Journal: Journal of the American Society of Nephrology : JASN

Article Title: Transcription Factor Trps1 Promotes Tubular Cell Proliferation after Ischemia-Reperfusion Injury through cAMP–Specific 3′,5′-Cyclic Phosphodiesterase 4D and AKT

doi: 10.1681/ASN.2016010009

Figure Lengend Snippet: Trps 1 regulates the expression of Pde4d and Akt, but not Gucy2c, after I/R injury. The mRNA levels of (A) Pde4d and (B) Gucy2c in renal tissues after moderate I/R injury were detected by quantitative PCR. Data are expressed as means±SD (n=6 per group at each time point). (C) The expression of Pde4d, Akt, and pAkt in renal tissues in the Trps1 siRNA and control siRNA groups was detected by Western blotting, and (D–F) the relative levels to β-actin were quantified. Data are expressed as means±SD (n=6 per group at each time point). *P<0.05 versus control. (G) In the moderate I/R model, immunohistochemical analyses for Pde4d and pAkt expression in renal tissues were performed at 3 days after I/R injury.

Article Snippet: NRK-52E cells were transfected with Trps1-siRNA, Trps1 overexpression vectors, Pde4d-siRNA, or control siRNA (sc-35438; Santa Cruz Biotechnology) using jetPRIME (Polyplus Transfection) as described previously by Yang et al . 44 Cells incubated with jetPRIME alone were used as the negative control.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemical staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Transcription Factor Trps1 Promotes Tubular Cell Proliferation after Ischemia-Reperfusion Injury through cAMP–Specific 3′,5′-Cyclic Phosphodiesterase 4D and AKT

doi: 10.1681/ASN.2016010009

Figure Lengend Snippet: Trps1 mediates H/R-induced activation of the Pde4d promoter. The psiCHECKTM-2 dual luciferase reporter plasmids were used, and the Pde4d 3′-UTR promoter fragment was cloned to produce psiCHECK-Pde4d. NRK-52E cells were cultured to approximately 80% confluence in a 24-well plate and then, cotransfected with psiCHECK-Pde4d and Trps1 overexpression vector (or Trps1-siRNA) for 48 hours. The cells were cultured under hypoxic conditions (1% O2, 94% N2, and 5% CO2) in FBS- and antibiotic-free medium for 4 hours at 37°C to induce hypoxic injury; then, they were returned to 5% CO2 and 95% air for reoxygenation for 24 hours (H/R). Luciferase activities in the cells were determined by the dual luciferase assay kit. Data are expressed as means±SD (n=6). *P<0.05 versus psiCHECK-Pde4d; #P<0.05 versus psiCHECK-Pde4d.

Article Snippet: NRK-52E cells were transfected with Trps1-siRNA, Trps1 overexpression vectors, Pde4d-siRNA, or control siRNA (sc-35438; Santa Cruz Biotechnology) using jetPRIME (Polyplus Transfection) as described previously by Yang et al . 44 Cells incubated with jetPRIME alone were used as the negative control.

Techniques: Activation Assay, Luciferase, Clone Assay, Cell Culture, Over Expression, Plasmid Preparation

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Transcription Factor Trps1 Promotes Tubular Cell Proliferation after Ischemia-Reperfusion Injury through cAMP–Specific 3′,5′-Cyclic Phosphodiesterase 4D and AKT

doi: 10.1681/ASN.2016010009

Figure Lengend Snippet: Trps1 promotes the proliferation of rat renal tubule cells, which is mediated by the Pde4d/PI3K/AKT signaling pathway. (A) The PCNA levels were detected by Western blotting, and (B) the relative levels to β-actin were quantified in Trps1 normal and Trps1 overexpression NRK-52E cells at 0 and 24 hours after H/R. Transfer with Pde4d siRNA and the PI3K inhibitor wortmannin resulted in a decreased proportion of the PCNA protein level. Data are expressed as means±SD (n=6). *P<0.05 versus the counterpart at 0 hours after H/R; #P<0.05 versus control at 24 hours after H/R. (C) The PCNA levels were detected by Western blotting, and (D) the relative levels to β-actin were quantified in the rat I/R model with Trps1 overexpression. Pde4d-specific inhibitor resulted in a decreased proportion of the PCNA protein level. Data are expressed as means±SD (n=6). #P<0.05 versus DMSO.

Article Snippet: NRK-52E cells were transfected with Trps1-siRNA, Trps1 overexpression vectors, Pde4d-siRNA, or control siRNA (sc-35438; Santa Cruz Biotechnology) using jetPRIME (Polyplus Transfection) as described previously by Yang et al . 44 Cells incubated with jetPRIME alone were used as the negative control.

Techniques: Western Blot, Over Expression

Journal: Oncology Letters

Article Title: Knockdown of phosphodiesterase 4D inhibits nasopharyngeal carcinoma proliferation via the epidermal growth factor receptor signaling pathway

doi: 10.3892/ol.2014.2422

Figure Lengend Snippet: Expression of PDE4D detected in clinical samples and cell lines. (A) The expression of PDE4D was detected in 40 cases of NPC and 21 cases of NNET by western blot analysis. PDE4D expression was found to be significantly higher in the NPC cases when compared with NNET cases (P<0.05). (B) A correlation was identified between PDE4D and the clinical parameters of NPC. Early clinical stage NPC tissues exhibited higher PDE4D expression than normal tissues (P<0.01 vs. control). (C) Expression of PDE4D in five NPC cell lines and NP69. Each experiment was performed in triplicate and the mean value was calculated. * P<0.05 and ** P<0.01. PDE4D, phosphodiesterase 4D; NNET, normal nasopharyngeal epithelial tissues; NPC, nasopharyngeal carcinoma.

Article Snippet: PDE4D-targeted shRNA lentiviral particles (LV-PDE4D shRNA; sc-44004-v) and control shRNA lentiviral particles (ctr-shRNA; sc-108080) (both Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were infected into the CNE2 cells according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot

Journal: Oncology Letters

Article Title: Knockdown of phosphodiesterase 4D inhibits nasopharyngeal carcinoma proliferation via the epidermal growth factor receptor signaling pathway

doi: 10.3892/ol.2014.2422

Figure Lengend Snippet: Knockdown of PDE4D inhibits the growth of NPC cells, which is reversed by EGF stimulation. (A) Western blot analysis results and graph presenting the infection efficiency of PDE4D-targeted shRNA lentiviral particles in CNE2 and 5–8F cells. Following infection, PDE4D expression was significantly inhibited in the two cell lines. (B) Effect of PDE4D-targeted shRNA lentiviral particles on cell proliferation measured by MTT assay following infection in CNE2 and 5–8F cells. The effect of EGF stimulation on the proliferation of the NPC cells, which were infected with PDE4D-targeted shRNA lentiviral particles, is also shown. * P<0.05 and ** P<0.01 vs. C and LV+E. PDE4D, phosphodiesterase 4D; NPC, nasopharyngeal carcinoma; EGF, epidermal growth factor; shRNA, short hairpin RNA; OD, optical density; C, cells infected with control shRNA lentiviral particles; LV, cells infected with PDE4D-targeted shRNA lentiviral particles; LV+E, cells infected with PDE4D-targeted shRNA lentiviral particles followed by 100 ng/ml EGF treatment; ctr-shRNA, control shRNA lentiviral particles.

Article Snippet: PDE4D-targeted shRNA lentiviral particles (LV-PDE4D shRNA; sc-44004-v) and control shRNA lentiviral particles (ctr-shRNA; sc-108080) (both Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were infected into the CNE2 cells according to the manufacturer’s instructions.

Techniques: Western Blot, Infection, shRNA, Expressing, MTT Assay

Journal: Oncology Letters

Article Title: Knockdown of phosphodiesterase 4D inhibits nasopharyngeal carcinoma proliferation via the epidermal growth factor receptor signaling pathway

doi: 10.3892/ol.2014.2422

Figure Lengend Snippet: Effect of PDE4D on cell cycle arrest and colony formation in CNE2 cells. (A) Knockdown of PDE4D induced cell cycle arrest in the G 0 /G 1 phase in the CNE2 cells, which was reversed by EGF stimulation. * P<0.05 vs. C and LV+E. (B) Results of the colony formation assay showed that knockdown of PDE4D inhibited colony formation in the CNE2 cells, which was reversed by EGF stimulation. ** P<0.01 vs. C and LV+E. PDE4D, phosphodiesterase 4D; EGF, epidermal growth factor; shRNA, short hairpin RNA; C, cells infected with control shRNA lentiviral particles; LV, cells infected with PDE4D-targeted shRNA lentiviral particles; LV+E, cells infected with PDE4D-targeted shRNA lentiviral particles followed by 100 ng/ml EGF treatment.

Article Snippet: PDE4D-targeted shRNA lentiviral particles (LV-PDE4D shRNA; sc-44004-v) and control shRNA lentiviral particles (ctr-shRNA; sc-108080) (both Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were infected into the CNE2 cells according to the manufacturer’s instructions.

Techniques: Colony Assay, shRNA, Infection

Journal: Oncology Letters

Article Title: Knockdown of phosphodiesterase 4D inhibits nasopharyngeal carcinoma proliferation via the epidermal growth factor receptor signaling pathway

doi: 10.3892/ol.2014.2422

Figure Lengend Snippet: Western blot analysis of EGFR signaling pathway proteins of the CNE2 cells following infection with LV-PDE4D shRNA and EGF stimulation. (A) Knockdown of PDE4D inactivates EGFR and AKT in CNE2 cells, and EGF stimulation reverses the efficiency of LV-PDE4D shRNA. (B) Various time points following stimulation with 100 ng/ml of EGF. The phosphorylation of EGFR and AKT for C+E compared with LV+E cells. After 10 min, EGF was found to promote the phosphorylation of of EGFR and AKT, however, following PDE4D knockdown, EGF stimulation decreased. Each experiment was performed in triplicate and the mean value was calculated. PDE4D, phosphodiesterase 4D; NPC, nasopharyngeal carcinoma; EGFR, epidermal growth factor receptor; shRNA, short hairpin RNA; C, cells infected with control shRNA lentiviral particles; LV, cells infected with PDE4D-targeted shRNA lentiviral particles; LV+E, cells infected with PDE4D-targeted shRNA lentiviral particles followed by 100 ng/ml EGF treatment; C+E, control cells stimulated by 100 ng/ml EGF; p-EGFR, phosphorylated EGFR; p-AKT, phosphorylated AKT.

Article Snippet: PDE4D-targeted shRNA lentiviral particles (LV-PDE4D shRNA; sc-44004-v) and control shRNA lentiviral particles (ctr-shRNA; sc-108080) (both Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were infected into the CNE2 cells according to the manufacturer’s instructions.

Techniques: Western Blot, Infection, shRNA

Journal: Oncology Letters

Article Title: Knockdown of phosphodiesterase 4D inhibits nasopharyngeal carcinoma proliferation via the epidermal growth factor receptor signaling pathway

doi: 10.3892/ol.2014.2422

Figure Lengend Snippet: Knockdown of PDE4D inhibits tumor growth of NPC cells in nude mice. (A) CNE2 cells infected with LV-PDE4D shRNA and ctr-shRNA were injected subcutaneously into nude mice. At two weeks post-implantation, the LV-PDE4D shRNA-infected cells produced smaller tumors than those of the ctr-shRNA group. The growth curve represents the tumor volumes. Data are presented as the mean ± standard error of the mean of five mice. (B) At two weeks post-transplantation, the transplanted tumors of the two groups (each of five mice) were sectioned and stained for p-EGFR and p-AKT. Images were captured under inverted microscope at ×200 magnification. * P<0.05 and ** P<0.01. shRNA, short hairpin RNA; p-EGFR, phosphorylated epidermal growth factor receptor; p-AKT, phosphorylated AKT; ctr-shRNA, control shRNA lentiviral particles.

Article Snippet: PDE4D-targeted shRNA lentiviral particles (LV-PDE4D shRNA; sc-44004-v) and control shRNA lentiviral particles (ctr-shRNA; sc-108080) (both Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were infected into the CNE2 cells according to the manufacturer’s instructions.

Techniques: Infection, shRNA, Injection, Produced, Transplantation Assay, Staining, Inverted Microscopy