empty vector ev expression plasmid  (Addgene inc)

Journal: Cancer Management and Research

Article Title: E2F1 induces LSINCT5 transcriptional activity and promotes gastric cancer progression by affecting the epithelial-mesenchymal transition

doi: 10.2147/CMAR.S171652

Figure Lengend Snippet: E2F1 upregulates LSINCT5 expression in GC cells. Notes: qRT-PCR analysis of the E2F1 expression levels in GC tissues and paired ANTs ( A ) and in metastatic and nonmetastatic GC tissues ( B ). ( C ) qRT-PCR analysis of E2F1 expression in a series of human GC cell lines and a normal gastric epithelial cell line (GES-1). ( D ) ChIP assays using E2F1 antibody demonstrated endogenous E2F1 binding to the LSINCT5 gene promoter, and ectopic expression or siRNA knockdown increased or reduced E2F1 enrichment at the LSINCT5 promoter, respectively. ( E ) A dual-luciferase reporter assay was performed through cotransfection of the LSINCT5 promoter fragment (pGL3-LSINCT5) and an E2F1-overexpression construct. ( F ) We examined the LSINCT5 core promoter region for transcription factor binding sites, and identified four tandem putative E2F1-binding sites. Reporter assays were conducted in cells transfected with various LSINCT5 promoter constructs in which different E2F1-binding elements were deleted (WT, del). Luciferase activity is presented relative to that of the pGL3 vector (a promoter-less vector). ( G ) qRT-PCR analysis of the LSINCT5 expression levels following the transfection of MGC803 and BGC823 cells with a pMax-E2F1 expression vector and siRNA-E2F1, respectively. ( H ) Analysis of the relationship between LSINCT5 and E2F1 mRNA levels (ΔCt value) in GC tissues. Bars: SD; * P <0.05 and ** P <0.01. Abbreviations: ANTs, adjacent normal tissues; Ts, tumor tissues; ChIP, chromatin immunoprecipitation; del, deletion; GC, gastric cancer; qRT-PCR, quantitative real-time polymerase chain reaction; WT, wild type; NC, negative control.

Article Snippet: The pMax-E2F1 expression vector was purchased from Addgene (plasmid #16007) (Cambridge, MA, USA), and siRNAs specific for E2F1 (#AM16708) and LSINCT5 (#4392420) were chemically synthesized (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Reporter Assay, Cotransfection, Over Expression, Construct, Transfection, Activity Assay, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

Journal: Cancer Management and Research

Article Title: E2F1 induces LSINCT5 transcriptional activity and promotes gastric cancer progression by affecting the epithelial-mesenchymal transition

doi: 10.2147/CMAR.S171652

Figure Lengend Snippet: Primers used in this study

Article Snippet: The pMax-E2F1 expression vector was purchased from Addgene (plasmid #16007) (Cambridge, MA, USA), and siRNAs specific for E2F1 (#AM16708) and LSINCT5 (#4392420) were chemically synthesized (Invitrogen, Carlsbad, CA, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: Frontiers in Oncology

Article Title: RPL21 siRNA Blocks Proliferation in Pancreatic Cancer Cells by Inhibiting DNA Replication and Inducing G1 Arrest and Apoptosis

doi: 10.3389/fonc.2020.01730

Figure Lengend Snippet: Expression analysis for genes that as the G1-S phase and DNA replication regulators after RPL21 knockdown. (A,B) The validation of transcriptome analysis by quantitative real-time PCR (qPCR). Human pancreatic cancer PANC-1 cells were transfected with siL21-Mix (40 nM, 72 h), the up-regulated genes ( AHR , THBS1 , DDIT3 , and MKNK2 ) in transcriptome sequencing were confirmed by qPCR. The down-regulated genes ( E2F1 , PCNA , CCND1 , CCNE1 MCM2 , MCM4 , MCM5 , MCM7 , and KIAA0101 ) in transcriptome sequencing were confirmed by qPCR. (C) The PANC-1 and BxPC-3 cells were transfected with Mock-siRNA, siL21-1 and siL21-2 (40 nM, 72 h). The equal amounts (15 μg) of each protein sample were analyzed by western blot with antibodies of E2F1 , CCND1 , CCNE1 , MCM2 -7 and GAPDH . The GAPDH served as an internal control. The control, negative control (NC), siL21-1 and siL21-2 represented the untransfected, Mock-siRNA transfected, siL21-1 and siL21-2 transfected, respectively. Three independent experiments were of similar results.

Article Snippet: The E2F1 expression vector pCMV-E2F1 was constructed using the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, United States).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Sequencing, Western Blot, Negative Control

Journal: Frontiers in Oncology

Article Title: RPL21 siRNA Blocks Proliferation in Pancreatic Cancer Cells by Inhibiting DNA Replication and Inducing G1 Arrest and Apoptosis

doi: 10.3389/fonc.2020.01730

Figure Lengend Snippet: RPL21 regulates the cell cycle and DNA replication via E2F1 in PANC-1 and BxPC-3 cells. (A,C) The luciferase reporter vectors ( CCND1 , CCNE1 and MCM2 - 7 ) were constructed using pGL-3 vectors. 1 μg reporter vector was co-transfected with 1 μg pCMV-E2F1 vector or 1 μg empty pcDNA3.1 vector (control) for each well (6-well plates) containing 6 × 10 5 PANC-1 and BxPC-3 cells. Luciferase activity was measured 24 h after the transfection. (B,D) The PANC-1 and BxPC-3 cells were transfected with siRNAs with lipofectamine 2000 reagent. The control, NC, siL21-1 and siL21-2 represented the untransfected, Mock-siRNA transfected, siL21-1 and siL21-2 transfected, respectively. The cells after transfection were seeded in 6-well plates at 6 × 10 5 cells/well, and 2 g E2F1 luciferase reporters (E2F1-promoter) were transfected using lipofectamine 2000 reagent. Luciferase activity was measured 24 h after the transfection. Each bar represents the mean ± SD of triplicate analysis. * indicates P < 0.05 compared to the control as determined by the Student’s t -test.

Article Snippet: The E2F1 expression vector pCMV-E2F1 was constructed using the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, United States).

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

Journal: Frontiers in Oncology

Article Title: RPL21 siRNA Blocks Proliferation in Pancreatic Cancer Cells by Inhibiting DNA Replication and Inducing G1 Arrest and Apoptosis

doi: 10.3389/fonc.2020.01730

Figure Lengend Snippet: Model of E2F1 -mediated pancreatic cancer cell proliferation.

Article Snippet: The E2F1 expression vector pCMV-E2F1 was constructed using the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, United States).

Techniques: