Journal: Nature communications

Article Title: E2F1-dependent miR-421 regulates mitochondrial fragmentation and myocardial infarction by targeting Pink1.

doi: 10.1038/ncomms8619

Figure Lengend Snippet: Figure 6 | miR-421 is a transcriptional target of E2F1. (a) Mouse miR-421 promoter region contains a potential E2F1-binding site. (b) E2F1 activates miR-421 promoter activity. Cardiomyocytes were treated with the adenoviral b-gal or E2F1, the constructs of the empty vector (pGL-4.17), the wild type promoter or the promoter with mutations in the binding site (mutant), respectively. Luciferase activity was assayed. Data are shown as mean±s.e.m. of four independent experiments. Analysis was performed with one-way analysis of variance (ANOVA) followed by Tukey–Kramer post hoc analysis. *Po0.05. (c) E2F1 promotes miR-421 expression. Cardiomyocytes were infected with adenoviral b-gal or E2F1. E2F1 expression was analysed by immunoblot (left panel). Quantitative analysis of expression levels of E2F1 protein shown in lower panel. MiR-421 levels were analysed by qRT–PCR (right panel). Data are shown as mean±s.e.m. of four independent experiments. *Po0.05 versus untreated group in Student’s t-test. (d) MiR-421 levels in E2F1-deficient mice. E2F1-knockout mice (KO), wild-type littermates (WT). Data are shown as mean±s.e.m. of six independent experiments. *Po0.05 versus WT in Student’s t-test. (e) ChIP analysis of E2F1 binding to the promoter of miR-421. (f) Knockdown of E2F1 attenuates the increase of miR-421 promoter activity induced by H2O2. Cardiomyocytes were treated with the adenoviral E2F1-siRNA or E2F1-sc, the constructs of the empty vector (pGL-4.17), the wild type (WT) promoter, then were treated with H2O2. Luciferase activity was assayed. Data are shown as mean±s.e.m. of three independent experiments. Analysis was performed with one-way ANOVA followed by Tukey–Kramer post hoc analysis. *Po0.05.

Article Snippet: Mouse E2F1 cDNA was purchased from Origene.

Techniques: Binding Assay, Activity Assay, Construct, Plasmid Preparation, Mutagenesis, Luciferase, Expressing, Infection, Western Blot, Quantitative RT-PCR, Knock-Out, Knockdown

Journal: Nature communications

Article Title: E2F1-dependent miR-421 regulates mitochondrial fragmentation and myocardial infarction by targeting Pink1.

doi: 10.1038/ncomms8619

Figure Lengend Snippet: Figure 7 | E2F1 promotes mitochondrial fragmentation and apoptosis. (a) Cardiomyocytes were exposed to H2O2. Cells were harvested at the indicated time for the analysis of E2F1 levels by immunoblot. Data are shown as mean±s.e.m. of three independent experiments. *Po0.05 versus untreated group in Student’s t-test. (b,c) Enforced expression of E2F1 induces mitochondrial fragmentation and apoptosis. Cardiomyocytes were infected with adenoviral E2F1 or b-gal. Forty-eight hours after infection mitochondrial fragmentation (b) and apoptosis (c) were analysed. Data are shown as mean±s.e.m. of three independent experiments. Analysis was performed with one-way analysis of variance (ANOVA) followed by Tukey–Kramer post hoc analysis. *Po0.05 versus untreated group. (d) Cardiomyocytes were infected with adenoviral E2F1-siRNA or E2F1-sc, and then exposed to H2O2. E2F1 levels were detected by immunoblot. Data are shown as mean±s.e.m. of three independent experiments. Analysis was performed with one-way ANOVA followed by Tukey–Kramer post hoc analysis. *Po0.05. (e) Knockdown of E2F1 reduces mitochondrial fragmentation and apoptosis. Cardiomyocytes were treated as described in d, mitochondrial fragmentation (upper panel) and apoptosis (lower panel) were analysed. Data are shown as mean±s.e.m. of three independent experiments. Analysis was performed with one-way ANOVA followed by Tukey–Kramer post hoc analysis. *Po0.05. (f,g) E2F1-knockout mice attenuates mitochondrial fragmentation and apoptosis on I/R. WT and E2F1-knockout mice were subjected to I/R as described in Methods. Mitochondrial fragmentation (f) and apoptosis (g) were analysed. Data are shown as mean±s.e.m. of six independent experiments. Analysis was performed with one-way ANOVA followed by Tukey–Kramer post hoc analysis. *Po0.05 versus WT þ I/R. (h) E2F1-knockout mice attenuates myocardial infarction sizes on I/R. WTand E2F1-knockout mice were subjected to I/R as described in f. The upper panels are representative photos of midventricular myocardial slices. The lower panel shows infarct sizes. Data are shown as mean±s.e.m. of six independent experiments. Analysis was performed with one-way ANOVA followed by Tukey–Kramer post hoc analysis. *Po0.05 versus WT þ I/R. Bar ¼ 2 mm.

Article Snippet: Mouse E2F1 cDNA was purchased from Origene.

Techniques: Western Blot, Expressing, Infection, Knockdown, Knock-Out

Journal:

Article Title: The Epstein-Barr Virus Immediate-Early Protein BZLF1 Induces Expression of E2F-1 and Other Proteins Involved in Cell Cycle Progression in Primary Keratinocytes and Gastric Carcinoma Cells

doi: 10.1128/JVI.76.24.12543-12552.2002

Figure Lengend Snippet: Representative cellular genes activated by BZLF1 expressiona

Article Snippet: The primary antibodies included anti-E2F-1 rabbit polyclonal antibody (diluted 1:500; C20 [Santa Cruz]), anti-cyclin E mouse monoclonal antibody (1:500; BF683 [Santa Cruz]), Cdc25A mouse monoclonal antibody (1:500; F-6 [Santa Cruz]), SLBP rabbit polyclonal antibody (1:2,000; a generous gift of William Marzluff, University of North Carolina at Chapel Hill), and anti-BZLF1 mouse monoclonal antibody (1:200; AZ-69 [Argene]).

Techniques: Activation Assay

Journal:

Article Title: The Epstein-Barr Virus Immediate-Early Protein BZLF1 Induces Expression of E2F-1 and Other Proteins Involved in Cell Cycle Progression in Primary Keratinocytes and Gastric Carcinoma Cells

doi: 10.1128/JVI.76.24.12543-12552.2002

Figure Lengend Snippet: BZLF1 induces E2F-1 in a cell type-dependent manner. (A) Telomerase-immortalized human keratinocytes and normal human fibroblasts were infected with AdLacZ or AdBZLF1. At 48 h postinfection, the levels of E2F-1, BZLF1, and β-actin were quantitated by immunoblot analysis. (B) AGS gastric carcinoma cells and primary tonsil keratinocytes were infected as in panel A, and the level of E2F-1 was determined by immunoblot analysis. NS, nonspecific cross-reacting band. (C) FACS analysis was performed to compare the level of BZLF1 expression in Ad-BZLF1-infected AGS cells or telomerase-immortalized keratinocytes to that in the constitutively BZLF1-positive population of EBV-AGS cells. Neg., negative; Pos., positive.

Article Snippet: The primary antibodies included anti-E2F-1 rabbit polyclonal antibody (diluted 1:500; C20 [Santa Cruz]), anti-cyclin E mouse monoclonal antibody (1:500; BF683 [Santa Cruz]), Cdc25A mouse monoclonal antibody (1:500; F-6 [Santa Cruz]), SLBP rabbit polyclonal antibody (1:2,000; a generous gift of William Marzluff, University of North Carolina at Chapel Hill), and anti-BZLF1 mouse monoclonal antibody (1:200; AZ-69 [Argene]).

Techniques: Infection, Western Blot, Expressing

Journal:

Article Title: The Epstein-Barr Virus Immediate-Early Protein BZLF1 Induces Expression of E2F-1 and Other Proteins Involved in Cell Cycle Progression in Primary Keratinocytes and Gastric Carcinoma Cells

doi: 10.1128/JVI.76.24.12543-12552.2002

Figure Lengend Snippet: BZLF1 increases the expression of E2F-1-responsive genes in some cell types. (A) Telomerase-immortalized keratinocytes or normal human fibroblasts were mock infected or infected with the AdLacZ or AdBZLF1 vector. Immunoblot analysis was performed 2 days later to quantitate expression of cyclin E, SLBP, or Cdc25A, as indicated. (B) AGS and primary tonsil keratinocytes were infected with the AdLacZ or AdBZLF1 vector, and immunoblot analysis was performed 2 days later to quantitate expression of cyclin E and SLBP as indicated.

Article Snippet: The primary antibodies included anti-E2F-1 rabbit polyclonal antibody (diluted 1:500; C20 [Santa Cruz]), anti-cyclin E mouse monoclonal antibody (1:500; BF683 [Santa Cruz]), Cdc25A mouse monoclonal antibody (1:500; F-6 [Santa Cruz]), SLBP rabbit polyclonal antibody (1:2,000; a generous gift of William Marzluff, University of North Carolina at Chapel Hill), and anti-BZLF1 mouse monoclonal antibody (1:200; AZ-69 [Argene]).

Techniques: Expressing, Infection, Plasmid Preparation, Western Blot

Journal:

Article Title: The Epstein-Barr Virus Immediate-Early Protein BZLF1 Induces Expression of E2F-1 and Other Proteins Involved in Cell Cycle Progression in Primary Keratinocytes and Gastric Carcinoma Cells

doi: 10.1128/JVI.76.24.12543-12552.2002

Figure Lengend Snippet: BZLF1 induction of E2F-1 is not associated with apoptosis. Telomerase-immortalized human keratinocytes were mock infected or infected with AdLacZ or AdBZLF1. At 48 h postinfection, the binding of Annexin V-fluorescein isothiocyanate was quantitated by FACS analysis.

Article Snippet: The primary antibodies included anti-E2F-1 rabbit polyclonal antibody (diluted 1:500; C20 [Santa Cruz]), anti-cyclin E mouse monoclonal antibody (1:500; BF683 [Santa Cruz]), Cdc25A mouse monoclonal antibody (1:500; F-6 [Santa Cruz]), SLBP rabbit polyclonal antibody (1:2,000; a generous gift of William Marzluff, University of North Carolina at Chapel Hill), and anti-BZLF1 mouse monoclonal antibody (1:200; AZ-69 [Argene]).

Techniques: Infection, Binding Assay

Journal: Stem Cell Research & Therapy

Article Title: EIF5A2 enhances stemness of epithelial ovarian cancer cells via a E2F1/KLF4 axis

doi: 10.1186/s13287-021-02256-2

Figure Lengend Snippet: EIF5A2 modulates the expression of KLF4 through the transcription factor E2F1. a The mRNA levels of 9 candidate genes in SKOV3 cells with EIF5A2 knockdown were detected by qRT-PCR. b Knockdown of EIF5A2 substantially downregulated PLK1 and E2F1 expression in SKOV3 cells detected by western blotting. c Heatmap of EIF5A2, PLK1, and E2F1 mRNA expression of 28 MCAs samples detected by qRT-PCR. d The association between relative EIF5A2 and E2F1 mRNA levels in 28 MCAs samples was analyzed according to the Spearman-correlation factor. e The association between relative EIF5A2 and E2F1 mRNA levels from TCGA database. f Expression of KLF4 in SKOV3 cells transfected with empty vector, EIF5A2 plasmid, si-E2F1, or EIF5A2 plasmid plus si-E2F1. g The WT box indicates that there are 3 potential E2F1 binding sites within 2000 bp upstream of the KLF4 promoter region predicted by JASPAR, and the MUT box indicates the corresponding mutation sequence. The double-luciferase reporter gene experiment confirmed that only Site B transcriptional activity was significantly increased among the 3 binding sites, and the difference in luciferase activity disappeared after mutation. h Expression of pRb, Rb in SKOV3 cells after EIF5A2 downregulation and in HO-8910 cells after EIF5A2 upregulation. i Knockdown of EIF5A2 substantially downregulated Notch1, Hes1, and Hey1 expression in SKOV3 cells detected by western blotting and qRT-PCR

Article Snippet: The membranes were blocked with 5% fat-free milk dissolved in Tris-buffered saline supplemented with Tween 20 (TBST) for 1 h at room temperature and incubated with primary antibodies against EIF5A2 (1:1000; ab150439; Rabbit; Abcam), E2F1 (1:2000; #3742; Rabbit; Cell Signaling Technology), KLF4 (1:1000; #12173; Rabbit; Cell Signaling Technology), ALDH1A1 (1:1000; #54135; Rabbit; Cell Signaling Technology), and OCT-4 (1:3000; Mouse monoclonal; Proteintech) overnight at 4 °C, GAPDH (1:2000; Rabbit; Multi Sciences) was used as the control.

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Binding Assay, Mutagenesis, Sequencing, Luciferase, Activity Assay

Journal: Cell reports

Article Title: LncRNA SLNCR phenocopies the E2F1 DNA binding site to promote melanoma progression

doi: 10.1016/j.celrep.2025.115608

Figure Lengend Snippet: (A) Unbiased TF target enrichment analysis of RNA-seq data from A375 cells overexpressing SLNCR . (B) DATNA of TCGA RNA-seq samples of patients with melanoma. (C) Kaplan-Meier plots for patients with high levels of SLNCR and high levels of E2F1 (top left), high SLNCR and low E2F1 (bottom left), low SLNCR and high E2F1 (top right), and low SLNCR and low E2F1 (bottom right) identified using the TCGA SKCM STAR normalized dataset. Log rank p values ( p ) are indicated. (D) Matrigel invasion assays of A375 cells transfected with SLNCR overexpression or empty control plasmid and infected with scramble or E2F1-knockdown (KD) short hairpin RNA (shRNA). (E) Proliferation assay of A375 transfected as in (D). (F) Matrigel invasion assays of WM1575 (left) and WM1976 (right) infected with scramble or E2F1-KD shRNA. (G) Proliferation assay of WM1575 (left) and WM1976 (right) infected as in (F). In (A)–(G), **** p < 0.0001. For (D)–(G), data are represented as mean ± SD. Scale bar represents 100 μm. See also and .

Article Snippet: Protein production plasmids were created by introducing synthetic gene fragments containing wildtype, L132E and R166H mutant E2F1 DBD (residues 110–194) with an N-terminal His6-tag followed by a TEV protease cleavage site into pET28a SpyCatcherSnoopCatcher (gifted from Mark Howarth, Addgene plasmid #72324) by Gibson assembly.

Techniques: RNA Sequencing, Transfection, Over Expression, Control, Plasmid Preparation, Infection, Knockdown, shRNA, Proliferation Assay

Journal: Cell reports

Article Title: LncRNA SLNCR phenocopies the E2F1 DNA binding site to promote melanoma progression

doi: 10.1016/j.celrep.2025.115608

Figure Lengend Snippet: (A) Schematic presentation of SLNCR fragments assayed for E2F1 binding by pairwise Y3H. Predicted E2F1 BSs are marked with red triangles. (B) Predicted E2F1 BSs mapped onto SLNCR secondary structure. (C) REMSA with biotin-labeled BS1–5 RNA oligonucleotides as single-strand, double-strand, or hybrid fragments, incubated with purified E2F1. (D) REMSA using biotin-labeled BS1–5 (double-strand), BS1+2, BS2+3, and BS4+5 RNA oligonucleotides with E2F1 with and without competition by unlabeled versions of the same RNA oligonucleotides. For (C) and (D), gray lines indicate separate gels, and black lines were added for better visualization. See also .

Article Snippet: Protein production plasmids were created by introducing synthetic gene fragments containing wildtype, L132E and R166H mutant E2F1 DBD (residues 110–194) with an N-terminal His6-tag followed by a TEV protease cleavage site into pET28a SpyCatcherSnoopCatcher (gifted from Mark Howarth, Addgene plasmid #72324) by Gibson assembly.

Techniques: Binding Assay, Labeling, Incubation, Purification

Journal: Cell reports

Article Title: LncRNA SLNCR phenocopies the E2F1 DNA binding site to promote melanoma progression

doi: 10.1016/j.celrep.2025.115608

Figure Lengend Snippet: (A) REMSA of recombinant E2F1 incubated with biotin-labeled 42-mer dsDNA (left) or 60-mer BS2+3 RNA (right) probes. The labeled 42-mer dsDNA was competed by titrating increasing amounts of unlabeled 60-mer BS2+3 RNA, and vice versa, the labeled 60-mer BS2+3 RNA was competed by titrating increasing amounts of unlabeled 42-mer dsDNA. In each experiment, the third lane from the left has 10 μM unlabeled oligonucleotide of the same species as the labeled nucleotide to show specific binding. (B) REMSA of recombinant E2F1 DBD incubated with biotin-labeled 42-mer dsDNA (left) or 60-mer BS2+3 SLNCR RNA (right) probes. The labeled 42-mer dsDNA was competed by titrating increasing amounts of unlabeled BS2+3 RNA, and vice versa, the labeled 60-mer BS2+3 RNA was competed by titrating increasing amounts of unlabeled 42-mer dsDNA. In each experiment, the third lane from left has 18μM unlabeled DNA binding site and 5 μM of unlabeled 60-mer BS2+3 RNA to show specific binding. (C) Representative structure models of E2F1 DBD +60-mer RNA from 5 MD runs. (D) Representative structure models of E2F1 DBD +42-mer dsDNA from 3 MD runs. (E) Averaged residue interaction energies of E2F1 DBD with the 15-mer reference dsDNA (gray), the 42-mer DNA 5′ motif (light blue), the 42-mer dsDNA 3′ motif (dark blue), and 60-mer RNA (red) calculated by MMGBSA. Gray dashed line indicates −9.8 kcal/mol threshold for strong interactions. (F) Number of wild-type (WT) and mutant E2F1 DBD interactions at the base, phosphate, and sugar moieties of 15-mer dsDNA, 42-mer dsDNA 5′ and 3′ motifs, and 60-mer BS2+3 RNA. See also and , , , , , , , and .

Article Snippet: Protein production plasmids were created by introducing synthetic gene fragments containing wildtype, L132E and R166H mutant E2F1 DBD (residues 110–194) with an N-terminal His6-tag followed by a TEV protease cleavage site into pET28a SpyCatcherSnoopCatcher (gifted from Mark Howarth, Addgene plasmid #72324) by Gibson assembly.

Techniques: Recombinant, Incubation, Labeling, Binding Assay, Residue, Mutagenesis

Journal: Cell reports

Article Title: LncRNA SLNCR phenocopies the E2F1 DNA binding site to promote melanoma progression

doi: 10.1016/j.celrep.2025.115608

Figure Lengend Snippet: (A–C) Amount of protein contacts per nucleotide calculated by MMGBSA at base, phosphate, or sugar moieties. (A) Calculated protein contacts for the E2F1 DBD +15-mer dsDNA model. The top graph shows contacts with the sense strand (5′ to 3′), and the bottom graph shows protein contacts with the antisense strand (3′ to 5′). The E2F1 BS is shaded. (B) Calculated protein contacts for the E2F1 DBD +42-mer dsDNA model. The top graph shows contacts with the sense strand (5′ to 3′), and the bottom graph shows protein contacts with the antisense strand (3′ to 5′). The 5′ and 3′ motifs are shaded. (C) Calculated protein contacts for the E2F1 DBD +60-mer BS2+3 RNA model. Base-pairing interactions as predicted by RNAFold are shown as dot-and-bracket annotations below the sequence. BS2 and BS3 are shaded. (D) Table summarizing the protein contacts with base, phosphate, and sugar moieties in each MD model. See also .

Article Snippet: Protein production plasmids were created by introducing synthetic gene fragments containing wildtype, L132E and R166H mutant E2F1 DBD (residues 110–194) with an N-terminal His6-tag followed by a TEV protease cleavage site into pET28a SpyCatcherSnoopCatcher (gifted from Mark Howarth, Addgene plasmid #72324) by Gibson assembly.

Techniques: Sequencing

Journal: Cell reports

Article Title: LncRNA SLNCR phenocopies the E2F1 DNA binding site to promote melanoma progression

doi: 10.1016/j.celrep.2025.115608

Figure Lengend Snippet: (A) REMSA of γ-P-labeled 42-mer dsDNA (left) or biotin-labeled 60-mer BS2+3 RNA, (right) incubated with WT or mutant (R166H or L132E) E2F1 DBD or full-length E2F1 and DP1. (B and C) Averaged residue interaction energies calculated by MMGBSA of WT and mutant E2F1 DBD with 15-mer reference dsDNA (B) or 60-mer BS2+3 RNA (C). Gray dashed line indicates −9.8 kcal/mol threshold for strong interactions. WT systems are first presented in and are re-plotted here for comparison with the mutants. See also .

Article Snippet: Protein production plasmids were created by introducing synthetic gene fragments containing wildtype, L132E and R166H mutant E2F1 DBD (residues 110–194) with an N-terminal His6-tag followed by a TEV protease cleavage site into pET28a SpyCatcherSnoopCatcher (gifted from Mark Howarth, Addgene plasmid #72324) by Gibson assembly.

Techniques: Labeling, Incubation, Mutagenesis, Residue, Comparison

Journal: Cell reports

Article Title: LncRNA SLNCR phenocopies the E2F1 DNA binding site to promote melanoma progression

doi: 10.1016/j.celrep.2025.115608

Figure Lengend Snippet: (A) REMSA using biotin-labeled 60-mer BS2+3 RNA incubated with recombinant E2F1 or DP1 or both with increasing amounts of DP1. (B) Representative structure from 3 independent simulations of E2F1 DBD +DP1 DBD +15-mer dsDNA showing the arrangement of the ternary system. E2F1 (salmon), DP1 (gray), and 15-mer reference dsDNA (blue). (C) Model structure of the E2F1 DBD +60-mer SLNCR RNA with DP1 docked to fit the interaction interface of E2F1 (salmon), DP1 (gray), and RNA (orange). (D) Averaged residue interaction energies of DP1 DBD calculated using MMGBSA of the E2F1 DBD +DP1 DBD +15-mer dsDNA and E2F1 DBD +DP1 DBD +15-mer dsRNA models. The RNA in the latter system was created by directly converting 15-mer dsDNA (by adding O2′) to get a 15-mer dsRNA backbone. Gray dashed line indicates −9.8 kcal/mol threshold for strong interactions. (E) Amount of protein contacts per nucleotide calculated by MMGBSA of the E2F1 DBD +DP1 DBD +15-mer dsDNA model at base, phosphate, or sugar moieties contributed by E2F1 DBD (left) and those contributed by DP1 DBD (right). The top graphs show contacts with the sense strand (5′ to 3′), and the bottom graphs show protein contacts with the anti-sense strand (3′ to 5′). The E2F1 BS is shaded. (F) Amount of protein contacts per nucleotide calculated by MMGBSA of the E2F1 DBD +DP1 DBD +15-mer dsRNA model as described in (E). See also and and .

Article Snippet: Protein production plasmids were created by introducing synthetic gene fragments containing wildtype, L132E and R166H mutant E2F1 DBD (residues 110–194) with an N-terminal His6-tag followed by a TEV protease cleavage site into pET28a SpyCatcherSnoopCatcher (gifted from Mark Howarth, Addgene plasmid #72324) by Gibson assembly.

Techniques: Labeling, Incubation, Recombinant, Residue

Journal: mBio

Article Title: Genome-Wide Profiling of Cervical RNA-Binding Proteins Identifies Human Papillomavirus Regulation of RNASEH2A Expression by Viral E7 and E2F1

doi: 10.1128/mBio.02687-18

Figure Lengend Snippet: RNASEH2A is regulated by viral E7 via E2F1 and its expression in association with E2F1 and PCNA in cervical and endocervical cancer and head and neck squamous cell carcinoma. (A) Read coverage maps showing the RNA expression levels of all 7 cervical cancer samples and 7 normal samples for RNASEH2A. Distributions of RNASEH2A reads (blue for normal, red for cancer) along with RNASEH2A transcript structure were visualized using the Integrative Genomics Viewer (IGV). (B) Correlation between RNASEH2A and HPV16 E7 mRNAs in 48 clinical cervical lesion samples, including 25 CIN2-CIN3 and 23 cervical cancer tissue samples. (C) siRNA-specific knockdown of HPV16 or HPV18 E7 expression in CaSki (HPV16 + ) cells or HPV18-immortalized human foreskin keratinocytes (HFK18) with respect to expression of RNASEH2A, PCNA, and NOVA1 was evaluated by Western blotting. si-NS, nonspecific siRNA; si-E7, HPV16 or HPV18 E7-specific siRNA. Knockdown efficiency of E7 expression was evaluated by analysis of increased expression of p53 due to the E7 siRNA also targeting the overlapped bicistronic E6 RNA ; tubulin served as a sample loading control. (D) siRNA-specific knockdown of HPV16 or HPV18 E6 or E7 in CaSki (HPV16 + ) or HeLa (HPV18 + ) cells with respect to RNASEH2A expression was evaluated by TaqMan RT-qPCR. si-NS, nonspecific siRNA; si-E6, HPV16 or HPV18 E6-specific siRNA; si-E7, HPV16 or HPV18 E7-specific siRNA. (E and F) Specific-siRNA knockdown (E) or ectopic expression of E2F1 (F) in CaSki or HeLa cells with respect to RNASEH2A expression was evaluated by TaqMan RT-qPCR. si-NS, nonspecific siRNA; si-E2F1, E2F1-specific siRNA; p, control vector; p-E2F1, E2F1-expression vector. * , P < 0.05; * * , P < 0.01; ** * , P < 0.001. The relative expression levels of RNASEH2A in panels D, E, and F are shown as means ± SD for each treatment group determined in duplicate from three independent experiments, with the level in the si-NS (D and E) or vector control (F) group being set to 1. (G) Increased coexpression of RNASEH2A, PCNA, and E2F1 in cervical squamous cell carcinoma, endocervical adenocarcinoma (CESC), and head and neck squamous cell carcinoma (HNSC). Data were extracted from the TCGA data sets using TCGA2STAT R package version 1.2 ( https://CRAN.R-project.org/package=TCGA2STAT ).

Article Snippet: CaSki and HeLa cells were also transfected with 2 μg of an E2F1 expression vector (pRcCMV-HA-E2F1; Addgene, Watertown, MA) with X-tremeGENE HP DNA transfection reagent (Roche), and total RNA from the cells with E2F1 overexpression was analyzed for RNASEH2A expression by real-time RT-qPCR.

Techniques: Expressing, RNA Expression, Western Blot, Quantitative RT-PCR, Plasmid Preparation

Journal: The Journal of Cell Biology

Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1

doi: 10.1083/jcb.201411128

Figure Lengend Snippet: miR-9-3p down-regulates E2F1 by directly targeting its 3′ UTR. (A) WT 3′ UTR of E2F1, E2F3, MDM2, or PDK1 or mutant 3′ UTR of E2F1 mRNA was cloned into pMIR-REPORT downstream of Firefly luciferase, and the resulting plasmid was designated pMIR-3′-UTR-E2F1, pMIR-3′-UTR-E2F3, pMIR-3′-UTR-MDM2, pMIR-3′-UTR-PDK1, or pMIR-3′-UTR-E2F1 mutant, respectively (the underlines represent the mutated seed regions). (B) Nontargeted or miR-9-3p mimic was transfected into MCF-7 cells together with pMIR-3′-UTR-E2F1, pMIR-3′-UTR-E2F3, pMIR-3′-UTR-MDM2, pMIR-3′-UTR-PDK1, or pMIR-3′-UTR-E2F1 mutant and a control Renilla luciferase expression vector. 60 h after transfection, cells were harvested and assayed for relative luciferase units. (C) MCF-7 cells were transfected with nontargeted miRNA, miR-9-3p mimic, or miR-9-3p antagomir. 60 h after transfection, cells were harvested, and E2F1 expression was analyzed using RT-qPCR at the mRNA level (left) or Western blotting at the protein level (right). (D, top) MCF-7 cells were treated with PP242 or rapamycin as indicated and analyzed for E2F1 expression as described in C. (Bottom) 36 h after transfection with a miR-9-3p antagomir gradient, MCF-7 cells were treated with 200-nM PP242 for an additional 24 h, and E2F1 expression was analyzed via Western blotting. Error bars represent mean values ± SEM. ns, not significant.

Article Snippet: Plasmid myc-Rictor (plasmid 1860; ) coding for human Rictor fused to Myc in pRK-5 and plasmid pSG5L HA E2F1 (plasmid 10736; ) coding for human E2F1 fused to HA in pSG5L were obtained from Addgene.

Techniques: Mutagenesis, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot

Journal: The Journal of Cell Biology

Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1

doi: 10.1083/jcb.201411128

Figure Lengend Snippet: miR-9-3p acts downstream of mTORC2 and triggers apoptosis by targeting E2F1. (A) MCF-7 cells were transfected with a pool of two siRNAs (1:1) targeting different regions of E2F1 mRNA. After 36 h, cells were serum starved or treated with 400-µM 5-FU for an additional 24 h, harvested, and labeled with Annexin V–FITC and propidium iodide for analysis of apoptosis. The data shown are from a single representative experiment out of three repeats. (B) MCF-7 cells were transfected separately with the two siRNAs and treated as in A, followed by trypan blue staining (top) or Western blotting (bottom). (C) MCF-7 cells were transfected with vehicle or WT E2F1 in the absence or presence of PP242, as indicated. 12 h after transfection, cells were serum starved for an additional 24 h and harvested for either trypan blue staining (top) or Western blotting (bottom). (D) MCF-7 cells were sequentially transfected with Rictor siRNA and WT E2F1. After 24 h of serum starvation, the effects of ectopic E2F1 expression on Rictor knockdown were monitored via trypan blue staining (top) and Western blotting (bottom). (E) MCF-7 cells were sequentially transfected with miR-9-3p and E2F1, followed by assay as described in C. Error bars represent mean values ± SEM. C, control; NC, negative control.

Article Snippet: Plasmid myc-Rictor (plasmid 1860; ) coding for human Rictor fused to Myc in pRK-5 and plasmid pSG5L HA E2F1 (plasmid 10736; ) coding for human E2F1 fused to HA in pSG5L were obtained from Addgene.

Techniques: Transfection, Labeling, Staining, Western Blot, Expressing, Knockdown, Control, Negative Control

Journal: The Journal of Cell Biology

Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1

doi: 10.1083/jcb.201411128

Figure Lengend Snippet: mTORC2 mediates pri-miR-9-2 / miR-9-3p /E2F1 signaling and apoptosis via c-Myc. (A) The effect of silencing c-Myc on the pri-miR-9-2 / miR-9-3p level was assayed using RT-qPCR (left and middle). Protein expression of c-Myc was analyzed via Western blotting (right). (B) MCF-7 cells were treated with PP242 or rapamycin (left) or transfected with two different siRNAs for Rictor and Raptor (right) as indicated, and their effects on c-Myc expression were monitored using Western blotting. (C) MCF-7 cells were treated with 200-nM PP242 or 10-nM rapamycin for 24 h and subjected to chromatin extraction. Sheared chromatin was immunoprecipitated with a c-Myc antibody and the two binding promoter regions of hsa-miR-9-2 (one of three miR-9-3p –coding DNAs) augmented using RT-qPCR. (D and E) The effect of c-Myc silencing on the pri-miR-9-2 / miR-9-3p level regulated by PP242 (D) or Rictor knockdown (E) was analyzed using RT-qPCR. (F–I) The effect of c-Myc silencing on apoptosis induced upon Rictor depletion or PP242 treatment was detected with a light microscope (F), trypan blue staining (G), or Western blotting (H and I), as indicated. Bar, 50 µm. (J) In MDA-MB-231 cells, the effect of c-Myc silencing on apoptosis induced upon 200-nM PP242 treatment (left) or Rictor depletion (right) was detected with Western blot analysis of PARP cleavage. C, control; NC, negative control; TSS, transcription start site. Error bars represent mean values ± SEM.

Article Snippet: Plasmid myc-Rictor (plasmid 1860; ) coding for human Rictor fused to Myc in pRK-5 and plasmid pSG5L HA E2F1 (plasmid 10736; ) coding for human E2F1 fused to HA in pSG5L were obtained from Addgene.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transfection, Extraction, Immunoprecipitation, Binding Assay, Knockdown, Light Microscopy, Staining, Control, Negative Control

Journal: The Journal of Cell Biology

Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1

doi: 10.1083/jcb.201411128

Figure Lengend Snippet: mTORC2 modulates c-Myc/ miR-9-3p /E2F1 to promote survival in tumor xenografts and a mouse genetic model. (A and B) Images (A) and tumor growth curves (B) of MDA-MB-231 xenografts in BALB/c nude mice treated with vehicle, rapamycin, or PP242 daily by gavage. Treatment groups comprised five mice each. Each data point signifies the estimated tumor areas. Bar, 1 in. (C) Tumor weights of MDA-MB-231 xenografts in nude mice treated with vehicle, rapamycin, or PP242. (D) MDA-MB-231 xenografts were subjected to TUNEL labeling, with the percentage of apoptotic cells calculated under a light microscope. ***, P < 0.001 for comparison of PP242 therapy versus control therapy. Bars, 50 µm. (E and F) MDA-MB-231 xenografts were analyzed for expression of the indicated proteins via Western blotting (E) or miR-9-3p via RT-qPCR (F). (G–J) B lymphocytes from mice conditionally deficient in the Rictor gene were harvested and analyzed for cell death rate with Annexin V labeling (G) and trypan blue staining (H), expression of miR-9-3p with RT-qPCR (I), or cleavage of PARP and expression of c-Myc and E2F1 using Western blotting (J). The figures shown are from a single representative experiment out of three repeats. Error bars represent mean values ± SEM.

Article Snippet: Plasmid myc-Rictor (plasmid 1860; ) coding for human Rictor fused to Myc in pRK-5 and plasmid pSG5L HA E2F1 (plasmid 10736; ) coding for human E2F1 fused to HA in pSG5L were obtained from Addgene.

Techniques: TUNEL Assay, Labeling, Light Microscopy, Comparison, Control, Expressing, Western Blot, Quantitative RT-PCR, Staining

Journal: The Journal of Cell Biology

Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1

doi: 10.1083/jcb.201411128

Figure Lengend Snippet: Antagomir-9-3p restores AZD8055-suppressed growth and survival of tumor xenografts. (A and B) BALB/c nude mice were treated with captisol or AZD8055 by gavage and intratumorally injected with antagomirs of NC and miR-9-3p . Each treatment group comprised eight mice. (A) Representative images of mice (top) and MDA-MB-231 xenografts (bottom). (B) Tumor growth curves (left) and tumor weights (right) of MDA-MB-231 xenografts. (C and D) MDA-MB-231 xenografts treated as described in A were either analyzed for the indicated proteins by Western blotting (C) or subjected to TUNEL labeling (D). Images of DAB/hematoxylin staining under a light microscope (40×) are shown. Bar, 50 µm. Arrowheads indicate apoptotic cells. (E) A schematic diagram illustrates the currently defined mTORC2–CIP2A–PP2A–c-Myc– miR-9-3p –E2F1 pathway. Error bars represent mean values ± SEM. NC, negative control.

Article Snippet: Plasmid myc-Rictor (plasmid 1860; ) coding for human Rictor fused to Myc in pRK-5 and plasmid pSG5L HA E2F1 (plasmid 10736; ) coding for human E2F1 fused to HA in pSG5L were obtained from Addgene.

Techniques: Injection, Western Blot, TUNEL Assay, Labeling, Staining, Light Microscopy, Negative Control

Journal: PLoS Pathogens

Article Title: EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation

doi: 10.1371/journal.ppat.1005844

Figure Lengend Snippet: A) 10 million human peripheral blood mononuclear cells (PBMC) were infected with BAC-GFP-EBV for 4 hours. Cells were harvested at indicated time periods, and total RNA was isolated and reverse transcribed to cDNA according to the manufacturer’s instructions. Quantitative Real-time PCR was performed to detect E2Fs transcription levels. B) Bar graph shows log2 fold changes of E2F1 and E2F6 mRNA expression in detail that were analyzed previously. C-D) 10 million HEK-293 or Saos-2 cells were co-transfected with Myc-EBNA3C and control plasmid or Flag-tagged E2F1, E2F2, E2F3a, E2F4, E2F6 expression plasmids. Transfected cells were harvested and lysed at 36 hours post-transfection. About 5% of total lysates were reserved as the input. After incubating with 1 μg of mouse anti-Flag (M2) antibody, Immunoprecipitations were prepared for detection. Input and immunoprecipitated proteins were identified with 10% SDS-PAGE and analyzed by western blot using specific antibodies.

Article Snippet: Rabbit anti-E2F1 (C-20) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Infection, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Transfection, Control, Plasmid Preparation, Immunoprecipitation, SDS Page, Western Blot

Journal: PLoS Pathogens

Article Title: EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation

doi: 10.1371/journal.ppat.1005844

Figure Lengend Snippet: A-F) 10 million HEK-293 or Saos-2 cells were transfected with pGL2 control vector, full-length E2F1 promoter construct, Flag-E2F6, either increasing doses of EBNA3C or E2F6 expression plasmids. Thirty-six hours post-transfection, cells were harvested and carried out for luciferase reporter assays. Expression of transfected constructs were detected by western blot. G-H) 10 million HEK-293 or Saos-2 cells were transfected with pGL2 control vector, full-length E2F1 promoter construct, Flag-E2F1, Flag-E2F6, and EBNA3C expression plasmids. Luciferase reporter assays were carried out at 36 hours post-transfection as above. Expression levels of transfected constructs were also examined by western blot.

Article Snippet: Rabbit anti-E2F1 (C-20) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Control, Plasmid Preparation, Construct, Expressing, Luciferase, Western Blot

Journal: PLoS Pathogens

Article Title: EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation

doi: 10.1371/journal.ppat.1005844

Figure Lengend Snippet: A-B) Cell lysates from indicated B-cells (BJAB, BJAB7, BJAB10, LCL1, and LCL2) were immunoprecipitated with E2F1 specific antibody. Then immunoprecipitations were analyzed by western blot to identify expressions of endogenous EBNA3C, E2F6 proteins. C-D) 10 million HEK-293 or Saos-2 cells were electroporated with indicated combinations of Myc-E2F6, Flag-E2F1, EBNA3C expression constructs. The cell lysates were immunoprecipitated using anti-Myc antibody and the immunoprecipitations were examined with western blot.

Article Snippet: Rabbit anti-E2F1 (C-20) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immunoprecipitation, Western Blot, Expressing, Construct

Journal: PLoS Pathogens

Article Title: EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation

doi: 10.1371/journal.ppat.1005844

Figure Lengend Snippet: A-B) HEK-293 cells were co-transfected with appropriate combinations of Myc-EBNA3C, Flag-E2F6, and pGL2-E2F1 promoter reporter plasmid or pGL2 control plasmid. Then the ChIP assay was performed with E2F6 antibody and recovered DNA was quantitated by Real-time PCR using primers specific for transfected E2F1 promoter (A) or endogenous E2F1 promoter (B). NC, negative control. C) BJAB, BJAB7, LCL2 cells were harvested directly and performed ChIP assay as previously mentioned. The recovered DNA was detected with primers specific for endogenous E2F1 promoter. Shown are the results from independent experiments. Error bars indicate standard deviations.

Article Snippet: Rabbit anti-E2F1 (C-20) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, Negative Control

Journal: PLoS Pathogens

Article Title: EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation

doi: 10.1371/journal.ppat.1005844

Figure Lengend Snippet: EBNA3C interacts with E2F6 specifically and enhances the stability of E2F6. EBNA3C can also compete with E2F1 for E2F6 binding. E2F6 recruited together with EBNA3C binds E2F1 promoter and inhibits its activity, which contributes to B-cell proliferation by reducing the expression of E2F1. This mechanism describes that the contribution of E2F6 in EBNA3C-related oncogenic activity important for EBV-transformed B-cell proliferation.

Article Snippet: Rabbit anti-E2F1 (C-20) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Activity Assay, Expressing, Transformation Assay