e2f1 expression vector e2f1 ha  (Addgene inc)

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: