Journal:

Article Title: Reexamination of the electrophile response element sequences and context reveals a lack of consensus in gene function

doi: 10.1016/j.bbagrm.2010.05.003

Figure Lengend Snippet: The constitutive and HNE-inducible activity of putative EpRE sequences in the 5′-flanking region of human GCLC gene. Oligos containing the corresponding EpRE were cloned into pGL-3 promoter vector and the reporters were transfected into HBE1 cells. After being treated with/without 15 μM HNE for 24h, cells were collected and the luciferase activity was measured. The luciferase activity was normalized with co-transfected β-Gal activity. N=3, * P< 0.05 compared with vehicle vector (v: pGL-3 promoter vector), # P< 0.05 compared with control treatment.

Article Snippet: Luciferase activity assay kit, pGL-3 bas ic vector, and pGL-3 promoter vector, were from Promega (Madison, WI).

Techniques: Activity Assay, Clone Assay, Plasmid Preparation, Transfection, Luciferase, Control

Journal:

Article Title: Reexamination of the electrophile response element sequences and context reveals a lack of consensus in gene function

doi: 10.1016/j.bbagrm.2010.05.003

Figure Lengend Snippet: Effect of EpRE location on EpRE activity. Reporters were constructed as described in the Experimental Procedurals. The reporters were transfected into HBE 1 cells and the luciferase activity was measured. The relative luciferase activity was calculated by using the vehicle vector (pGL-3 basic vector) without HNE exposure as the control. N=3, *, P< 0.05 compared with corresponding reporter without HNE exposure; #, P< 0.05 compared with GCLC-Luc without HNE exposure. GCLC-Luc, −3802/+85 GCLC-luc; m4 GCLC-Luc, −3802/+85 GCLC-Luc with EpRE 4 mutated; in EpRE 4/n1-Luc, EpRE 4/n6-Luc, EpRE 4 was relocated to the position of EpRE n1, and n6 of m4 GCLC-Luc, respectively; in EpRE n1/E4-Luc, EpRE n6/E4-Luc and EpRE 3/E4-Luc, EpRE 4 in GCLC-Luc was replaced with EpRE n1, n6, and EpRE 3, respectively.

Article Snippet: Luciferase activity assay kit, pGL-3 bas ic vector, and pGL-3 promoter vector, were from Promega (Madison, WI).

Techniques: Activity Assay, Construct, Transfection, Luciferase, Plasmid Preparation, Control

Journal:

Article Title: Upregulation of cytochromes P 450 2B in rat liver by orphenadrine

doi: 10.1038/sj.bjp.0705305

Figure Lengend Snippet: HepG2 cells were transfected with the cyp2b10 gene PBREM-luciferase reporter construct or cotransfected with the PBREM-luciferase reporter construct and a plasmid encoding the mCAR. In addition, all cells were cotransfected with a plasmid encoding β-galactosidase. Transfected cells were treated with androsten-3α-o1 (AND; 8 μM) to suppress endogenous CAR activation, ORPH (0.1 mM) or TCPOBOP (250 nM), or combinations of these reagents. Results are fold induction of reporter-gene activity in AND-treated cells after correction for pGL3 alone; data derived from three to four separate transfections. Different from AND-treated cells: **P<0.01, *P<0.05.

Article Snippet: The β -galactosidase plasmid, reagents for luciferase assay and pGL-3 basic vector were from Promega.

Techniques: Transfection, Luciferase, Construct, Plasmid Preparation, Activation Assay, Activity Assay, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA-944 Affects Cell Growth by Targeting EPHA7 in Non-Small Cell Lung Cancer

doi: 10.3390/ijms17101493

Figure Lengend Snippet: EPHA7 is a direct target of miR-944 in NSCLC. ( A ) The expressions of EPHA7 were examined by qRT-PCR in NSCLC cell lines; ( B ) The expressions of EPHA7 were examined by western blot in NSCLC tissues and noncancerous lung tissues; ( C ) The relationships between miR-944 and EPHA7 protein levels were analyzed using Pearson’s method in NSCLC tissues, R = −0.48, p < 0.05; ( D ) The mRNA and protein levels of EPHA7 were measured in EPLC-32M1 and XLA-07 cells transfected with the miR-944 mimic and negative control (NC); ( E ) Sequences of the putative miR-944 binding sites; ( F ) The relative luciferase activity was determined using luciferase reporters with EPHA7-3′UTR in EPLC-32M1 and A549 cells transfected with the miR-944 mimic and negative control (NC); ( G ) The EPHA7 expression was analyzed using qRT-PCR and western blot in EPLC-32M1 and XLA-07 cells transfected with the siRNA (si-EPHA7) and negative control (NC); ( H ) The proliferation rates were analyzed using proliferation assay in EPLC-32M1 and XLA-07 cells after transfection with the siRNA (si-EPHA7) and negative control (NC). GAPDH was used as an internal control ( B , D , G ). The significance was evaluated by t -test ( A , B , D , F – H ), * p < 0.05.

Article Snippet: For generation of EPHA7 luciferase reporter plasmid (pGL-EPHA7), the 3′-untranslated region (3′-UTR) containing the miR-944 binding site was cloned between the NheI and BglII restriction sites of the pGL-3 basic vector (Promega, Madison, WI, USA) using a PCR-amplified fragment.

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Negative Control, Binding Assay, Luciferase, Activity Assay, Expressing, Proliferation Assay, Control