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: Cell Death & Disease

Article Title: E2F1 induces TINCR transcriptional activity and accelerates gastric cancer progression via activation of TINCR/ STAU1 / CDKN2B signaling axis

doi: 10.1038/cddis.2017.205

Figure Lengend Snippet: E2F1 is overexpressed in GC tissues and cell lines. ( a ) Analysis of E2F1 mRNA expression in GC and paired ANTs based on GSE51575 microarray database. ( b ) The ROC curve for prediction of GC based on E2F1 expression level in GSE51575, using corresponding adjacent non-tumorous tissues as a control. ( c ) IHC analysis of E2F1 protein expression in GC tumor tissues (T) and paired ANTs. Pictures of representative areas were presented at different staining intensities in ANT and T. ( d ) Analysis of the expression pattern of E2F1 in gastric tissues detected by IHC. Stages I–IV, TNM stages. Statistical analyses were performed using Student’s paired t -test and one-way ANOVA. ( e ) Real-time RT-PCR and western blot analyzed the expression of E2F1 in a series of human GC cell lines and normal gastric epithelial cell line (GES-1). ( f ) Kaplan–Meier survival plots demonstrating the good prognostic effect of E2F1 upregulation correlated with a worse FPS and OS in GC patients ( n =876)

Article Snippet: pmaxGFP-E2F1 expression vector was purchased from Addgene (plasmid #16007) (Cambridge, USA).

Techniques: Expressing, Microarray, Staining, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: E2F1 induces TINCR transcriptional activity and accelerates gastric cancer progression via activation of TINCR/ STAU1 / CDKN2B signaling axis

doi: 10.1038/cddis.2017.205

Figure Lengend Snippet: Functional roles of E2F1 in vitro and in vivo . E2F1 knockdown in GC cells transfected with siRNAs against E2F1 or E2F1 upregulation by pmaxGFP-E2F1 vector. E2F1 depletion inhibits GC cell growth, as detected by the ( a ) MTT assay and ( c ) colony-formation assay, whereas ectopic expression of E2F1 promotes GC cell growth, as examined by the ( b ) MTT assay and ( d ) colony-formation assay. Bars: S.D.; * P <0.05, ** P <0.01. ( e ) Cell cycle analyses in the BGC823 and MGC803 cell lines. Relative to scrambled siRNA-transfected cells, E2F1 knockdown induced significantly increased the number of cells in the G0/G1 phase and reduced the number of cells in the S phase. Relative to empty vector-transfected cells, E2F1 upregulation promotes cell cycle progression. Representative FACS images and statistics based on three independent experiments. Bars: S.D.; * P <0.05, ** P <0.01. ( f ) Representative data showed that overexpression of E2F1 significantly promote tumor growth in nude mice xenograft model. MGC803 cells were transfected with empty vector or E2F1 expression vector and then injected into mouse flanks. Tumor growth was measured every 2 days after injection, and tumors were harvested at day 16 and weighed. ( g ) Detection of the cell proliferation markers PCNA in xenograft tumors by IHC

Article Snippet: pmaxGFP-E2F1 expression vector was purchased from Addgene (plasmid #16007) (Cambridge, USA).

Techniques: Functional Assay, In Vitro, In Vivo, Transfection, Plasmid Preparation, MTT Assay, Colony Assay, Expressing, Over Expression, Injection

Journal: Cell Death & Disease

Article Title: E2F1 induces TINCR transcriptional activity and accelerates gastric cancer progression via activation of TINCR/ STAU1 / CDKN2B signaling axis

doi: 10.1038/cddis.2017.205

Figure Lengend Snippet: E2F1 upregulate TINCR expression in GC cells. ( a ) A dual-luciferase reporter assay was performed by co-transfection of the TINCR promoter fragment (TINCR-pGL3) with overexpression of E2F1. ( b ) Reporter assay in cells transfected with various TINCR promoters constructs with deletion in different binding elements for E2F1 (WT, wild type; D, deletion type). Luciferase activity was expressed as relative to that of the pGL3 vector (a promoter-less vector) ( c ) ChIP assay demonstrated endogenous E2F1 binding to the TINCR gene promoter, and the ectopic expression or siRNA knockdown, respectively, increased or reduced E2F1 enrichment on the TINCR promoter * P <0.05, ** P <0.01. ( d ) qPCR analysis of TINCR expression levels following the treatment of BGC823 and MGC803 cells with siRNA-E2F1 and pmaxGFP-E2F1 expression vector, respectively. Bars: S.D.; * P <0.05, ** P <0.01. ( e ) Analysis of the relationship between TINCR expression (ΔCt value) and E2F1 mRNA level (ΔCt value) in 80 GC tissues

Article Snippet: pmaxGFP-E2F1 expression vector was purchased from Addgene (plasmid #16007) (Cambridge, USA).

Techniques: Expressing, Luciferase, Reporter Assay, Cotransfection, Over Expression, Transfection, Construct, Binding Assay, Activity Assay, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: E2F1 induces TINCR transcriptional activity and accelerates gastric cancer progression via activation of TINCR/ STAU1 / CDKN2B signaling axis

doi: 10.1038/cddis.2017.205

Figure Lengend Snippet: TINCR promotes cells proliferation and regulates CDKN2B expression in MGC803 and AGS cells; and is involved in the E2F1-mediated promotion of viability. ( a ) MTT assays were performed to determine the cell viability of siRNAs- TINCR -transfected GC cells. ( b ) EDU (red)/DAPI (blue) immunostaining assay was used to confirm the results of MTT assay. The data represent the mean±S.D. from three independent experiments. * P <0.05, ** P <0.01. ( c ) The simultaneous depletion of TINCR could partly 'rescue' the proliferation effects induced by overexpressed E2F1 in MGC803 cells. Error bars represent S.D., n =3. * P <0.05; ** P <0.01. ( d ) TINCR depletion upregulates the expression of CDKN2B in GC cells, and the simultaneous overexpression of E2F1 could partly 'rescue' the CDKN2B expression. The expression of CDKN2B protein in MGC803 cells was analyzed by western blotting

Article Snippet: pmaxGFP-E2F1 expression vector was purchased from Addgene (plasmid #16007) (Cambridge, USA).

Techniques: Expressing, Transfection, Immunostaining, MTT Assay, Over Expression, Western Blot

Journal: Cell Death & Disease

Article Title: E2F1 induces TINCR transcriptional activity and accelerates gastric cancer progression via activation of TINCR/ STAU1 / CDKN2B signaling axis

doi: 10.1038/cddis.2017.205

Figure Lengend Snippet: Summary diagram describes that E2F1 and TINCR regulates GC cell proliferation

Article Snippet: pmaxGFP-E2F1 expression vector was purchased from Addgene (plasmid #16007) (Cambridge, USA).

Techniques: