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:

Journal: Scientific reports

Article Title: An E2F1/MiR-17-92 Negative Feedback Loop mediates proliferation of Mouse Palatal Mesenchymal Cells.

doi: 10.1038/s41598-017-05479-7

Figure Lengend Snippet: Figure 2. Role of E2F1 in regulating the proliferation of PMCs. (A) The expression of E2F1-3 in palatal shelves on E12, E13 and E14 determined by Westernblot; Blots were cropped for figure construction. (B) Expression of E2F1-3 on E13.5 was measured by immunohistochemical staining (left panel, magnification ×100). The positive rate of E2F1 and E2F3 in palatal shelves was significantly higher than E2F2 (right panel, n = 6, *P < 0.01). (C) MTT assay showing the growth curve of PMCs with or without E2F1 knockdown. Values are mean ± SD of triplicate experiments in each group. *P < 0.01. (D) Left panel: immunofluorescence staining of Ki-67 and E2F1 in PMCs. Right panel: the quantification of Ki67 and E2F1 double positive cells. *P < 0.01.

Article Snippet: Vectors of shE2F1 (Origene TG509487), shSCR (vectors against a scrambled sequence, negative control (Origene TR30013), and E2F1 expression vectors (Addgene plasmid 10736) were transfected into PMCs with TurboFectin 8.0 (Origene, Rockville, MD, USA) following the manufacturer’s protocol.

Techniques: Expressing, Immunohistochemical staining, Staining, MTT Assay, Knockdown, Immunofluorescence

Journal: Scientific reports

Article Title: An E2F1/MiR-17-92 Negative Feedback Loop mediates proliferation of Mouse Palatal Mesenchymal Cells.

doi: 10.1038/s41598-017-05479-7

Figure Lengend Snippet: Figure 3. Positive regulation of miR-17-92 by E2F1. (A) The expression of miR-17-92 members in control and E2F1 overexpressing cells was analyzed by RT-qPCR. Values represent means ± SD. Error bars, SD. *P < 0.05. (B) To detect the direct regulation of E2F1 on miR-17-92, a ChIP assay was performed with 3 putative binding sites of E2F1 on the miR-17-92 promoter. Input DNA that was not enriched by immunoprecipitation was amplified as a positive control. The miR-106a ORF region was used as negative control. The gels were cropped for figure construction.

Article Snippet: Vectors of shE2F1 (Origene TG509487), shSCR (vectors against a scrambled sequence, negative control (Origene TR30013), and E2F1 expression vectors (Addgene plasmid 10736) were transfected into PMCs with TurboFectin 8.0 (Origene, Rockville, MD, USA) following the manufacturer’s protocol.

Techniques: Expressing, Control, Quantitative RT-PCR, Binding Assay, Immunoprecipitation, Amplification, Positive Control, Negative Control

Journal: Scientific reports

Article Title: An E2F1/MiR-17-92 Negative Feedback Loop mediates proliferation of Mouse Palatal Mesenchymal Cells.

doi: 10.1038/s41598-017-05479-7

Figure Lengend Snippet: Figure 4. Negative regulation of E2F1 by miR-17-92. (A) The relative expression of E2F1-3 mRNA was evaluated by qPCR. Values represent means ± SD. Error bars, SD. *P < 0.05. (B) Protein levels of E2F1-3 were measured by Western blot analysis. GAPDH was used as the internal control. Blots were cropped for figure construction. (C) The location and sequences of predicted target sites in the 3′UTR of mouse E2F1 for miR- 17/20a. The seed sequences of miR-17/20a. The sequences of predicted target sites are conserved across species including human and mouse. (D) Luciferase assay was used to validate the predicted target sites of miR-17/20a on 3′UTR of E2F1. Data were presented as relative luciferase activities compared to normoxia after normalizing to Renilla luciferase activities. Experiments were performed in triplicate (*P < 0.05).

Article Snippet: Vectors of shE2F1 (Origene TG509487), shSCR (vectors against a scrambled sequence, negative control (Origene TR30013), and E2F1 expression vectors (Addgene plasmid 10736) were transfected into PMCs with TurboFectin 8.0 (Origene, Rockville, MD, USA) following the manufacturer’s protocol.

Techniques: Expressing, Western Blot, Control, Luciferase

Journal: Scientific reports

Article Title: An E2F1/MiR-17-92 Negative Feedback Loop mediates proliferation of Mouse Palatal Mesenchymal Cells.

doi: 10.1038/s41598-017-05479-7

Figure Lengend Snippet: Figure 5. MiR-17-92 negatively regulates E2F1-induced cell cycle transition of PMCs. (A) MTT assay showing the role of miR-17-92 on the growth of PMCs. Values are mean ± SD of triplicate experiments in each group. *P < 0.01. (B) Cell cycle of PMCs transfected with miR-17-92 MI or miR SCR was analysed by flow cytometry. (C) Quantification analysis of cells in G0/G1 phase. Values are mean ± SD of triplicate experiments in each group. *P < 0.01. (D) Quantification analysis of cells in S phase. Values are mean ± SD of triplicate experiments in each group. *P < 0.01.

Article Snippet: Vectors of shE2F1 (Origene TG509487), shSCR (vectors against a scrambled sequence, negative control (Origene TR30013), and E2F1 expression vectors (Addgene plasmid 10736) were transfected into PMCs with TurboFectin 8.0 (Origene, Rockville, MD, USA) following the manufacturer’s protocol.

Techniques: MTT Assay, Transfection, Flow Cytometry

Journal: Scientific reports

Article Title: An E2F1/MiR-17-92 Negative Feedback Loop mediates proliferation of Mouse Palatal Mesenchymal Cells.

doi: 10.1038/s41598-017-05479-7

Figure Lengend Snippet: Figure 6. Schematic cartoon illustrating the negative feedback loop between E2F1 and miR-17-92 and its regulation on the cell cycle of PMCs.

Article Snippet: Vectors of shE2F1 (Origene TG509487), shSCR (vectors against a scrambled sequence, negative control (Origene TR30013), and E2F1 expression vectors (Addgene plasmid 10736) were transfected into PMCs with TurboFectin 8.0 (Origene, Rockville, MD, USA) following the manufacturer’s protocol.

Techniques: