Journal: PLoS ONE

Article Title: Repression of Androgen Receptor Transcription through the E2F1/DNMT1 Axis

doi: 10.1371/journal.pone.0025187

Figure Lengend Snippet: (A) Histology of prostate tissue taken from both K5-E2F1 transgenic and wild type mice. (B) Prostate epithelial cell lines established from both transgenic and wild type mice were analyzed by western blot for the expression of E2F1, cell cycle genes (Cyclin E and PCNA), the epithelial cell specific marker E-cadherin (E-cad), and Androgen Receptor (AR). Actin is shown as a loading control. (C) A trypan blue exclusion assay was implemented to measure the viability of cell lines obtained from the mouse models. Each point represents the mean of three independent experiments with the standard deviation.

Article Snippet: The gel was transferred to Optitran nitrocellulose membrane (Schleicher & Schuell Biosciences Inc. Keene, NH) by electrophoresis for 1 hour at 45 V. The membrane was blocked in 10% nonfat dry milk in TBST (10 mM Tris, 250 mM NaCl, 1% Tween 20) for 1 hour at room temperature and immunoblotted with primary antibodies for AR (N-20, Santa Cruz, Santa Cruz, CA), E2F1 (KH-95, Pharmingen, Franklin Lakes, NJ), Rb (Pharmingen), PCNA (C-20, Santa Cruz), E-Cadherin (H-108 Santa Cruz), Cyclin E (M-20, Santa Cruz), DNMT1 (Raw M0231S prep gift from Dr. Sriharsa Pradhan, New England BioLabs Inc., Ipswich, MA), β-actin (C-11, Santa Cruz), or actin (AC-40 Sigma, St. Louis, MO).

Techniques: Transgenic Assay, Western Blot, Expressing, Marker, Trypan Blue Exclusion Assay, Standard Deviation

Journal: PLoS ONE

Article Title: Repression of Androgen Receptor Transcription through the E2F1/DNMT1 Axis

doi: 10.1371/journal.pone.0025187

Figure Lengend Snippet: ( A) Northern blot analysis of stably transfected mouse prostate epithelial cells (PrE) with either pCDNA3 (control vector) or E2F1 shown as E2F1-1 and E2F1-2 to detect AR and E2F1 gene transcription. The 28S and 18S ribosomal bands are shown for loading comparison. ( B) Western blot analysis of whole cell lysates harvested from PrE cells stably transfected with pcDNA3 (control) or E2F1 for the detection of AR, Cyclin E, and PCNA. β-actin is shown as a loading control. ( A and B) The northern blot and western is representative of 3 separate experiments. LNCaP cells were co-transfected with 1 µg of ARE-Luc ( C) and 500 ng of either empty pcDNA3 vector or E2F1 in the presence of 10 −9 M R1881. were normalized to β-galactosidase (B-gal) from a co-transfected CMV promoter driven B-gal reporter construct. The histogram represents the mean value of three independent experiments with the indicated standard deviation. The western depicts the expression levels for E2F1 and AR relative to tubulin for the transfection and treatment conditions. * indicates P<0.05 for the indicated comparison in brackets.

Article Snippet: The gel was transferred to Optitran nitrocellulose membrane (Schleicher & Schuell Biosciences Inc. Keene, NH) by electrophoresis for 1 hour at 45 V. The membrane was blocked in 10% nonfat dry milk in TBST (10 mM Tris, 250 mM NaCl, 1% Tween 20) for 1 hour at room temperature and immunoblotted with primary antibodies for AR (N-20, Santa Cruz, Santa Cruz, CA), E2F1 (KH-95, Pharmingen, Franklin Lakes, NJ), Rb (Pharmingen), PCNA (C-20, Santa Cruz), E-Cadherin (H-108 Santa Cruz), Cyclin E (M-20, Santa Cruz), DNMT1 (Raw M0231S prep gift from Dr. Sriharsa Pradhan, New England BioLabs Inc., Ipswich, MA), β-actin (C-11, Santa Cruz), or actin (AC-40 Sigma, St. Louis, MO).

Techniques: Northern Blot, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Construct, Standard Deviation, Expressing

Journal: PLoS ONE

Article Title: Repression of Androgen Receptor Transcription through the E2F1/DNMT1 Axis

doi: 10.1371/journal.pone.0025187

Figure Lengend Snippet: ( A) mPrE cells were co-transfected with 1 µg of mARp-Luc, E2F-Luc or CRE-Luc luciferase reporter constructs with 0.5 µg of empty vector (pcDNA3), wild type E2F1, dominant negative E2F1 (DN E2F1) or SV-40 Large T antigen (Tag). After 72 hours, cells were harvested and assayed for luciferase expression. The results are shown as averages of three independent experiments. Assays were done in triplicate and mean values are shown with standard deviation. The results were normalized to β-galactosidase (B-gal) expression from a co-transfected CMV promoter driven B-gal reporter construct. (B) LNCaP cells were co-transfected with either 1 µg of hAR-Luc or DHFR-Luc and 0.5 µg of either wild type E2F1or E2F1 1–284 mutant constructs. The histograms represent the mean value of three independent experiments with the indicated standard deviation. were normalized to β-galactosidase (B-gal) expression from a co-transfected CMV promoter driven by a B-gal reporter construct. * indicates P<0.05 for the indicated comparison in brackets.

Article Snippet: The gel was transferred to Optitran nitrocellulose membrane (Schleicher & Schuell Biosciences Inc. Keene, NH) by electrophoresis for 1 hour at 45 V. The membrane was blocked in 10% nonfat dry milk in TBST (10 mM Tris, 250 mM NaCl, 1% Tween 20) for 1 hour at room temperature and immunoblotted with primary antibodies for AR (N-20, Santa Cruz, Santa Cruz, CA), E2F1 (KH-95, Pharmingen, Franklin Lakes, NJ), Rb (Pharmingen), PCNA (C-20, Santa Cruz), E-Cadherin (H-108 Santa Cruz), Cyclin E (M-20, Santa Cruz), DNMT1 (Raw M0231S prep gift from Dr. Sriharsa Pradhan, New England BioLabs Inc., Ipswich, MA), β-actin (C-11, Santa Cruz), or actin (AC-40 Sigma, St. Louis, MO).

Techniques: Transfection, Luciferase, Construct, Plasmid Preparation, Dominant Negative Mutation, Expressing, Standard Deviation, Mutagenesis

Journal: PLoS ONE

Article Title: Repression of Androgen Receptor Transcription through the E2F1/DNMT1 Axis

doi: 10.1371/journal.pone.0025187

Figure Lengend Snippet: The use of either a dominant negative E2F1 construct or a shRNA to knock down DNMT1 both result in the downregulation of DNMT1 and subsequent rescue of AR expression. E2F1 overexpression experiments suggest that elevated E2F1 levels increase DNMT1 protein expression that associates directly with the AR promoter or possibly in complex with E2F1 to repress AR.

Article Snippet: The gel was transferred to Optitran nitrocellulose membrane (Schleicher & Schuell Biosciences Inc. Keene, NH) by electrophoresis for 1 hour at 45 V. The membrane was blocked in 10% nonfat dry milk in TBST (10 mM Tris, 250 mM NaCl, 1% Tween 20) for 1 hour at room temperature and immunoblotted with primary antibodies for AR (N-20, Santa Cruz, Santa Cruz, CA), E2F1 (KH-95, Pharmingen, Franklin Lakes, NJ), Rb (Pharmingen), PCNA (C-20, Santa Cruz), E-Cadherin (H-108 Santa Cruz), Cyclin E (M-20, Santa Cruz), DNMT1 (Raw M0231S prep gift from Dr. Sriharsa Pradhan, New England BioLabs Inc., Ipswich, MA), β-actin (C-11, Santa Cruz), or actin (AC-40 Sigma, St. Louis, MO).

Techniques: Dominant Negative Mutation, Construct, shRNA, Expressing, Over Expression

Journal: Cell Death & Disease

Article Title: E2F1 mediated DDX11 transcriptional activation promotes hepatocellular carcinoma progression through PI3K/AKT/mTOR pathway

doi: 10.1038/s41419-020-2478-0

Figure Lengend Snippet: a E2F1 was speculated as an upstream factor of DDX11 based on differential E2F1expression analysis between normal tissues and tumor tissues ( a left panel) and Pearson analysis of the correlation between DDX11 and E2F1 expression in TCGA-LIHC cohort ( a right panel). b , c qPCR and western blot ( d ) analysis of the E2F1 and DDX11 expression in HepG2 or SMMC7721 cells transfected with E2F1 plasmid and E2F1 siRNA, respectively. e The sequence logo of a potential E2F1 binding site in JASPAR and a diagram of mutant sites in the DDX11 sequence. f , g Dual-luciferase reporter assay was performed by co-transfection of the DDX11 promoter fragment (WT or MUT pGL3-DDX11) and an E2F1-overexpression construct into HEK293 cells. h ChIP-seq analysis of E2F1 binding in DDX11 gene locus based on the data from GSE95305. i , j ChIP assay was performed using E2F1 antibody to determine the E2F1 binding to the DDX11 gene promoter. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: E2F1 antibody and IgG control antibody were purchased from Proteintech Company (Wuhan, China).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Sequencing, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Cotransfection, Over Expression, Construct, ChIP-sequencing

Journal: Cell Death & Disease

Article Title: E2F1 mediated DDX11 transcriptional activation promotes hepatocellular carcinoma progression through PI3K/AKT/mTOR pathway

doi: 10.1038/s41419-020-2478-0

Figure Lengend Snippet: HepG2 or SMMC7721 cells were transfected with negative control (NC), siRNA targeting E2F1 (siRNA-E2F1), or siRNA-E2F1 & DDX11 overexpression plasmid (DDX11). a – c Cell proliferation of HepG2 or HuH-6 cells was assessed by CCK-8 assay, colony formation assay or EDU staining assay. Scale bars, 50 μm. d Cell invasion ability of HepG2 or SMMC7721 cells in different groups was analyzed by transwell assay. Scale bars, 50 μm. e Expression levels of PI3K/p-PI3K(Tyr458), Akt/p-Akt (Ser473), and mTOR/p-mTOR (Ser2448) in SMMC7721 transfected with negative control, siRNA-E2F1, negative control plasmid, or DDX11 plasmid were analyzed by western blot. f Expression levels of PI3K/p-PI3K(Tyr458), Akt/p-Akt (Ser473), and mTOR/p-mTOR (Ser2448) in SMMC7721 transfected with negative control, E2F1 plasmid, negative control siRNA, or DDX11 siRNA were analyzed by western blot. The representative result of at least three independent experiments was shown. g Schematic representation showing E2F1/DDX11 axis mediated aggressive behaviors in HCC through activating PI3K/AKT/mTOR signaling pathway. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: E2F1 antibody and IgG control antibody were purchased from Proteintech Company (Wuhan, China).

Techniques: Transfection, Negative Control, Over Expression, Plasmid Preparation, CCK-8 Assay, Colony Assay, Staining, Transwell Assay, Expressing, Western Blot

Journal: Oncotarget

Article Title: E2F1-induced upregulation of long noncoding RNA LINC00668 predicts a poor prognosis of gastric cancer and promotes cell proliferation through epigenetically silencing of CKIs

doi: 10.18632/oncotarget.6745

Figure Lengend Snippet: A. Analysis of LINC00668 expression levels in GC cell lines (SGC-7901, BGC-823 and MGC-803) compared with the normal gastric epithelium cell line (GES-1) by qRT-PCR. B. Description of E2F1 binding sites in the promoter region of LINC00668. The position of ChIP primers F. was indicated by arrows. C. Western blot assays detected the expression of E2F1 after the transfection of the overexpression plasmid and si-RNA. D. The expression of LINC00668 and ANRIL was determined in E2F1 and si-E2F1 systems in BGC-823 and SGC-7901 cells. E. The luciferase assays were performed to detect the promoter activity of LINC00668. Induction of LINC00668 promoter activity by E2F1 in BGC-823 cells. Then, deletion analysis of the promoter activity was performed to determine the role of the E2F1 site in E2F1-mediated regulation of LINC00668. F. Enrichment of E2F1 in the LINC00668 promoter after E2F1 overexpression. E2F1 was immunoprecipitated, and the promoter region containing E2F1-binding sequences were quantified by qRT-PCR. The ChIP primers are detailed in . G. Immunohistochemistry was used to detect the expression of the E2F1 protein in GC and corresponding non-tumor tissues. Bar, 100μm. H. The immunoreactivity of the E2F1 protein in GC tissues showed a statistically significant positive correlation with the relative level of LINC00668 expression. Error bars indicate the means±S.E.M. * P < 0.05, ** P < 0.01. n.s., not significant.

Article Snippet: E2F1 antibody was obtained from Cell Signaling Technology.

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Western Blot, Transfection, Over Expression, Plasmid Preparation, Luciferase, Activity Assay, Immunoprecipitation, Immunohistochemistry

Journal: Oncotarget

Article Title: E2F1-induced upregulation of long noncoding RNA LINC00668 predicts a poor prognosis of gastric cancer and promotes cell proliferation through epigenetically silencing of CKIs

doi: 10.18632/oncotarget.6745

Figure Lengend Snippet: A. MTT assays were performed to determine the cell proliferation of BGC-823 and SGC-7901 cells after the transfection of the overexpression plasmid and si-RNA of LINC00668. B. Representative results of the colony formation of BGC-823 and SGC-7901 cells transfected with the overexpression plasmid and si-RNA of LINC00668. C. At 48 h after transfection, the cell cycle was analyzed by flow cytometry. The bar chart represents the percentage of cells in G1-G0, S, or G2-M phase, as indicated. D. BGC-823 transfected with Vector/E2F1/E2F1 + si-NC and transfected with E2F1 followed by transfection with si-LINC00668. Forty-eight hours after transfection, the cells were analyzed by flow cytometry and MTT assays. E. Scramble or shLINC00668 was transfected into BGC-823 cells, which were injected in nude mice ( n = 6), respectively. Tumor volumes were calculated after injection every two days. Bars indicate the SD. Tumor weights are represented as the means of tumor weights ±SD. * P < 0.05, ** P < 0.01.

Article Snippet: E2F1 antibody was obtained from Cell Signaling Technology.

Techniques: Transfection, Over Expression, Plasmid Preparation, Flow Cytometry, Injection

Journal: Oncotarget

Article Title: E2F1-induced upregulation of long noncoding RNA LINC00668 predicts a poor prognosis of gastric cancer and promotes cell proliferation through epigenetically silencing of CKIs

doi: 10.18632/oncotarget.6745

Figure Lengend Snippet: A. Immunohistochemistry was used to detect the expression of p57 protein in 30 GC pairs and the corresponding non-tumor tissues. Bar, 100μm. The immunoreactivity of the p57 protein in GC tissues showed a statistically significant negative correlation with the relative level of LINC00668 expression. B. Western blot assays detected the expression of p57 after the transfection of the overexpression plasmid. C. BGC-823 transfected with Vector/p57/p57 + Vector and transfected with LINC00668 followed by transfection with p57. Forty-eight hours after transfection, the cells were analyzed by MTT assays and flow cytometry. D. A proposed model of gastric cancer cell cycle progression and growth control that is mediated by E2F1-regulated LINC00668.* P < 0.05, ** P < 0.01.

Article Snippet: E2F1 antibody was obtained from Cell Signaling Technology.

Techniques: Immunohistochemistry, Expressing, Western Blot, Transfection, Over Expression, Plasmid Preparation, Flow Cytometry, Control

Journal: Oncology Research

Article Title: SNX9 Orchestrates Lung Metastasis via EGFR-ERK Signaling and Actin Cytoskeleton Remodeling in Breast Cancer

doi: 10.32604/or.2026.082536

Figure Lengend Snippet: Antibodies list and details.

Article Snippet: E2F1 , A2067 , 1:1000 , Abclonal/Woburn, Massachusetts, USA.

Techniques: Immunofluorescence, Staining, Immunohistochemistry, Immunoprecipitation

Journal: Oncology Research

Article Title: SNX9 Orchestrates Lung Metastasis via EGFR-ERK Signaling and Actin Cytoskeleton Remodeling in Breast Cancer

doi: 10.32604/or.2026.082536

Figure Lengend Snippet: Silencing of SNX9 increases the G2/M fraction of breast cancer cells. ( A ) Cell cycle distribution of SNX9-KD MCF-7 and MDA-MB-231 cells was measured by flow cytometry. Related cell cycle markers were investigated by WB in ( B ) SNX9-OE MCF-7 cells, ( C ) SNX9-OE BT-549 cells, ( D ) SNX9-KD MDA-MB-231 cells, and ( E ) SNX9-KD MCF-7 cells. Panel A was analyzed by one-way ANOVA . The results are mean ± SD. * p < 0.05, ** p < 0.01, and **** p < 0.0001 versus the corresponding control. Abbreviations: SNX9, sorting nexin 9; E2F1, the adenoviral early region 2 binding transcription factor 1; E2F6, the adenoviral early region 2 binding transcription factor 6; CDK2, cyclin-dependent kinase 2; CDK6, Cyclin-dependent kinase 6; RB, retinoblastoma, P indicates phosphorylated form.

Article Snippet: E2F1 , A2067 , 1:1000 , Abclonal/Woburn, Massachusetts, USA.

Techniques: Flow Cytometry, Control, Binding Assay

Journal: Islets

Article Title: 7-Ketocholesterol accelerates pancreatic β-cell senescence by inhibiting the SIRT1/CDK4–Rb–E2F1 signaling pathway

doi: 10.1080/19382014.2023.2219105

Figure Lengend Snippet: 7-KC or H 2 O 2 caused MIN6 cell senescence through the SIRT1–CDK4–Rb – E2F1 signaling pathway. (A – H) MIN6 cells were treated with 7-KC or H 2 O 2 for 24 h, then SIRT1 (B), CDK4 (C), KMTb/EZH2 (D), E2F1 (E), p -Rb (F), γ-H2A.X (G), and p-p38MAPK (H) expression was detected by western blotting (A). *P < 0.05, **P < 0.01 vs. Normal control.

Article Snippet: The antibodies against E2F1 (A19579), insulin (a19066) were from ABclonal (Wuhan, China).

Techniques: Expressing, Western Blot, Control