e2f3  (Bioss)

Journal: Cell reports. Medicine

Article Title: Signaling-induced systematic repression of miRNAs uncovers cancer vulnerabilities and targeted therapy sensitivity.

doi: 10.1016/j.xcrm.2023.101200

Figure Lengend Snippet: Figure 4. Drug prediction by miRNA sequencing uncovers drug sensitivities (A) Heatmap and unsupervised clustering according to the miRNA expression pattern of 12 included cancer models. (B) Overview of predicted activated pathways and corresponding PRSs for analyzed samples. The most frequent active pathways include WNT, TGF-b, and neurotophin (MAPK). (C) Overview of the top predicted target genes that are part of KEGG pathways. The most frequently predicted activated target genes are the cancer-associated genes E2F3 and CDK6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit Polyclonal anti-E2F3 antibody Proteintech Cat# 27615-1-AP; RRID: AB_2880926 CDK6 (D4S8S) Rabbit mAb Cell Signaling Technology Cat# 13331; RRID: AB_2721897 Activin Receptor Type IIA antibody Biozol Cat# GTX107815; RRID: AB_2036157 Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb Cell Signaling Technology Cat# 3108; RRID: AB_490941 Smad2 (D43B4) XP Rabbit mAb Cell Signaling Technology Cat# 5339; RRID: AB_10626777 IGF-I Receptor b (D23H3) XP Rabbit mAb Cell Signaling Technology Cat# 9750; RRID: AB_10950969 Cyclin D2 (D52F9) Rabbit mAb Cell Signaling Technology Cat# 3741; RRID: AB_2070685 c-Cbl (C49H8) Rabbit mAb Cell Signaling Technology Cat# 2179; RRID: AB_823449 HER4/ErbB4 (111B2) Rabbit mAb Cell Signaling Technology Cat# 4795; RRID: AB_2099883 Rac1/2/3 Antibody Cell Signaling Technology Cat# 2465; RRID: AB_2176152 GAPDH (D16H11) XP Rabbit mAb Cell Signaling Technology Cat# 5174; RRID: AB_10622025 Mouse anti-alpha-Tubulin (clone B-5-1-2) Sigma Cat# T5168; RRID: AB_477579 HRP Rabbit anti-Mouse (polyclonal) Abcam Cat# ab6728; RRID: AB_955440 HRP Goat anti-Rabbit (polyclonal) Abcam Cat# ab6721; RRID: AB_955447 Biological samples Normal and tumor tissue from cancer patients from different entities University Hospital Heidelberg or Dresden N/A Chemicals, peptides, and recombinant proteins Advanced DMEM/F-12 Thermo Fisher Scientific Cat# 12634028 Glucose Thermo Fisher Scientific Cat# 15023021 L-glutamine Thermo Fisher Scientific Cat# 25030024 HEPES Sigma-Aldrich Cat# H0887 Heparin Sigma-Aldrich Cat# H3149 BSA PAN-Biotech Cat# P06-10200 EGF, recombinant human R&D Systems Cat# 236-EG FGF basic, recombinant human R&D Systems Cat# 233-FB TGF beta 1, recombinant human Immunotools Cat# 11343160 Cultrex Reduced Growth Factor Basement Membrane Extract R&D Systems Cat# 3433-010-01 B-27TM Supplement Thermo Fisher Scientific Cat# A3582801 Penicillin-Streptomycin (10.000 U/ml) Thermo Fisher Scientific Cat# 15140148 Nicotinamide Sigma-Aldrich Cat# 72340-100G N-acetyl-L-cysteine Sigma-Aldrich Cat# A7250-25G SB 202190 Sigma-Aldrich Cat# S7067-5MG A 83-01 Sigma-Aldrich Cat# SML0788-5MG Gastrin Sigma-Aldrich Cat# SCP0152-1MG Prostaglandin E2 Sigma-Aldrich Cat# P5640-1MG Primocin InvivoGen Cat# ant-pm-1 Y-27632 Stem Cell Technologies Cat# 72302 RPMI1640 with GlutaMax Thermo Fisher Scientific Cat# 61870036 FBS PAN-Biotech Cat# P40-47500 DMSO Sigma-Aldrich Cat# 472301 PBS Life Technologies Cat# 14190169 0.9% NaCl solution B.Braun Cat# 3570350 (Continued on next page) e1 Cell Reports Medicine 4, 101200, October 17, 2023

Techniques: Sequencing, Expressing

Journal: Cell Reports Medicine

Article Title: Signaling-induced systematic repression of miRNAs uncovers cancer vulnerabilities and targeted therapy sensitivity

doi: 10.1016/j.xcrm.2023.101200

Figure Lengend Snippet: Drug prediction by miRNA sequencing uncovers drug sensitivities (A) Heatmap and unsupervised clustering according to the miRNA expression pattern of 12 included cancer models. (B) Overview of predicted activated pathways and corresponding PRSs for analyzed samples. The most frequent active pathways include WNT, TGF-β, and neurotophin (MAPK). (C) Overview of the top predicted target genes that are part of KEGG pathways. The most frequently predicted activated target genes are the cancer-associated genes E2F3 and CDK6 . (D) Selected cancer models from the miRNA sequencing approach with strong expandability potential were employed for western blotting with antibodies for top predicted target genes. For ACVR2A, phosphorylation of the downstream activator SMAD2 was observed in all samples with predicted ACVR2A activity. (E) Summary of prediction accuracy and protein presence on the western blot level for all depicted top-hit proteins. (F) 10 of 12 samples were utilized for drug response assays with either the standard of care (SOC) reagents of each entity or the miRNA expression-based top targeted predicted drugs (PDs). In addition, randomly picked, targeted non-predicted drugs (NPDs) were included in selected drug screens. For each sample, fold changes between the pan-cancer Z score of SOC drugs and PDs are indicated. (G) Summary of all executed drug screens for all samples, depicted as individual pan-cancer Z scores for all included SOC, PD, and NPD therapies. (H) In vivo validation of drug screens. CRC02-SP and CRC03-SP cultures were transplanted subcutaneously into immunodeficient NSG mice. Total times from starting of the treatment until final tumor size are depicted as Kaplan-Meier curves. CRC02 xenografts were treated with the IGFR inhibitor linsitinib (n = 7). CRC03 xenografts were treated with the CDK4/6 inhibitor palbociclib (n = 6). ∗∗p < 0.01, Wilcoxon rank-sum test; ∗p < 0.05, log rank (Mantel-Cox) test.

Article Snippet: Rabbit Polyclonal anti-E2F3 antibody , Proteintech , Cat# 27615-1-AP; RRID: AB_2880926.

Techniques: Sequencing, Expressing, Western Blot, Phospho-proteomics, Activity Assay, In Vivo, Biomarker Discovery

Journal: Cell Reports Medicine

Article Title: Signaling-induced systematic repression of miRNAs uncovers cancer vulnerabilities and targeted therapy sensitivity

doi: 10.1016/j.xcrm.2023.101200

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal anti-E2F3 antibody , Proteintech , Cat# 27615-1-AP; RRID: AB_2880926.

Techniques: Recombinant, Membrane, Protease Inhibitor, Isolation, Proliferation Assay, Luminescence Assay, Expressing, Sequencing, Comparison, Software

Journal: Animals : an Open Access Journal from MDPI

Article Title: SV40T/E6E7-Induced Proliferation Is Involved in the Activity of E2F3 in Bovine Mammary Epithelial Cells

doi: 10.3390/ani12141790

Figure Lengend Snippet: Involvement of E2F signaling pathway in the proliferation of the immortalized BMECs. ( A ) Western blotting analysis of CDK4, CDK6, cyclin D1, cyclin D3, and CDK2 in primary and immortalized BMECs. ( B ) Western blotting analysis of E2F1, E2F2, and E2F3 in primary and immortalized BMECs. Primary BMECs or BMECs immortalized by E6E7 and SV40T were collected at early (E) and late (L) passages of culture. Data are based on triplicate experiments.

Article Snippet: The following primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, Shanghai, China): rabbit anti-E2F3 polyclonal antibody (1:200; sc-878), mouse anti-Cdc2 monoclonal antibody (1:200; sc-166885), and rabbit anti-p21 Cip1 polyclonal antibody (1:200; sc-397).

Techniques: Western Blot

Journal: Molecular Medicine Reports

Article Title: miR-145 suppresses ovarian cancer progression via modulation of cell growth and invasion by targeting CCND2 and E2F3

doi: 10.3892/mmr.2019.10004

Figure Lengend Snippet: CCND2 and E2F3 are the targets of miR-145 in ovarian cancer cells. (A) The schematic construction of WT and Mut 3′-UTR of CCND2 and E2F3. (B) SKOV3 cells were co-transfected with WT or Mut 3′-UTR of CCND2 and E2F3, followed by transfection with the miR-145 or NC mimics. The luciferase activities were examined 48 h after transfection. (C) SKOV3 cells were transfected with miR-145 or the control mimics, and CCND2 and E2F3 mRNA were detected by qPCR. (D) The expression levels of CCND2 and E2F3 were detected in ovarian cancer tissues and normal ovarian tissues using western blot analysis. (E) The protein level of CCND2 and E2F3 were detected in SKOV3 cells transfected with miR-145 or the NC mimics. (F) Densitometric evaluation of band intensities. Experiments were performed in triplicate. *P<0.05 and **P<0.01 vs. control. CCND2, cyclin D2; E2F3, E2F transcription factor 3; miR, microRNA; WT, wild type, Mut, mutant; UTR, untranslated region; NC/ctrl, negative control; FC, fold change.

Article Snippet: Following blocking using 5% non-fat milk for 2 h at room temperature, the membranes were incubated with anti-CCND2 monoclonal antibody (cat. no. SC-56305; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-E2F3 polyclonal antibody (cat. no. SC-879; 1:500; Santa Cruz Biotechnology, Inc.), anti-B cell lymphoma 2 (Bcl-2; cat. no. 4223; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-Bcl-2-associated X protein (Bax; cat. no. 5023; 1:1,000; Cell Signaling Technology, Inc.) and anti-β-actin (cat. no. 4970; 1:2,000; Cell Signaling Technology, Inc.) overnight at 4°C, which was followed by incubation with anti-mouse (cat. no. 7076; 1:4,000; Cell Signaling Technology, Inc.) or anti-rabbit (cat. no. 7074; 1:5,000; Cell Signaling Technology, Inc.) horseradish peroxidase-conjugated immunoglobulin G at 4°C for 2 h. The blots were developed using an enhanced chemiluminescent system (Amersham; GE Healthcare, Chicago, IL, USA), and the density of bands was detected using ImageJ 1.42q software (National Institutes of Health, Bethesda, MD, USA).

Techniques: Transfection, Luciferase, Control, Expressing, Western Blot, Mutagenesis, Negative Control

Journal: Molecular Medicine Reports

Article Title: miR-145 suppresses ovarian cancer progression via modulation of cell growth and invasion by targeting CCND2 and E2F3

doi: 10.3892/mmr.2019.10004

Figure Lengend Snippet: Restoration of CCND2 and E2F3 expression reverses the effects of miR-145. (A) SKOV3 and ES-2 cells were transfected with CCND2 and E2F3 overexpression plasmids, and the expression was then examined by western blot analysis. The 2 cells lines were also cotransfected with miR-145 mimic + CCND2 and E2F3 overexpression plasmids or the control vector. (B) Cell viability was measured by Cell Counting Kit-8 assay. (C) SKOV3 and ES-2 cells were stained with propidium iodide and subjected to flow cytometry to determine the distribution of cells at each phase of the cell cycle, and were compared with the NC group. The proportion of G1 phase decreased in the cells transfected with CCND2 and E2F3 overexpression plasmids. (D) Quantification of cell cycle distribution results presented as the mean ± standard error of the mean of 3 independent experiments. *P<0.05 and **P<0.01. (E) Wound healing assay was performed at 24 h post-transfection (magnification, 200×). (E-a) SKOV3 cells at 0 h. Following 24 h, the migration of SKOV3 cells transfected with (E-b) NC was not significantly altered compared with cells transfected with (E-c) miR-145, E2F3 and CCND2. The results indicated that the effects of miR-145 were reversed by CCND2 and E2F3. (F) Invasion assays were performed. Experiments were performed in triplicate. *P<0.05 and **P<0.01 vs. the control. CCND2, cyclin D2; E2F3, E2F transcription factor 3; miR, microRNA; NC, negative control.

Article Snippet: Following blocking using 5% non-fat milk for 2 h at room temperature, the membranes were incubated with anti-CCND2 monoclonal antibody (cat. no. SC-56305; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-E2F3 polyclonal antibody (cat. no. SC-879; 1:500; Santa Cruz Biotechnology, Inc.), anti-B cell lymphoma 2 (Bcl-2; cat. no. 4223; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-Bcl-2-associated X protein (Bax; cat. no. 5023; 1:1,000; Cell Signaling Technology, Inc.) and anti-β-actin (cat. no. 4970; 1:2,000; Cell Signaling Technology, Inc.) overnight at 4°C, which was followed by incubation with anti-mouse (cat. no. 7076; 1:4,000; Cell Signaling Technology, Inc.) or anti-rabbit (cat. no. 7074; 1:5,000; Cell Signaling Technology, Inc.) horseradish peroxidase-conjugated immunoglobulin G at 4°C for 2 h. The blots were developed using an enhanced chemiluminescent system (Amersham; GE Healthcare, Chicago, IL, USA), and the density of bands was detected using ImageJ 1.42q software (National Institutes of Health, Bethesda, MD, USA).

Techniques: Expressing, Transfection, Over Expression, Western Blot, Control, Plasmid Preparation, Cell Counting, Staining, Flow Cytometry, Wound Healing Assay, Migration, Negative Control

Journal: Oncotarget

Article Title: MicroRNA-432 functions as a tumor suppressor gene through targeting E2F3 and AXL in lung adenocarcinoma

doi: 10.18632/oncotarget.7884

Figure Lengend Snippet: ( A and B ) Predicted miR-432 target sequences in the 3′UTRs of E2F3 and AXL ( W ). The miR-432 mutant ( M ) contained four altered nucleotides in the seed sequence. A549 ( C ) and H1299 ( D ) cells transfected with miR-432 mimics or miR-432 inhibitors, and the expression levels of E2F3(left) and AXL(right) were detected by Western blotting. GAPDH was used as a control. The quantification of each protein band in the result of Western blotting was done using LAS-3000 with MultiGauge software (Fuji film). HEK-293T cells were co-transfected with miR-432 mimics or mimics control with WT/Mut 3′-UTR of E2F3 ( E ) and AXL ( F ). Relative luciferase activity was evaluated. Experiments were performed in triplicate. ( G and H ) The same assay above was also performed in A549. * p < 0.05 compared with mimis control. After co-transfection with miR-432 inhibitor or its control and WT/Mut 3′-UTR of E2F3 ( I ) and AXL ( J ) in H1299, the luciferase activity was detected. * p < 0.05 compared with inhibitor control.

Article Snippet: The slides were incubated with rabbit polyclonal anti-E2F3 antibody (1:100 dilution, Abcam, Cambridge, MA, USA), anti-AXL antibody (1:100 dilution, Abcam, Cambridge, MA, USA) or mouse monoclonal anti-Ki67 antibody (1:100 dilution, Dako, Carpinteria, CA, USA).

Techniques: Mutagenesis, Sequencing, Transfection, Expressing, Western Blot, Control, Software, Luciferase, Activity Assay, Cotransfection

Journal: Oncotarget

Article Title: MicroRNA-432 functions as a tumor suppressor gene through targeting E2F3 and AXL in lung adenocarcinoma

doi: 10.18632/oncotarget.7884

Figure Lengend Snippet: Effects of miR-432 inhibitor transfection on the cisplatin sensitivity were assayed by MTS method in A549 ( A ) and H1299 ( B ). Inhibitor control + Cisplatin vs miR-432 inhibitor + Cisplatin:* p < 0.05; ** p < 0.01. The cell vitality ( C ) and apoptotic fraction ( D ) were analyzed in A549 and H1299 cells after cisplatin treatment at various concentrations with or without miR-432 mimics transfection. Cisplatin + mimics control vs Cispliatin + miR-432 mimics: * p < 0.05; ** p < 0.01. Effects of cisplatin treatment at different concentrations and various time of on miR-432 expression in A549 ( E ) and H1299 ( F ). The expression of E2F3 and AXL mRNA was detected by RT-qPCR in A549 ( G ) and H1299 ( H ) after cisplatin (5 μg/ml) treatment. Western blotting was performed to analyse the expression of AXL (upper) and E2F3 (lower) in A549 ( I ) and H1299 ( J ) as indicated treatments. * p < 0.05.

Article Snippet: The slides were incubated with rabbit polyclonal anti-E2F3 antibody (1:100 dilution, Abcam, Cambridge, MA, USA), anti-AXL antibody (1:100 dilution, Abcam, Cambridge, MA, USA) or mouse monoclonal anti-Ki67 antibody (1:100 dilution, Dako, Carpinteria, CA, USA).

Techniques: Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot

Journal: Oncotarget

Article Title: MicroRNA-432 functions as a tumor suppressor gene through targeting E2F3 and AXL in lung adenocarcinoma

doi: 10.18632/oncotarget.7884

Figure Lengend Snippet: The cell vitality was assayed by MTS after over-expressing E2F3 or AXL in miR-432 mimics transfected A549 ( A ) and H1299 ( B ) cells. E2F3 or AXL expression vector vs empty vector: * p < 0.05. The effects of silencing E2F3 and AXL on miR-432 inhibitor in A549 ( C ) and H1299 ( D ) cells were detected by MTS. siRNA targeting E2F3 or AXL vs NC: * p < 0.05. In the presence of Cisplatin, the effects of over-expressing E2F3 or AXL on the miR-432 mimics transfected A549 ( E ) and H1299 ( F ) cells was detected by MTS. E2F3 or AXL expression vector vs empty vector: * p < 0.05. The same experiments were performed to evaluate the effect of silencing E2F3 or AXL on cisplatin sensitivity in miR-432 inhibitor-transfected A549 ( G ) and H1299 ( H ) cells. siRNA targeting E2F3 or AXL vs NC: * p < 0.05.

Article Snippet: The slides were incubated with rabbit polyclonal anti-E2F3 antibody (1:100 dilution, Abcam, Cambridge, MA, USA), anti-AXL antibody (1:100 dilution, Abcam, Cambridge, MA, USA) or mouse monoclonal anti-Ki67 antibody (1:100 dilution, Dako, Carpinteria, CA, USA).

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: Oncotarget

Article Title: MicroRNA-432 functions as a tumor suppressor gene through targeting E2F3 and AXL in lung adenocarcinoma

doi: 10.18632/oncotarget.7884

Figure Lengend Snippet: ( A ) The percentage of specimens showing low or high miR-32 expression in relation to the expression levels of E2F3 and AXL; * p < 0.05. ( B ) Two representative cases are shown.

Article Snippet: The slides were incubated with rabbit polyclonal anti-E2F3 antibody (1:100 dilution, Abcam, Cambridge, MA, USA), anti-AXL antibody (1:100 dilution, Abcam, Cambridge, MA, USA) or mouse monoclonal anti-Ki67 antibody (1:100 dilution, Dako, Carpinteria, CA, USA).

Techniques: Expressing