Journal: Blood and Lymphatic Cancer: Targets and Therapy

Article Title: Crosstalk Between FOXN3 and E2F5 Reveals a Novel Tumor Suppressive Pathway in Acute Myeloid Leukemia via MAPK Signaling: Implications for Potential Future Targeted Therapy

doi: 10.2147/BLCTT.S571966

Figure Lengend Snippet: Identification of E2F5 as a transcriptional target of FOXN3. (A ) Genomic distribution of FOXN3 binding sites, represented through pie charts. ( B ) Distribution of binding sites relative to transcription initiation peaks within 5 kb upstream and downstream of the transcription start site (TSS). ( C and D ) RT-qPCR ( C ) and Western blotting ( D ) analyses of E2F5 expression in THP1 cells following FOXN3 overexpression. ( E ) Scatter plot illustrating the correlation between FOXN3 and E2F5 expression levels in AML patient samples from the GSE13159 dataset, indicating a negative correlation between the two genes. ( F ) Scatter plot confirming the negative correlation between FOXN3 and E2F5 expression in AML patient samples collected from our institution. ( G ) Visualization of representative FOXN3 binding peaks in the genome browser using karyoploteR, specifically in the E2F5 promoter region. The tracks display both input and immunoprecipitation (IP) data for the Kasumi1 and THP1 cell lines. The highlighted region denotes the E2F5 promoter area, where significant binding peaks were detected in the ChIP-seq experiment. ( H ) ChIP-PCR analysis demonstrating FOXN3 binding to the E2F5 promoter. Significant enrichment of FOXN3 binding is observed at the E2F5 promoter compared to the IgG control, with the SIRT6 promoter serving as a positive control ( P = 0.001). ( I ) Results from the luciferase reporter assay illustrating the repressive effect of FOXN3 on E2F5 promoter activity. The relative luciferase activity is significantly lower in the presence of FOXN3 ( P < 0.01). Data are presented as mean ± SD of three independent experiments. Statistical significance is indicated as follows: * P < 0.05, ** P < 0.01.

Article Snippet: The primary antibodies used were as follows: FOXN3 (ab129453, 1:2500, Abcam, UK), E2F5 (A16042, 1:500, ABclonal, China), CDC42 (2466T, 1:1000, Cell Signaling Technology, MA), HDAC1 (34589T, 1:1000, Cell Signaling Technology, MA), PCNA (WL03213, 1:500, Wanleibio, China), CyclinE1 (WL01072, 1:1000, Wanleibio, China), CDK2 (WL01543, 1:500, Wanleibio, China), CyclinD1 (WL01435a, 1:400, Wanleibio, China), CDK6 (WL03460, 1:500, Wanleibio, China), Bcl-2 (WL01556, 1:1000, Wanleibio, China), EZH2 (WL00795, 1:1000, Wanleibio, China), p-ERK (WLP1512, 1:1000, Wanleibio, China), ERK (WL01864, 1:1000, Wanleibio, China), p-38 (WL00764, 1:1000, Wanleibio, China), and GAPDH (5174, 1:1000, Cell Signaling Technology, MA).

Techniques: Binding Assay, Quantitative RT-PCR, Western Blot, Expressing, Over Expression, Immunoprecipitation, ChIP-sequencing, Control, Positive Control, Luciferase, Reporter Assay, Activity Assay

Journal: Blood and Lymphatic Cancer: Targets and Therapy

Article Title: Crosstalk Between FOXN3 and E2F5 Reveals a Novel Tumor Suppressive Pathway in Acute Myeloid Leukemia via MAPK Signaling: Implications for Potential Future Targeted Therapy

doi: 10.2147/BLCTT.S571966

Figure Lengend Snippet: Aberrant upregulation of E2F5 expression and its association with poor outcomes in AML. ( A ) RT-qPCR analysis showing elevated E2F5 mRNA expression in AML patients compared to healthy controls. ( B ) E2F5 mRNA levels are significantly higher in AML compared to hematopoietic stem cells (HSCs), as demonstrated using the online Bloodpool dataset. ( C ) RT-qPCR analysis revealing increased E2F5 mRNA expression in AML cell lines compared to control cell lines. ( D ) Correlation between higher E2F5 expression and shorter overall survival (OS) in AML, determined using the median value as the cutoff to distinguish between high and low E2F5 expression levels from the online GEPIA dataset. Data are presented as mean ± SD of three independent experiments. Statistical significance is indicated as follows:* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The primary antibodies used were as follows: FOXN3 (ab129453, 1:2500, Abcam, UK), E2F5 (A16042, 1:500, ABclonal, China), CDC42 (2466T, 1:1000, Cell Signaling Technology, MA), HDAC1 (34589T, 1:1000, Cell Signaling Technology, MA), PCNA (WL03213, 1:500, Wanleibio, China), CyclinE1 (WL01072, 1:1000, Wanleibio, China), CDK2 (WL01543, 1:500, Wanleibio, China), CyclinD1 (WL01435a, 1:400, Wanleibio, China), CDK6 (WL03460, 1:500, Wanleibio, China), Bcl-2 (WL01556, 1:1000, Wanleibio, China), EZH2 (WL00795, 1:1000, Wanleibio, China), p-ERK (WLP1512, 1:1000, Wanleibio, China), ERK (WL01864, 1:1000, Wanleibio, China), p-38 (WL00764, 1:1000, Wanleibio, China), and GAPDH (5174, 1:1000, Cell Signaling Technology, MA).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Blood and Lymphatic Cancer: Targets and Therapy

Article Title: Crosstalk Between FOXN3 and E2F5 Reveals a Novel Tumor Suppressive Pathway in Acute Myeloid Leukemia via MAPK Signaling: Implications for Potential Future Targeted Therapy

doi: 10.2147/BLCTT.S571966

Figure Lengend Snippet: Inhibition of cell proliferation, induction of apoptosis, and cell cycle arrest following E2F5 knockdown in AML cells. ( A ) CCK-8 assay was conducted to assess the proliferation of Kasumi1 and THP1 cells following E2F5 knockdown. ( B ) Flow cytometry analysis evaluated the induction of apoptosis in Kasumi1 and THP1 cells after E2F5 knockdown. ( C ) Cell cycle analysis was performed on Kasumi1 and THP1 cells with E2F5 knockdown using flow cytometry. Data are presented as mean ± SD of three independent experiments. Statistical significance is indicated as follows: * P < 0.05, ** P < 0.01, and **** P < 0.0001.

Article Snippet: The primary antibodies used were as follows: FOXN3 (ab129453, 1:2500, Abcam, UK), E2F5 (A16042, 1:500, ABclonal, China), CDC42 (2466T, 1:1000, Cell Signaling Technology, MA), HDAC1 (34589T, 1:1000, Cell Signaling Technology, MA), PCNA (WL03213, 1:500, Wanleibio, China), CyclinE1 (WL01072, 1:1000, Wanleibio, China), CDK2 (WL01543, 1:500, Wanleibio, China), CyclinD1 (WL01435a, 1:400, Wanleibio, China), CDK6 (WL03460, 1:500, Wanleibio, China), Bcl-2 (WL01556, 1:1000, Wanleibio, China), EZH2 (WL00795, 1:1000, Wanleibio, China), p-ERK (WLP1512, 1:1000, Wanleibio, China), ERK (WL01864, 1:1000, Wanleibio, China), p-38 (WL00764, 1:1000, Wanleibio, China), and GAPDH (5174, 1:1000, Cell Signaling Technology, MA).

Techniques: Inhibition, Knockdown, CCK-8 Assay, Flow Cytometry, Cell Cycle Assay

Journal: Blood and Lymphatic Cancer: Targets and Therapy

Article Title: Crosstalk Between FOXN3 and E2F5 Reveals a Novel Tumor Suppressive Pathway in Acute Myeloid Leukemia via MAPK Signaling: Implications for Potential Future Targeted Therapy

doi: 10.2147/BLCTT.S571966

Figure Lengend Snippet: Enhancement of cell proliferation and cell cycle following E2F5 overexpression in AML cells. ( A ) CCK-8 assay evaluated the proliferation of Kasumi1 and THP1 cells following E2F5 overexpression. ( B ) Cell cycle analysis was conducted on Kasumi1 and THP1 cells with E2F5 overexpression using flow cytometry. Data are presented as mean ± SD of three independent experiments. Statistical significance is indicated as follows: ** P < 0.05, *** P < 0.01, and *** P < 0.0001.

Article Snippet: The primary antibodies used were as follows: FOXN3 (ab129453, 1:2500, Abcam, UK), E2F5 (A16042, 1:500, ABclonal, China), CDC42 (2466T, 1:1000, Cell Signaling Technology, MA), HDAC1 (34589T, 1:1000, Cell Signaling Technology, MA), PCNA (WL03213, 1:500, Wanleibio, China), CyclinE1 (WL01072, 1:1000, Wanleibio, China), CDK2 (WL01543, 1:500, Wanleibio, China), CyclinD1 (WL01435a, 1:400, Wanleibio, China), CDK6 (WL03460, 1:500, Wanleibio, China), Bcl-2 (WL01556, 1:1000, Wanleibio, China), EZH2 (WL00795, 1:1000, Wanleibio, China), p-ERK (WLP1512, 1:1000, Wanleibio, China), ERK (WL01864, 1:1000, Wanleibio, China), p-38 (WL00764, 1:1000, Wanleibio, China), and GAPDH (5174, 1:1000, Cell Signaling Technology, MA).

Techniques: Over Expression, CCK-8 Assay, Cell Cycle Assay, Flow Cytometry

Journal: Blood and Lymphatic Cancer: Targets and Therapy

Article Title: Crosstalk Between FOXN3 and E2F5 Reveals a Novel Tumor Suppressive Pathway in Acute Myeloid Leukemia via MAPK Signaling: Implications for Potential Future Targeted Therapy

doi: 10.2147/BLCTT.S571966

Figure Lengend Snippet: Partial rescue of proliferation inhibition and induction of apoptosis by E2F5 upregulation in AML cells overexpressing FOXN3. ( A ) Assessment of cell proliferation in leukemic cells co-transfected with lentiviruses for FOXN3 overexpression and E2F5 overexpression using the CCK-8 assay. ( B ) Flow cytometry analysis evaluated apoptosis in leukemic cells co-transfected with FOXN3 and E2F5 overexpressing lentiviruses. ( C ) Cell cycle analysis was performed on leukemic cells co-transfected with FOXN3 and E2F5 overexpressing lentiviruses using flow cytometry. Data are presented as mean ± SD of three independent experiments. Statistical significance is indicated as follows:* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The primary antibodies used were as follows: FOXN3 (ab129453, 1:2500, Abcam, UK), E2F5 (A16042, 1:500, ABclonal, China), CDC42 (2466T, 1:1000, Cell Signaling Technology, MA), HDAC1 (34589T, 1:1000, Cell Signaling Technology, MA), PCNA (WL03213, 1:500, Wanleibio, China), CyclinE1 (WL01072, 1:1000, Wanleibio, China), CDK2 (WL01543, 1:500, Wanleibio, China), CyclinD1 (WL01435a, 1:400, Wanleibio, China), CDK6 (WL03460, 1:500, Wanleibio, China), Bcl-2 (WL01556, 1:1000, Wanleibio, China), EZH2 (WL00795, 1:1000, Wanleibio, China), p-ERK (WLP1512, 1:1000, Wanleibio, China), ERK (WL01864, 1:1000, Wanleibio, China), p-38 (WL00764, 1:1000, Wanleibio, China), and GAPDH (5174, 1:1000, Cell Signaling Technology, MA).

Techniques: Inhibition, Transfection, Over Expression, CCK-8 Assay, Flow Cytometry, Cell Cycle Assay

Journal: Blood and Lymphatic Cancer: Targets and Therapy

Article Title: Crosstalk Between FOXN3 and E2F5 Reveals a Novel Tumor Suppressive Pathway in Acute Myeloid Leukemia via MAPK Signaling: Implications for Potential Future Targeted Therapy

doi: 10.2147/BLCTT.S571966

Figure Lengend Snippet: Regulation of EZH2 and MAPK pathways by the FOXN3-E2F5 axis in AML. ( A ) Volcano plots illustrating differentially expressed genes in THP1 cells with FOXN3 overexpression compared to control THP1 cells. ( B ) The top twenty significant KEGG pathways related to differentially expressed genes in THP1 cells with FOXN3 overexpression compared to control THP1 cells. The red box highlights the signal pathway that are in agreement with the functional results. ( C ) Western blot analysis showing the protein levels of EZH2, ERK1/2, and phosphorylated ERK1/2 (p-ERK1/2) in cells co-transfected with lentiviruses for FOXN3 and E2F5 overexpression. The experiments were conducted using three independent replicates and the data are presented as mean ± SD. Statistical significance is indicated as follows: * P < 0.05, and ** P < 0.01.

Article Snippet: The primary antibodies used were as follows: FOXN3 (ab129453, 1:2500, Abcam, UK), E2F5 (A16042, 1:500, ABclonal, China), CDC42 (2466T, 1:1000, Cell Signaling Technology, MA), HDAC1 (34589T, 1:1000, Cell Signaling Technology, MA), PCNA (WL03213, 1:500, Wanleibio, China), CyclinE1 (WL01072, 1:1000, Wanleibio, China), CDK2 (WL01543, 1:500, Wanleibio, China), CyclinD1 (WL01435a, 1:400, Wanleibio, China), CDK6 (WL03460, 1:500, Wanleibio, China), Bcl-2 (WL01556, 1:1000, Wanleibio, China), EZH2 (WL00795, 1:1000, Wanleibio, China), p-ERK (WLP1512, 1:1000, Wanleibio, China), ERK (WL01864, 1:1000, Wanleibio, China), p-38 (WL00764, 1:1000, Wanleibio, China), and GAPDH (5174, 1:1000, Cell Signaling Technology, MA).

Techniques: Over Expression, Control, Functional Assay, Western Blot, Transfection

Journal: Blood and Lymphatic Cancer: Targets and Therapy

Article Title: Crosstalk Between FOXN3 and E2F5 Reveals a Novel Tumor Suppressive Pathway in Acute Myeloid Leukemia via MAPK Signaling: Implications for Potential Future Targeted Therapy

doi: 10.2147/BLCTT.S571966

Figure Lengend Snippet: The mechanism of FOXN3-E2F5-EZH2-MAPK regulatory axis in AML.

Article Snippet: The primary antibodies used were as follows: FOXN3 (ab129453, 1:2500, Abcam, UK), E2F5 (A16042, 1:500, ABclonal, China), CDC42 (2466T, 1:1000, Cell Signaling Technology, MA), HDAC1 (34589T, 1:1000, Cell Signaling Technology, MA), PCNA (WL03213, 1:500, Wanleibio, China), CyclinE1 (WL01072, 1:1000, Wanleibio, China), CDK2 (WL01543, 1:500, Wanleibio, China), CyclinD1 (WL01435a, 1:400, Wanleibio, China), CDK6 (WL03460, 1:500, Wanleibio, China), Bcl-2 (WL01556, 1:1000, Wanleibio, China), EZH2 (WL00795, 1:1000, Wanleibio, China), p-ERK (WLP1512, 1:1000, Wanleibio, China), ERK (WL01864, 1:1000, Wanleibio, China), p-38 (WL00764, 1:1000, Wanleibio, China), and GAPDH (5174, 1:1000, Cell Signaling Technology, MA).

Techniques:

Journal: BMC Cancer

Article Title: E2F5 status significantly improves malignancy diagnosis of epithelial ovarian cancer

doi: 10.1186/1471-2407-10-64

Figure Lengend Snippet: Clinical characteristics and age distribution of study samples (serum samples) used for E2F5 and CA125 expression studies.

Article Snippet: Primary antibodies directed against the AREB6 (AVIVA Systems Biology, San Diego, California, USA), E2F5 (Acris GmbH, Hiddenhausen, Germany), PAX8 (Abcam Limited, Cambridge, UK), and ELF3 (Orbigen Inc, San Diego, CA) were chosen as the four TFs suggested by the bioinformatics analysis.

Techniques: Expressing

Journal: BMC Cancer

Article Title: E2F5 status significantly improves malignancy diagnosis of epithelial ovarian cancer

doi: 10.1186/1471-2407-10-64

Figure Lengend Snippet: Summary of Immunohistochemistry results for E2F5 antibody tested on OEC

Article Snippet: Primary antibodies directed against the AREB6 (AVIVA Systems Biology, San Diego, California, USA), E2F5 (Acris GmbH, Hiddenhausen, Germany), PAX8 (Abcam Limited, Cambridge, UK), and ELF3 (Orbigen Inc, San Diego, CA) were chosen as the four TFs suggested by the bioinformatics analysis.

Techniques: Immunohistochemistry

Journal: BMC Cancer

Article Title: E2F5 status significantly improves malignancy diagnosis of epithelial ovarian cancer

doi: 10.1186/1471-2407-10-64

Figure Lengend Snippet: Validation of potential target (AREB6, PAX8, ELF3) using western blots (A) and expression of E2F5 in serum from normal, benign and, malignant patient samples (B) . (A) Target validation using western blots of AREB6, PAX8, ELF3, in serum of healthy volunteers (N) (n = 2), patients with benign ovarian cysts (B1, B2) (n = 2), and patients with late- (L1, L2) (n = 2) and early-stage (E1-E3) (n = 3) serous adenocarcinoma. AREB6 was overexpressed in late stage disease, but was not discriminatory for early cancer. PAX8 and ELF3 were equally expressed in all serum samples. (B) Expression of E2F5 in serum from normal (n = 56), benign (n = 40) and, malignant (n = 48) serum obtained from patients with cancer of the ovary.

Article Snippet: Primary antibodies directed against the AREB6 (AVIVA Systems Biology, San Diego, California, USA), E2F5 (Acris GmbH, Hiddenhausen, Germany), PAX8 (Abcam Limited, Cambridge, UK), and ELF3 (Orbigen Inc, San Diego, CA) were chosen as the four TFs suggested by the bioinformatics analysis.

Techniques: Biomarker Discovery, Western Blot, Expressing

Journal: BMC Cancer

Article Title: E2F5 status significantly improves malignancy diagnosis of epithelial ovarian cancer

doi: 10.1186/1471-2407-10-64

Figure Lengend Snippet: Details of E2F5/CA125 expression pattern on different subtypes of OEC performed using western blotting technique.

Article Snippet: Primary antibodies directed against the AREB6 (AVIVA Systems Biology, San Diego, California, USA), E2F5 (Acris GmbH, Hiddenhausen, Germany), PAX8 (Abcam Limited, Cambridge, UK), and ELF3 (Orbigen Inc, San Diego, CA) were chosen as the four TFs suggested by the bioinformatics analysis.

Techniques: Expressing, Western Blot

Journal: BMC Cancer

Article Title: E2F5 status significantly improves malignancy diagnosis of epithelial ovarian cancer

doi: 10.1186/1471-2407-10-64

Figure Lengend Snippet: Diagnostic accuracy developed for ovarian cancer detection with 13 features using Kstar classification based on SMOTE algorithms.

Article Snippet: Primary antibodies directed against the AREB6 (AVIVA Systems Biology, San Diego, California, USA), E2F5 (Acris GmbH, Hiddenhausen, Germany), PAX8 (Abcam Limited, Cambridge, UK), and ELF3 (Orbigen Inc, San Diego, CA) were chosen as the four TFs suggested by the bioinformatics analysis.

Techniques: Diagnostic Assay