e2f3  (Santa Cruz Biotechnology)

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Critical functions of microRNA‐30a‐5p‐E2F3 in cardiomyocyte apoptosis induced by hypoxia/reoxygenation

doi: 10.1002/kjm2.12309

Figure Lengend Snippet: miR‐30a‐5p targets E2F3 in cardiomyocytes. A, The common targets of miR‐30‐5p in five bioinformatic analysis datasets. B, Conserved binding sites of miR‐30a‐5p on the Wt and Mut 3′‐UTR of E2F3. C, A luciferase reporter construct with the Wt or Mut miR‐30a‐5p binding sequence from the 3′‐UTR of the E2F3 was transfected into H9c2 cells with miR‐30a‐5p mimics and mimic negative control (miR‐ctrl). ** P < .01, compared with miR‐ctrl. D, The mRNA level of E2F3 was detected by qRT‐PCR assay. E, The protein level of E2F3 in H9c2 cells transfected with miR‐30a‐5p mimics was detected by western blot analysis. F, The protein level of E2F3 in H9c2 cells treated H/R was detected by western blot analysis (upper panel). The expressions of E2F3 in myocardial tissue from I/R injury group and Sham group were measured by western blot (lower panel). G, H9c2 cardiomyocytes were transfected with vector or pcDNA3.1‐E2F3. The expression of E2F3 was detected by western blotting assay. H, H9c2 cardiomyocytes were transfected with vector or pcDNA3.1‐E2F3. Transfected H9c2 cardiomyocytes were subjected to H/R treatment. The proliferation of H9c2 cardiomyocytes was determined by CCK‐8 assay. I, Transfected H9c2 cardiomyocytes were subjected to H/R treatment. Cell apoptosis was detected by Annexin VFITC/PI double staining in H/R‐treated H9c2 cells. N = 3 per group. ** P < .01, compared with vector, ## P < .01, compared with H/R vector. E2F3, E2F transcription factor 3; H/R, hypoxia/reoxygenation; Mut, mutant type; Wt, wild type

Article Snippet: Expression of E2F transcription factor 3 (E2F3) siRNA (si‐E2F3) was obtained from Genepharma (Shanghai, China).

Techniques: Binding Assay, Luciferase, Construct, Sequencing, Transfection, Negative Control, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Expressing, CCK-8 Assay, Double Staining, Mutagenesis

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Critical functions of microRNA‐30a‐5p‐E2F3 in cardiomyocyte apoptosis induced by hypoxia/reoxygenation

doi: 10.1002/kjm2.12309

Figure Lengend Snippet: Downregulation of E2F3 reverses the effects of miR‐30a‐5p in H/R‐treated H9C2 cells. A, qRT‐PCR analysis was performed to detect the level of E2F3 in H/R‐treated H9c2 cells following transfection with anti‐miR‐30a‐5p or cotransfection with anti‐miR‐30‐5p combination with si‐E2F3. B, Western blot analysis was performed to detect the level of E2F3 in H/R‐treated H9c2 cells. C, CCK‐8 assay was conducted using H/R‐treated H9c2 cells. D, Cell apoptosis was detected by Annexin VFITC/PI double staining in H/R‐treated H9c2 cells. E, Western blot analysis of Bax and Bcl‐2 protein expression in H/R‐treated H9c2 cells. N = 3 per group. ** P < .01, compared with control, ## P < .01, compared with H/R, && P < .01, compared with H/R anti‐miR‐30a‐5p. E2F3, E2F transcription factor 3; H/R, hypoxia/reoxygenation

Article Snippet: Expression of E2F transcription factor 3 (E2F3) siRNA (si‐E2F3) was obtained from Genepharma (Shanghai, China).

Techniques: Quantitative RT-PCR, Transfection, Cotransfection, Western Blot, CCK-8 Assay, Double Staining, Expressing, Control

Journal: Non-Coding RNA

Article Title: Truncated Isoforms of lncRNA ANRIL Are Overexpressed in Bladder Cancer, But Do Not Contribute to Repression of INK4 Tumor Suppressors

doi: 10.3390/ncrna1030266

Figure Lengend Snippet: ( a ) mRNA expression of E2F1 (dark grey bars) and E2F3 (light grey bars) was determined in UCCs and UCon by qRT-PCR and normalized to TBP . ( b ) Expression levels of E2F factors in UCCs and controls were correlated with that of ANRIL transcripts by calculating the Pearson correlation coefficient ( * p ≤ 0.05). ( c ) Relative overall ANRIL expression (“ANRIL all”) was determined by qRT-PCR after siRNA-mediated knockdown of E2F1 (left panel; * p ≤ 0.05) or transient ectopic expression (right panel). Efficiency of E2F1 knockdown and overexpression was verified on the protein level ( d ). Similarly, ANRIL expression was measured after siRNA-mediated knockdown of E2F3 (left panel) or transient ectopic expression (right panel). Again, expression modulation of E2F3 was checked on the protein level ( e ). The two different isoforms of E2F3 (A and B) were overexpressed separately by the respective plasmid. However, the siRNA targeted both isoforms as shown in ( f ). The siRNA knockdown in HT-1376 cells was less efficient.

Article Snippet: For siRNA-mediated knockdown cells were transfected with 10 nM ANRIL siRNA (Lincode SMART-Pool #R-188105-00, Thermo Scientific Dharmacon, Darmstadt, Germany) compared to a non-targeting control pool (#D-001320-10, Thermo Scientific Dharmacon), siRNA against E2F1 (5’- GGACCUUCGUAGCAUUGCAtt; Ambion, Darmstadt, Germany), siRNA against E2F3 targeting both isoforms (5’- GCGAUCUCUUCGAUGCUUAtt, Ambion) or a non-targeting control (5’-AGGUAGUGUAAUCGCCUUG-5’; Ambion) using Lipofectamine RNAiMAX (Life Technologies, Darmstadt, Germany), according to the manufacturer’s recommendations.

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Plasmid Preparation