Disruption

Journal: Oncotarget

Article Title: Nicotine induces EP4 receptor expression in lung carcinoma cells by acting on AP-2α: The intersection between cholinergic and prostanoid signaling

doi: 10.18632/oncotarget.18023

Figure Lengend Snippet: ( A ) EP4 SiRNA decreased the proliferation of A549 cells induced by nicotine (0.5 μM). ( B ) AH23848 decreased the proliferation of A549 cells induced by nicotine (0.5 μM). ( C ) EP4 SiRNA decreased the proliferation of H1838 cells induced by nicotine (0.5 μM). ( D ) AH23848 decreased the proliferation of H1838 cells induced by nicotine (0.5 μM). ( E ) Nicotine increased secretion of PGE 2 in dose-dependent manner in A549 cells in ELISA assay. * indicates significantly difference from control. ** indicates significance of combination treatment as compared with nicotine alone ( P < 0.05). Con indicates untreated control cells.

Article Snippet: The α7 nAChR (sc-42532), EP4 (sc-40173) and control (sc-37007) siRNAs, and polyclonal antibodies against AP-2α were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Oncotarget

Article Title: Nicotine induces EP4 receptor expression in lung carcinoma cells by acting on AP-2α: The intersection between cholinergic and prostanoid signaling

doi: 10.18632/oncotarget.18023

Figure Lengend Snippet: ( A ) Nicotine increased the expression of EP4 in dose-dependent manner in A549 cells. ( B ) Nicotine increased the expression of EP4 in time-dependent manner in A549 cells. ( C ) Nicotine increased the expression of EP4 in dose-dependent manner in H1838 cells. ( D ) Nicotine increased the expression of EP4 in time-dependent manner in H1838 cells. E. Nicotine increased EP4 mRNA expression as determined by real time RT-PCR. GAPDH served as internal control for normalization purposes. * indicates significant differences from control ( P < 0.05).

Article Snippet: The α7 nAChR (sc-42532), EP4 (sc-40173) and control (sc-37007) siRNAs, and polyclonal antibodies against AP-2α were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Oncotarget

Article Title: Nicotine induces EP4 receptor expression in lung carcinoma cells by acting on AP-2α: The intersection between cholinergic and prostanoid signaling

doi: 10.18632/oncotarget.18023

Figure Lengend Snippet: ( A ) Acetylcholine increased the expression of EP4 induced by nicotine in dose-dependent in A549 cells. ( B ) Acetylcholinesterase decreased the expression of EP4 induced by nicotine in dose-dependent in A549 cells. ( C ) a-bungarotoxin of a a7 nAChR blocker, not dihydro-β-erythroidine of a α4 nAChR inhibitor, decreased the expression of EP4 induced by nicotine in A549 cells. ( D ) α7 nAChR siRNA (100 nM) decreased the expression of EP4 induced by nicotine in A549 cells. ( E ) a-bungarotoxin, a a7 nAChR blocker, decreased the proliferation induced by nicotine in A549 cells and H1838 cells. ( F ) The specific inhibitors of PI3-K (wortmannin, 1 μM), JNK (SP600125, 20 μM) reduced expression of EP4 induced by nicotine in A549 cells. ( G ) The specific inhibitors of ERK (PD98095, 20 μM), not P38 MAPK (SB239063, 10 μM) had a minor effect on reduction of EP4 induced by nicotine in A549 cells. ( H ) The specific inhibitors of PKC (calphostin C, 0.5 μM), not PKA (H89, 10 μM) reduced expression of EP4 induced by nicotine in A549 cells. GAPDH served as internal control for normalization purposes.

Article Snippet: The α7 nAChR (sc-42532), EP4 (sc-40173) and control (sc-37007) siRNAs, and polyclonal antibodies against AP-2α were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Control

Journal: Oncotarget

Article Title: Nicotine induces EP4 receptor expression in lung carcinoma cells by acting on AP-2α: The intersection between cholinergic and prostanoid signaling

doi: 10.18632/oncotarget.18023

Figure Lengend Snippet: ( A ) The 5′-flanking region of the human EP4 gene wild type and deletion promoter constructs schematics are presented. These regions contain several transcription factor binding sites including AP-2. ( B ) Nicotine increased EP4 gene promoter activity in A549 cells, transfected with the full-length wild-type EP4 promoter (–1238/+1) luciferase reporter construct and other two EP4 deletion reporter constructs (–238/+1 and –197/+1), not with a shortest EP4 deletion reporter construct (–160/+1). ( C ) Nicotine decreased the binding ability of AP-2α to the Oligonucleotides which contains the AP-2α site. ( D ) Nicotine decreased the EP4 promoter DNA quantity binding to AP-2α protein. ( E ) AP-2α overexpression vector blocked nicotine-induced EP4 protein expression in A549 cells. ( F ) AP-2α overexpression vector blocked nicotine-induced promoter activity of EP4 in A549cells. ( G ) Site-directed mutation of AP-2α (–169 bp ) on EP4 promoter blocked nicotine-induced promoter activity of EP4 in A549cells. * indicates significance as compared with controls. ** indicates significance of combination treatment as compared with nicotine alone ( P < 0.05). Con , untreated control cells.

Article Snippet: The α7 nAChR (sc-42532), EP4 (sc-40173) and control (sc-37007) siRNAs, and polyclonal antibodies against AP-2α were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Construct, Binding Assay, Activity Assay, Transfection, Luciferase, Over Expression, Plasmid Preparation, Expressing, Mutagenesis, Control

Journal: Oncotarget

Article Title: Nicotine induces EP4 receptor expression in lung carcinoma cells by acting on AP-2α: The intersection between cholinergic and prostanoid signaling

doi: 10.18632/oncotarget.18023

Figure Lengend Snippet: The novel mechanism of nicotine increasing EP4 expression and NSCLC proliferation

Article Snippet: The α7 nAChR (sc-42532), EP4 (sc-40173) and control (sc-37007) siRNAs, and polyclonal antibodies against AP-2α were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing