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: Journal of Cancer

Article Title: E2F1 promotes cancer cell sensitivity to cisplatin by regulating the cellular DNA damage response through miR-26b in esophageal squamous cell carcinoma

doi: 10.7150/jca.33983

Figure Lengend Snippet: Regulatory mechanism of miR-26b by E2F1. A and B. The expression levels of miR-26b were measured by qRT-PCR in EC109 and Kyse450 cells for E2F1 overexpression, respectively. C and D. The expression levels of miR-26b were measured by qRT-PCR in both EC109 and Kyse450 cells for siRNA interference of E2F1 with or without cisplatin treatment. E. Bioinformatics predicted four potential E2F1 binding sites (sites 1 to 4) using the ChIP Mapper website. The miR-26b gene located in the intron region of CTDSP1. F. ChIP qRT-PCR of the potential binding sites in EC-109 cells. G. Luciferase reporter gene assay showed that pGL3 containing the site 3 fragment significantly increased luciferase activity when E2F1 was overexpressed. (*p<0.5, ** p<0.01).

Article Snippet: The E2F1 expression plasmid 408 pSG5L-HA-E2F1 was obtained from Addgene (#10736) .

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Binding Assay, Luciferase, Reporter Gene Assay, Activity Assay

Journal: Journal of Cancer

Article Title: E2F1 promotes cancer cell sensitivity to cisplatin by regulating the cellular DNA damage response through miR-26b in esophageal squamous cell carcinoma

doi: 10.7150/jca.33983

Figure Lengend Snippet: Analysis of E2F1 regarding the chemosensitization to cisplatin in vivo . A. Mice were injected subcutaneously with Si-E2F1 or negative control(NC) in EC109 cells, and received cisplatin treatment with equal dose (4 mg/kg). The final xenografts were separated and weighted. B. In silico analysis (TargetScan, miRanda and PicTar) revealed that miR-26b has a conserved binding site in the 3′UTR regions of ATM and Rb. C and D. Luciferase reporter assay indicated that miR-26b decreased the luciferase activity, but the luciferase activity almost rose to control levels when the binding sites were mutated. E. The reduced ATM, Rb, and E2F1 expression was analysis by Western blot in EC109 and Kyse450 cells transfected with NC or miR-26b mimics for 48 h. (** p<0.01).

Article Snippet: The E2F1 expression plasmid 408 pSG5L-HA-E2F1 was obtained from Addgene (#10736) .

Techniques: In Vivo, Injection, Negative Control, In Silico, Binding Assay, Luciferase, Reporter Assay, Activity Assay, Control, Expressing, Western Blot, Transfection

Journal: Journal of Cancer

Article Title: E2F1 promotes cancer cell sensitivity to cisplatin by regulating the cellular DNA damage response through miR-26b in esophageal squamous cell carcinoma

doi: 10.7150/jca.33983

Figure Lengend Snippet: Regulation pattern of E2F1/miR-26b molecular network on cisplatin treatment.

Article Snippet: The E2F1 expression plasmid 408 pSG5L-HA-E2F1 was obtained from Addgene (#10736) .

Techniques:

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Silencing of Sodium-Hydrogen Exchanger 1 Attenuates the Proliferation, Hypertrophy, and Migration of Pulmonary Artery Smooth Muscle Cells via E2F1

doi: 10.1165/rcmb.2011-0032OC

Figure Lengend Snippet: Effects of overexpression of E2F1 on inhibition by NHE1 siRNA of human PASMC proliferation. (A) Expression of E2F1. Protein was isolated from human PASMCs under conditions of normoxia, hypoxia, and hypoxia, and treated with NHE1 siRNA for Western blot analysis. Left, representative imaging. Right, quantitative data. *P < 0.05, compared with other groups. #P > 0.05, compared with normoxia. Cell proliferation data were obtained through direct cell counts (B) and the BrdU incorporation assay (C). After transfection with NHE1 siRNA or E2F1 cDNA, PASMCs were cultured in a hypoxia chamber for 24 hours and harvested for cell counts and a BrdU assay. *P < 0.05, compared with the control group (n = 9 for each group). (D) Quantitative data on the protein expression of NHE1 and E2F1. Proteins isolated from PASMCs treated with NHE1 siRNA and E2F1 cDNA were subjected to Western blot analysis, setting the control value as 1. Data were normalized to Glyceraldehyde-3 phosphate dehydrogenase (GAPDH). *P < 0.05, compared with each control group (n = 3 for each group). NHE1 + E2F1, NHE1 siRNA + E2F1 cDNA.

Article Snippet: E2F1 human cDNA (pCMV6-XL6-E2F1) was purchased from Origene (Rockville, MD), and purified according to our previously described methods ( 17 , 19 ).

Techniques: Over Expression, Inhibition, Expressing, Isolation, Western Blot, Imaging, BrdU Incorporation Assay, Transfection, Cell Culture, BrdU Staining, Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Silencing of Sodium-Hydrogen Exchanger 1 Attenuates the Proliferation, Hypertrophy, and Migration of Pulmonary Artery Smooth Muscle Cells via E2F1

doi: 10.1165/rcmb.2011-0032OC

Figure Lengend Snippet: Effects of NHE1 and E2F1 on hypertrophy of human PASMCs under condition of hypoxia. After transfection with NHE1 siRNA and growing under condition of hypoxia for 24 hours, PASMCs were removed from hypoxia, and a hypertrophy assay was performed. (A) Cell-size assay for cells treated with NHE1 siRNA. (B) NHE1 siRNA plus E2F1 cDNA under condition of hypoxia. (C) Protein/DNA ratio under condition of hypoxia. *P < 0.05, #P > 0.05 as compared with the normoxia control, and $P < 0.05 as compared with hypoxia control in A. *P < 0.05 as compared with the controls in B and C (n = 9 for each group). NHE1 + E2F1, NHE1 siRNA + E2F1 cDNA.

Article Snippet: E2F1 human cDNA (pCMV6-XL6-E2F1) was purchased from Origene (Rockville, MD), and purified according to our previously described methods ( 17 , 19 ).

Techniques: Transfection, Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Silencing of Sodium-Hydrogen Exchanger 1 Attenuates the Proliferation, Hypertrophy, and Migration of Pulmonary Artery Smooth Muscle Cells via E2F1

doi: 10.1165/rcmb.2011-0032OC

Figure Lengend Snippet: Effects of NHE1 and E2F1 on migration of human PASMCs under condition of hypoxia. After transfection with NHE1 siRNA and E2F1 cDNA and being cultured in 2% oxygen for 24 hours, a wound-healing assay and migration chamber assay were performed. (A) Wound-healing assay for cells treated with NHE1 siRNA. (B) NHE1 siRNA plus E2F1 cDNA under condition of hypoxia. (C) Representative imaging. (D) Migration chamber assay under condition of hypoxia. *P < 0.05, compared with control group. #P < 0.05, compared with other groups (n = 9 for each group). NHE1 + E2F1 = NHE1 siRNA + E2F1 cDNA. Gap = the scratched area by pipette tip.

Article Snippet: E2F1 human cDNA (pCMV6-XL6-E2F1) was purchased from Origene (Rockville, MD), and purified according to our previously described methods ( 17 , 19 ).

Techniques: Migration, Transfection, Cell Culture, Wound Healing Assay, Boyden Chamber Assay, Imaging, Control, Transferring

Journal: Cells

Article Title: The Role of Crosstalk between AR3 and E2F1 in Drug Resistance in Prostate Cancer Cells

doi: 10.3390/cells9051094

Figure Lengend Snippet: Differential gene expression in response to DTX-ENZ combined treatment. ( A ) Flowchart of RNA sample preparation and RNA-seq. ( B ) Analysis of common deregulated genes in double drugs v.s. Control or ENZ or DTX group. ( p < 0.05, FDR < 0.25, fold change >1.4) ( C ) Expression of E2F1 in R1-ADR cells 12 h after drug treatment were determined by qRT-PCR. Error bars, S.D. * p < 0.05. ( D ) Protein expression of E2F1 in R1-ADR cells 12 and 24 h after drug treatment were determined by western blot. ( E ) Re-expressed E2F1 partially rescued DTX+ENZ induced growth inhibition in R1-ADR cells. Cell growth was determined by CCK-8 assays 72 h after re-introduced exogenous E2F1 in the presence of DTX and ENZ. Error bars, S.D. * p < 0.05.

Article Snippet: The plasmids used in the present study are as follows: the E2F1 expression vector pCMVHA E2F1 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24225) [ ], the E2F1 DNA binding mutant vector pCMV E2F1 E132 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24224) [ ], the E2F1 promoter luciferase vector pGL2-AN was a gift from William Kaelin (Addgene, Cambridge, MA, USA, plasmid # 20950) [ ], and the AR expression vector and ARR2-Luc vector were generated as previously described [ , ].

Techniques: Gene Expression, Sample Prep, RNA Sequencing, Control, Expressing, Quantitative RT-PCR, Western Blot, Inhibition, CCK-8 Assay

Journal: Cells

Article Title: The Role of Crosstalk between AR3 and E2F1 in Drug Resistance in Prostate Cancer Cells

doi: 10.3390/cells9051094

Figure Lengend Snippet: AR3 and AR-FL differentially regulated E2F1 expression. ( A , B ) R1-ADR cells were infected with the lentivirus encoding the control shRNA, AR3 shRNA (shAR3) or AR-FL shRNA (shAR-FL) for 48 h. Protein levels of E2F1, AR-FL, and AR3 were determined by western blot ( A ) and mRNA levels were determined by qRT-PCR ( B ), Error bars, S.D. * p < 0.05. ( C ) Binding of AR3 or AR-FL to the putative ARE sites of human E2F1 gene regulatory region was analyzed by ChIP assays. Error bars, S.D. * p < 0.05. ( D ) The E2F1-promoter driven luciferase reporter (E2F1-LUC) activity was measured in HEK293T cells treated with 0.1nM DHT or/and 10 μM ENZ. ( E ) ARR2-promoter luciferase assay was performed in HEK293T cells treated with 0.1nM DHT or/and 10 μM ENZ.

Article Snippet: The plasmids used in the present study are as follows: the E2F1 expression vector pCMVHA E2F1 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24225) [ ], the E2F1 DNA binding mutant vector pCMV E2F1 E132 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24224) [ ], the E2F1 promoter luciferase vector pGL2-AN was a gift from William Kaelin (Addgene, Cambridge, MA, USA, plasmid # 20950) [ ], and the AR expression vector and ARR2-Luc vector were generated as previously described [ , ].

Techniques: Expressing, Infection, Control, shRNA, Western Blot, Quantitative RT-PCR, Binding Assay, Luciferase, Activity Assay

Journal: Cells

Article Title: The Role of Crosstalk between AR3 and E2F1 in Drug Resistance in Prostate Cancer Cells

doi: 10.3390/cells9051094

Figure Lengend Snippet: AR3 and AR-FL recruited different cofactors and differentially regulated E2F1 gene expression. ( A ) R1-ADR cells were infected with the lentivirus encoding the control shRNA, shAR3 or shAR-FL for 48 h. Cell lysates were then immunoprecipitated with an anti-AR or anti-E2F1 antibody followed by western blot. ( B ) HEK293T cells were cotransfected with AR-FL, AR3, and/or E2F1-HA. Cell lysates were then immunoprecipitated with an anti-AR or anti-HA antibody followed by western blot. ( C ) R1-ADR cells were infected with the lentivirus encoding the control shRNA or shAR3 or shAR-FL for 48 h and then treated with 10nM DHT for 2 h before collection. Cell lysates were then immunoprecipitated with an anti-AR or anti-Rb antibody followed by western blot. ( D ) E2F1-promoter luciferase assay was performed in HEK293T cells treated with 0.1nM DHT or/and 10 μM ENZ. ( E ) E2F1-promoter luciferase assays with mutant E2F1 were performed in HEK293T cells.

Article Snippet: The plasmids used in the present study are as follows: the E2F1 expression vector pCMVHA E2F1 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24225) [ ], the E2F1 DNA binding mutant vector pCMV E2F1 E132 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24224) [ ], the E2F1 promoter luciferase vector pGL2-AN was a gift from William Kaelin (Addgene, Cambridge, MA, USA, plasmid # 20950) [ ], and the AR expression vector and ARR2-Luc vector were generated as previously described [ , ].

Techniques: Gene Expression, Infection, Control, shRNA, Immunoprecipitation, Western Blot, Luciferase, Mutagenesis

Journal: Cells

Article Title: The Role of Crosstalk between AR3 and E2F1 in Drug Resistance in Prostate Cancer Cells

doi: 10.3390/cells9051094

Figure Lengend Snippet: E2F1 regulated AR expression. ( A ) AR-FL, AR3, and E2F1 protein level 4 days after DTX+ENZ treatment were determined by western blot. ( B , C ) R1-ADR cells were infected with the lentivirus encoding the control shRNA or E2F1 shRNA for 48 h. Expression level of E2F1, AR-FL, and AR3 were determined by qRT-PCR ( B ), Error bars, S.D. * p < 0.05 and western blot ( C ). ( D ) Binding of E2F1 to the regulatory regions of AR was analyzed by ChIP assay.

Article Snippet: The plasmids used in the present study are as follows: the E2F1 expression vector pCMVHA E2F1 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24225) [ ], the E2F1 DNA binding mutant vector pCMV E2F1 E132 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24224) [ ], the E2F1 promoter luciferase vector pGL2-AN was a gift from William Kaelin (Addgene, Cambridge, MA, USA, plasmid # 20950) [ ], and the AR expression vector and ARR2-Luc vector were generated as previously described [ , ].

Techniques: Expressing, Western Blot, Infection, Control, shRNA, Quantitative RT-PCR, Binding Assay

Journal: Cells

Article Title: The Role of Crosstalk between AR3 and E2F1 in Drug Resistance in Prostate Cancer Cells

doi: 10.3390/cells9051094

Figure Lengend Snippet: E2F1 and AR/ARv expression was recovered in R1-DDR cells. ( A ) RNA-seq analysis, heatmap of those commonly dysregulated genes in R1-ADR cells treated with DTX+ENZ (p < 0.05, FDR < 0.25, fold change >1.4), we compared R1-DDR cells (maintained in DTX+ENZ) to R1-ADR cells treated with DTX+ENZ (D+E). Missing values are in color “gray”. ( B ) protein level of AR/ARv and E2F1 in R1-DDR cells were determined by western blot. ( C ) R1-DDR cells infected with lentivirus encoding control or shE2F1 or shAR-FL or shAR3. Cell growth was determined by CCK-8 assays 3 days after infection.

Article Snippet: The plasmids used in the present study are as follows: the E2F1 expression vector pCMVHA E2F1 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24225) [ ], the E2F1 DNA binding mutant vector pCMV E2F1 E132 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24224) [ ], the E2F1 promoter luciferase vector pGL2-AN was a gift from William Kaelin (Addgene, Cambridge, MA, USA, plasmid # 20950) [ ], and the AR expression vector and ARR2-Luc vector were generated as previously described [ , ].

Techniques: Expressing, RNA Sequencing, Western Blot, Infection, Control, CCK-8 Assay

Journal: Cells

Article Title: The Role of Crosstalk between AR3 and E2F1 in Drug Resistance in Prostate Cancer Cells

doi: 10.3390/cells9051094

Figure Lengend Snippet: Auranofin inhibited the growth of double drug-resistant prostate cancer cells in vitro and in vivo. ( A ) R1-DDR (left panel) and LN-DDR (right panel) cells were treated with different doses of Auranofin for 72 h. Cell growth was determined by CCK8 assay. ( B ) R1-DDR and LN-DDR cells were treated with 2 μM Auranofin for 12 h, Protein level of E2F1, AR-FL, and AR3 in R1-DDR cells were determined by western blot. ( C ) Animals were treated with solvent or DTX (5 mg/kg once a week) + ENZ (10 mg/kg per day, 5 days per week) or auranofin (5 mg/kg per day, 5 days per week), n = 5 per group. Error bars, S.E.M. * p < 0.05 ( D ) TUNEL assay was performed to detect apoptosis of R1-DDR xenograft. Error bars, S.D. * p < 0.05. ( E ) Ki67 proliferation marker was examined by immunohistochemistry. ( F ) AR/ARv and E2F1 protein level in R1-DDR xenograft were determined by western blot.

Article Snippet: The plasmids used in the present study are as follows: the E2F1 expression vector pCMVHA E2F1 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24225) [ ], the E2F1 DNA binding mutant vector pCMV E2F1 E132 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24224) [ ], the E2F1 promoter luciferase vector pGL2-AN was a gift from William Kaelin (Addgene, Cambridge, MA, USA, plasmid # 20950) [ ], and the AR expression vector and ARR2-Luc vector were generated as previously described [ , ].

Techniques: In Vitro, In Vivo, CCK-8 Assay, Western Blot, Solvent, TUNEL Assay, Marker, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: E2F1 Induces KIF26A Transcription and Promotes Cell Cycle Progression via CDK–RB–E2Fs Feedback Loop in Breast Cancer

doi: 10.3389/fonc.2020.530933

Figure Lengend Snippet: KIF26A promote the effect of proliferation and cell cycle progression on BC cells in vitro . (A–D) Results from MTS assay showed that BCs exhibited cell proliferation transfected with KIF26A siRNA (A, B) , and promote cell proliferation with KIF26A overexpression (C, D) . (E, F) EDU assays confirmed the promotion effect of KIF26A on cell proliferation in BCs with KIF26A knockdown and overexpression. (G, H) In the soft agar of the colony formation assay, the colonies were smaller and fewer in KIF26A-knockdown BC cells, and larger in KIF26A overexpression cells, compared with the control. (I, J) KIF26A knockdown induced G0/G1 phase cell cycle arrest compared with that of Si-NC in BCs with flow cytometry detection, and the opposite effect on the KIF26A overexpression group. (K, L) KIF26A knockdown could promote cell apoptosis in BCs, and overexpression group got the opposite effect. * means P < 0.05. ** means P < 0.01. **** menas P < 0.0001.

Article Snippet: E2F1 overexpression vector (Bioasia company, Jinan, China) was cloned into the P-Enter expression vector.

Techniques: In Vitro, MTS Assay, Transfection, Over Expression, Colony Assay, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: E2F1 Induces KIF26A Transcription and Promotes Cell Cycle Progression via CDK–RB–E2Fs Feedback Loop in Breast Cancer

doi: 10.3389/fonc.2020.530933

Figure Lengend Snippet: Cloning of the KIF26A promoter and verification of the transcription factors (TFs). (A) Various fragments of −1978/−15 TSS were cloned, named P1 to P10. Deletions from P6 to P7 significantly decreased the luciferase activity. (B) KIF26A gene promoter contains various TF binding sites, including E2F1 and SP1 as the intersection elements. (C) KIF26A mRNA expression was higher in E2F1-overexpressing cells. (D) Luciferase assays suggested that E2F1, rather than other nine transcription factors and empty vector, and P6 co-transfection could much more remarkably enhance the luciferase activity in 293T and MCF-7 cells lines. : E2F1 was verified to transcriptionally activate KIF26A, and the core promoter region was further identified. * means P < 0.05.

Article Snippet: E2F1 overexpression vector (Bioasia company, Jinan, China) was cloned into the P-Enter expression vector.

Techniques: Clone Assay, Luciferase, Activity Assay, Binding Assay, Expressing, Plasmid Preparation, Cotransfection

Journal: Frontiers in Oncology

Article Title: E2F1 Induces KIF26A Transcription and Promotes Cell Cycle Progression via CDK–RB–E2Fs Feedback Loop in Breast Cancer

doi: 10.3389/fonc.2020.530933

Figure Lengend Snippet: E2F1 was verified to be involved in the activation of the KIF26A through directly binding to the KIF26A promotor area and the core promoter region was further identified by mutation vector. (A, C, E) The predicted E2F1 binding site with the promotor area include binding site 1 (−395/−385), binding site 2 (−390/−383) and binding site 3 (−372/−362). (B, D, F) We constructed mutational PGL3-basic vectors by replace two bases of E2F1 binding site accordingly, named as T1, T2 and T3. (G) The activity of the luciferase reporter gene that carries T1 was inhibited significantly obvious than T2, T3 and P6 in 293T and MCF-7 cells, indicating the core promotor area located within the −395 to −385 region relative to the Tss. (H–J) The products of chromatin immunoprecipitation (CHIP) assay demonstrated by agarose gel electrophoresis (H) and the RT-qPCR outcome (I, J) indicated that the sequence binding to the antibodies precipitated proteins were proved to include the KIF26A promoter region by E2F1 antibody and the input(positive control antibody), but not by non-specific IgG (negative control antibody). * means P < 0.05. ** means P < 0.01.

Article Snippet: E2F1 overexpression vector (Bioasia company, Jinan, China) was cloned into the P-Enter expression vector.

Techniques: Activation Assay, Binding Assay, Mutagenesis, Plasmid Preparation, Construct, Activity Assay, Luciferase, Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Quantitative RT-PCR, Sequencing, Positive Control, Negative Control

Journal: Frontiers in Oncology

Article Title: E2F1 Induces KIF26A Transcription and Promotes Cell Cycle Progression via CDK–RB–E2Fs Feedback Loop in Breast Cancer

doi: 10.3389/fonc.2020.530933

Figure Lengend Snippet: KIF26A could modulate the expression of the P21 pathway. (A–D) P21 pathway was influenced significantly at the protein level, not mRNA level. (E–G) The elevated E2F1 activated the KIF26A expression and inhibited p21 pathway. (H) Mode pattern: KIF26A, directly upregulated by E2F1, promoted cell proliferation and cell cycle progression via CDK–RB–E2Fs feedback loop in BC.

Article Snippet: E2F1 overexpression vector (Bioasia company, Jinan, China) was cloned into the P-Enter expression vector.

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: Feedback between E2F1 and CIP2A regulated by human papillomavirus E7 in cervical cancer: implications for prognosis

doi:

Figure Lengend Snippet: Primer sequences for real-time PCR in cervical cancer cells

Article Snippet: The E2F1 expression plasmid (pCMV-E2F1) and pCMV vector were bought from Addgene Company (Cambridge, MA, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: American Journal of Translational Research

Article Title: Feedback between E2F1 and CIP2A regulated by human papillomavirus E7 in cervical cancer: implications for prognosis

doi:

Figure Lengend Snippet: Effect of HPV E7 on the gene expression of CIP2A. A, B. Quantified real-time PCR and western blot analysis of CIP2A mRNA and protein levels after siRNA silencing of HPV E7 in HeLa and SiHa cells. C. Quantified real-time PCR analysis of mRNA levels of ETS1, ELK1 and E2F1 with siRNA silencing of HPV E7 in SiHa cells. Cells were transfected with siRNA targeting E7 (siE7) or negative control siRNA (NC). Data are mean SEM of triplicate experiments (*P<0.05, ***P<0.001).

Article Snippet: The E2F1 expression plasmid (pCMV-E2F1) and pCMV vector were bought from Addgene Company (Cambridge, MA, USA).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Negative Control

Journal: American Journal of Translational Research

Article Title: Feedback between E2F1 and CIP2A regulated by human papillomavirus E7 in cervical cancer: implications for prognosis

doi:

Figure Lengend Snippet: HPV E7 upregulated CIP2A expression via the transcription factor E2F1. A, C. Quantified real-time PCR and western blot analysis of E2F1 and CIP2A mRNA and protein levels after siRNA silencing of HPV E7 in HeLa and SiHa cells. B, D. Quantified real-time PCR and western blot analysis of CIP2A mRNA or/and protein levels after siRNA silencing or plasmid overexpression of E2F1 in HeLa and SiHa cells. For gene silencing, cells were transfected with siRNA targeting E7 (siE7), siRNA targeting E2F1 (siE2F1) or negative control siRNA (NC). For gene overexpression, cells were transfected with pCMV-E2F1 plasmid (OE-E2F1) or pCMV vector (PCMV). Data are mean SEM of triplicate experiments (*P<0.05, **P<0.01).

Article Snippet: The E2F1 expression plasmid (pCMV-E2F1) and pCMV vector were bought from Addgene Company (Cambridge, MA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Over Expression, Transfection, Negative Control

Journal: American Journal of Translational Research

Article Title: Feedback between E2F1 and CIP2A regulated by human papillomavirus E7 in cervical cancer: implications for prognosis

doi:

Figure Lengend Snippet: CIP2A upregulated E2F1 gene expression at the transcription level. A. Silencing or over-expression of CIP2A could decrease or increase transcription and thus translation of E2F1 in HeLa cells. B. Silencing or over-expression of CIP2A could decrease or increase the translation of E2F1 in SiHa cells. For gene silencing, cells were transfected with siRNA targeting CIP2A (siCIP2A) or negative control siRNA (NC). For gene overexpression, cells were transfected with pBabe-CIP2A plasmid (OE-CIP2A) or pBabe vector (pBabe). Data are mean SEM of triplicate experiments (*P<0.05).

Article Snippet: The E2F1 expression plasmid (pCMV-E2F1) and pCMV vector were bought from Addgene Company (Cambridge, MA, USA).

Techniques: Gene Expression, Over Expression, Transfection, Negative Control, Plasmid Preparation

Journal: American Journal of Translational Research

Article Title: Feedback between E2F1 and CIP2A regulated by human papillomavirus E7 in cervical cancer: implications for prognosis

doi:

Figure Lengend Snippet: Sub-cellular co-expression of E2F1 and CIP2A in vivo. A. Representative immunofluorescence staining of CIP2A (green) and E2F1 (red) protein in HeLa and SiHa cells by confocal laser scanning microscopy. The nucleus was stained blue by DAPI. B. Immunohistochemistry staining of E2F1 and CIP2A protein in paraffin-embedded cervical tissue. Positive staining for E2F1 was defined as brown stain in the nucleus and for CIP2A in the cytoplasm.

Article Snippet: The E2F1 expression plasmid (pCMV-E2F1) and pCMV vector were bought from Addgene Company (Cambridge, MA, USA).

Techniques: Expressing, In Vivo, Immunofluorescence, Staining, Confocal Laser Scanning Microscopy, Immunohistochemistry

Journal: American Journal of Translational Research

Article Title: Feedback between E2F1 and CIP2A regulated by human papillomavirus E7 in cervical cancer: implications for prognosis

doi:

Figure Lengend Snippet: Association of clinicopathological parameters with CIP2A and E2F1 expression

Article Snippet: The E2F1 expression plasmid (pCMV-E2F1) and pCMV vector were bought from Addgene Company (Cambridge, MA, USA).

Techniques:

Journal: American Journal of Translational Research

Article Title: Feedback between E2F1 and CIP2A regulated by human papillomavirus E7 in cervical cancer: implications for prognosis

doi:

Figure Lengend Snippet: Kaplan-Meier curves for survival of 127 patients with cervical carcinoma by CIP2A and E2F1 positivity and negativity. Culminative overall survival (A) and disease-free survival (B).

Article Snippet: The E2F1 expression plasmid (pCMV-E2F1) and pCMV vector were bought from Addgene Company (Cambridge, MA, USA).

Techniques:

Journal: American Journal of Translational Research

Article Title: Feedback between E2F1 and CIP2A regulated by human papillomavirus E7 in cervical cancer: implications for prognosis

doi:

Figure Lengend Snippet: Cox univariate analysis of survival in 127 patients with cervical cancer

Article Snippet: The E2F1 expression plasmid (pCMV-E2F1) and pCMV vector were bought from Addgene Company (Cambridge, MA, USA).

Techniques:

Journal: American Journal of Translational Research

Article Title: Feedback between E2F1 and CIP2A regulated by human papillomavirus E7 in cervical cancer: implications for prognosis

doi:

Figure Lengend Snippet: Cox multivariate analysis of survival in 127 patients with cervical cancer

Article Snippet: The E2F1 expression plasmid (pCMV-E2F1) and pCMV vector were bought from Addgene Company (Cambridge, MA, USA).

Techniques: