Journal: Cell death and differentiation

Article Title: E2F1-regulated miR-30b suppresses Cyclophilin D and protects heart from ischemia/reperfusion injury and necrotic cell death.

doi: 10.1038/cdd.2014.165

Figure Lengend Snippet: Figure 6 E2F1 promotes necrosis through miR-30b and CypD. (a) E2F1 levels are increased in cardiomyocytes exposed to H2O2. Cardiomyocytes were exposed to H2O2. Cells were harvested at the indicated times for the analysis of E2F1 levels by immunoblot. (b) Knockdown of E2F1 reduces necrotic cell death induced by H2O2. Cardiomyocytes were infected with adenoviral E2F1-siRNA or E2F1-sc. Twenty-four hours after infection, cells were treated with H2O2. PI exclusion was analyzed. *Po0.05 versus H2O2 alone. (c) The levels of E2F1 are increased in myocardial I/R. Mice were induced to undergo cardiac I/R at the indicated times as described in Materials and Methods. E2F1 levels were analyzed by immunoblot. (d and e) E2F1 knockout mice attenuates myocyte necrosis and myocardial infarction upon I/R. WTand E2F1 knockout mice were subjected to I/R as described in Materials and Methods. Myocyte necrosis (d) and myocardial infarction (e) were analyzed. *Po0.05 versus WT+I/R. (f and g) CypD TP attenuates the inhibitory effect of E2F1 knockdown on CypD expression and necrotic responses induced by H2O2. Cardiomyocytes were infected with adenoviral E2F1-siRNA or E2F1-sc, transfected with the TP (CypD-TP miR-30b) or the control (CypD-TP control) and then exposed to H2O2. CypD expression (f) was analyzed by immunoblot. Necrosis was assessed by PI exclusion assay (g). *Po0.05. GAPDH, glyceraldehyde 3-phosphate dehydrogenase

Article Snippet: Mouse E2F1 cDNA was from Origene (Rockville, MD, USA).

Techniques: Western Blot, Knockdown, Infection, Knock-Out, Expressing, Transfection, Control, Exclusion Assay

Journal: Advanced Science

Article Title: Novel Evolutionarily Conserved Oncogene COA4 is Driven by KRAS Mutant and Promotes Cancer Metastasis Through Dual Mitochondrial Metabolism‐Dependent and ‐Independent Mechanisms

doi: 10.1002/advs.202507533

Figure Lengend Snippet: E2F1 directly binds to COA4 promoter and promotes its expression under KRAS mutation. A,B) Western blot (A) and RT‐qPCR (B) analyses of COA4 expression in A549 cells following E2F1 overexpression or knockdown. ( n = 3; ** p < 0.01; **** p < 0.0001) C) Luciferase reporter assay confirming that E2F1 directly regulates COA4 transcription in 293T and A549 cells. ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001) D) Luciferase reporter assay demonstrating that KRAS enhances E2F1‐mediated transcriptional activation of the COA4 promoter in 293T cells. ( n = 3; **** p < 0.0001) E) ChIP‐qPCR analysis identifying the specific E2F1 binding region within the COA4 promoter in A549 cells. ( n = 3; *** p < 0.001) F) AlphaFold 3 molecular docking of the COA4 promoter with E2F1, supporting their direct interaction. G) Chromatin immunoprecipitation followed by qPCR (ChIP‐qPCR) was performed to evaluate E2F1 binding to the COA4 promoter in normal tissues and KRAS‐mutant tumor tissues. Data are presented as mean ± SD ( n = 6, *** p < 0.001) H) Spearman's rank correlation analysis of COA4 and E2F1 expression in LUAD using the GEPIA2 database. I. IHC analysis of E2F1 expression in LUAD tumor versus normal tissues ( n = 12 per group; **** p < 0.0001) J) ATP quantification in E2F1 ‐overexpressing A549 cells following COA4 knockdown.( n = 3; ** p < 0.01) K) Complex IV enzyme activity in E2F1 ‐overexpressing A549 cells after COA4 knockdown. ( n = 3; ** p < 0.01) L,M) Western blot (L) and RT‐qPCR (M) analyses of COA4 expression in KRAS G12C ‐overexpressing A549 cells following E2F1 knockdown. ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001) N) Western blot analysis of COA4 expression in A549 cells treated with the PI3K agonist 740Y‐P(15 µM) for 48 h (left), and in 740Y‐P‐treated A549 cells following E2F1 knockdown (right).( n = 3) O) Western blot analysis of COA4 expression in A549 and H23 cells treated with the PI3K inhibitor LY294002 following E2F1 overexpression. ( n = 3) P) RT‐qPCR analysis of COA4 mRNA levels in A549 cells treated with LY294002 following E2F1 overexpression (left), and in A549 cells treated with 740Y‐P following E2F1 knockdown (right). ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001) Q) Transwell assays assessing migration of COA4 ‐overexpressing A549 and H23 cells following E2F1 knockdown (top), and of COA4‐knockdown cells following E2F1 overexpression (bottom). Scale bar: 275 µm (n = 3; ** p < 0.01; **** p < 0.0001) R) Transwell assays analyzing migration of A549 and H23 cells treated with 740Y‐P following E2F1 knockdown (left), and with LY294002 following E2F1 overexpression (right). Scale bar: 275 µm ( n = 3) S) Transwell assays evaluating migration and invasion in A549 and H23 cells following KRAS G12D overexpression, E2F1 knockdown, and COA4 overexpression. ( n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001) T) Analysis of E2F1 expression in LUAD tissues: left, comparing KRAS wild‐type versus KRAS mutant samples using TCGA data; right, comparing LUAD tissues with high versus low KRAS expression using combined datasets ( GSE30219 , GSE37745 , GSE118370 , and GSE140797 ). U) Survival analysis based on COA4 and E2F1 expression levels using the GSE30219 database. V) Combined analysis of COA4 and E2F1 expression with survival status and overall survival time, performed using the linkET package on the GSE30219 dataset. The data are given as mean ± SD and compared by Student's t test (A–E, G, I–T), Spearman's rank correlation analysis (H). The Log‐rank (Mantel–Cox) test was used for survival analyses (U,V). Significance levels are indicated as follows: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Primary antibodies: ACTIN (1:5000, Proteintech, 81115‐1‐RR); Coa4 (1:2500, Invitrogen, PA5‐59076); CDC42 (1:1000, Proteintech, 10155‐1‐AP); E2F1 (1:1000, Proteintech, 12171‐1‐AP); MTCO1 (1:500, ABclonal, A17889); MTCO2 (1:3000, Proteintech, 55070‐1‐AP); MTCO3 (1:2000, Proteintech, 55082‐1‐AP); KRAS (1:10000, Proteintech, 12063‐1‐AP); FAK (1:5000, ABclonal, A11131); p‐FAK‐Y397 (1:300, ABclonal, AP0302).

Techniques: Expressing, Mutagenesis, Western Blot, Quantitative RT-PCR, Over Expression, Knockdown, Luciferase, Reporter Assay, Activation Assay, ChIP-qPCR, Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Migration

Journal: Oncology Research

Article Title: Competitive Sequestration of miR-1183 by lncRNA DDX11-AS1 Drives Gliomagenesis through E2F7 Activation

doi: 10.32604/or.2025.065380

Figure Lengend Snippet: E2F7 is a target of miR-1183. ( A ) The bioinformatics analysis tools were used to predict the binding site of miR-1183 in E2F7. ( B ) Correlation between miR-1183 and E2F7 expression. ( C ) Luciferase activity in U87 cells co-transfected with miR-1183 and WT/MUT E2F7 reporter constructs ( n = 6). ( D – G ) U87 and U251 cells transfected with miR-1183, miR-NC or E2F7-overexpression vectors were used; ( D , E ) Relative expression of E2F7 was determined by RT-qPCR ( n = 6); ( F , G ) CCK-8 assay of ( F ) U87 and ( G ) U251 cells ( n = 6); ( H , I ) U87 and U251 cells transfected with sh-NC, sh-DDX11-AS1 or sh-DDX11-AS1 followed with miR-1183 inhibitor treatment were used. ( H ) Relative expression of E2F7 was determined by RT-qPCR ( n = 6). ( I ) Immunoblotting assays detected E2F7 and GAPDH in U87 and U251 cells. The bar chart quantifies relative E2F7 protein abundance normalized to GAPDH ( n = 6). All data are represented as the mean ± SEM, * p < 0.05, sh-DDX11-AS1 vs. sh-NC; # p < 0.05, sh-DDX11-AS1 vs. sh-DDX11-AS1 + miR-1183 inhibitor (One-way ANOVA)

Article Snippet: Membranes was blocked using powdered milk (5%) in Tris-buffered saline comprising Tween 20 (0.1%) at room temperature for 1 h. Probing of the membranes was performed using rabbit anti-human E2F7 (1:1000; catalog #24489-1-AP; Proteintech, Rosemont, IL, USA) and rabbit anti-human GAPDH (1:5000; catalog #2118; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight.

Techniques: Binding Assay, Expressing, Luciferase, Activity Assay, Transfection, Construct, Over Expression, Quantitative RT-PCR, CCK-8 Assay, Western Blot, Quantitative Proteomics

Journal: The World Journal of Men's Health

Article Title: Docetaxel Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Mediated Apoptosis in Prostate Cancer Cells via Epigenetic Gene Regulation by Enhancer of Zeste Homolog 2

doi: 10.5534/wjmh.220073

Figure Lengend Snippet: Expressions of EZH2 and E2F1 in DU 145 cells. WB assay shows decreases of EZH2 and E2F1 expressions after docetaxel treatment (A). Using knockdown of E2F1, the expressions of E2F1 and EZH2 are decreased (B), and TRAIL (100 ng/mL) treatment causes significant cell death in siE2F-1 DU 145 cells ( *** p<0.001) (C). DR4: death receptor 4, DR5: death receptor 5, EZH2: enhancer of zeste homolog 2, ns: not significant, TRAIL: tumor necrosis factor-related apoptosis-inducing ligand, WB: western blot.

Article Snippet: Small interfering RNA (siRNA) against human E2F1 (siE2F1) (sc-29297) and control siRNA (scRNA) (sc-37007) were purchased from Santa Cruz Biotechnology.

Techniques: Knockdown, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Lack of CCAAT Enhancer Binding Protein Beta (C/EBPβ) in Uterine Epithelial Cells Impairs Estrogen-Induced DNA Replication, Induces DNA Damage Response Pathways, and Promotes Apoptosis

doi: 10.1128/mcb.00872-09

Figure Lengend Snippet: FIG. 6. C/EBP regulates the expression of E2F family genes in E-treated uterine epithelial cells. (A) LE cells were isolated from uteri of ovariectomized WT or C/EBP/ mice following treatment with E for 0, 12, and 15 h. (a to c) Expression levels of E2F1 (a), E2F2 (b), and E2F3 (c) were determined by real-time PCR. The fold changes indicate mRNA expression levels of the E2F genes relative to those of E-treated WT cells at 0 h. (d) Relative steady-state levels of mRNAs for E2Fs 1, 2, and 3 in primary LE cells obtained from uteri treated with E for 15 h are shown following normalization with the 36B4 mRNA level. Statistically significant differences (P 0.05) are indicated by asterisks. (B) Uteri from WT and C/EBP-null mice treated with E for 15 h were collected, and sections were subjected to IHC analysis using anti-E2F3 antibody. Positive staining for E2F3 is indicated in red, and DAPI-stained nuclei are shown in blue. (C) ChIP analysis. LE cells were isolated from uteri of ovariectomized WT mice following treatment with E for 12 h. ChIP was performed as described in Materials and Methods using antibodies against C/EBP, RNA polymerase II (RNAP II), and rabbit IgG. Relative levels of recruitment at various sites on the E2F3 promoter were determined by real-time PCR and normalized to input DNA and RNA polymerase II values.

Article Snippet: Sections were incubated with the following primary antibodies diluted in blocking solution (0.25% bovine serum albumin [BSA], 0.3% Triton X-100, sterile PBS) overnight at 4°C: BrdU and Ki67 (BD Pharmingen), phospho-Ser10 histone H3 (Upstate Biotechnology); cyclin E, cyclin A, and Rad18 (Abcam); cyclin D1 (LabVision NeoMarkers); E2F3 (Santa Cruz Biotechnology); p27 (BD Transduction Laboratories); and phospho(Ser1981)-ATM, phospho-(Ser139)-H2AX, caspase 3, and cleaved caspase 3 (Cell Signaling Technology).

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Staining

Journal: Molecular and Cellular Biology

Article Title: Lack of CCAAT Enhancer Binding Protein Beta (C/EBPβ) in Uterine Epithelial Cells Impairs Estrogen-Induced DNA Replication, Induces DNA Damage Response Pathways, and Promotes Apoptosis

doi: 10.1128/mcb.00872-09

Figure Lengend Snippet: FIG. 10. Molecular pathways regulated by C/EBP during E-in- duced proliferation of uterine epithelial cells. In mouse uterine epi- thelial cells, the expression of C/EBP is stimulated by E and opposed by simultaneous treatment with P. C/EBP controls the entry of E- stimulated epithelial cells into the S phase of the cell cycle by upregu- lating the expression of E2F3 and cyclin E. It also promotes the nuclear localization of cyclin E, which enables the formation of an active cyclin E-cdk2 complex critical for DNA replication. The lack of C/EBP allows the nuclear accumulation of p27, which contributes to the cell cycle arrest. In E-stimulated C/EBP-null uterine epithelial cells, stalled DNA replication activates a DNA damage response path- way involving ATM/ATR, Chk1/Chk2, Rad18, and p53. Activated p53 maintains the block in DNA replication by enhancing the synthesis of the cell cycle inhibitor p21 and also promotes the removal of damaged cells via caspase-dependent apoptosis.

Article Snippet: Sections were incubated with the following primary antibodies diluted in blocking solution (0.25% bovine serum albumin [BSA], 0.3% Triton X-100, sterile PBS) overnight at 4°C: BrdU and Ki67 (BD Pharmingen), phospho-Ser10 histone H3 (Upstate Biotechnology); cyclin E, cyclin A, and Rad18 (Abcam); cyclin D1 (LabVision NeoMarkers); E2F3 (Santa Cruz Biotechnology); p27 (BD Transduction Laboratories); and phospho(Ser1981)-ATM, phospho-(Ser139)-H2AX, caspase 3, and cleaved caspase 3 (Cell Signaling Technology).

Techniques: Expressing, Blocking Assay