anti e2f5  (Santa Cruz Biotechnology)

Journal: Journal of Translational Medicine

Article Title: Exosomes loaded with the anti-cancer molecule mir-1-3p inhibit intrapulmonary colonization and growth of human esophageal squamous carcinoma cells

doi: 10.1186/s12967-024-05997-9

Figure Lengend Snippet: miR-1-3p directly targets and down-regulates E2F5 expression. The differentially expressed genes in the Exo-miR-1-3p group compared with the Exo-NC group, shown in the volcano plot ( A ), and the KEGG pathway analysis of differentially expressed genes ( B ). ( C ) Predicted target genes of miR-1-3p with TargetScan and miRWalk were cross-analyzed with significantly expression down-regulated genes. ( D ) E2F5 mRNA level after Exo-miR-1-3p incubation. ( E ) Putative binding sequence of miR-1-3p to the E2F5 mRNA 3’UTR and results of dual luciferase reporter assays. E2F5 protein expression of KYSE150 and Eca109 cells transfected with miR-1-3p mimics ( F ) or miR-1-3p inhibitor ( G ). Immunohistochemical staining ( H ) and H-scores ( I ) of E2F5 in mouse lung tissues. * P < 0.05, *** P < 0.001

Article Snippet: Antibodies information used in this study is provided below: anti-TSG101 (1:2000, 14497-1-AP, Proteintech), anti-CD81 (66866-1-Ig), anti-CD9 (60232-1-Ig), anti-E2F5 (1:2000, bs-1734R, Bioss), anti-GAPDH (1:5000, 60004-1-Ig, Proteintech), anti-p-p38 (1:1000, 4511, CST), anti-p38 (1:5000, 66234-1-lg, Proteintech), anti-p-ERK (1:1000, AP0974, ABclonal), anti-ERK (1:700, A4782, ABclonal), anti-p-Akt (1:2000, 28731-1-AP, Proteintech), anti-p-mTOR (1:500, AF3308, Affinity), HRP Goat anti-Rabbit IgG (1: 5000, AS014, ABclonal), HRP Goat anti-Mouse IgG (1: 5000, AS003, ABclonal).

Techniques: Expressing, Incubation, Binding Assay, Sequencing, Luciferase, Transfection, Immunohistochemical staining, Staining

Journal: Journal of Translational Medicine

Article Title: Exosomes loaded with the anti-cancer molecule mir-1-3p inhibit intrapulmonary colonization and growth of human esophageal squamous carcinoma cells

doi: 10.1186/s12967-024-05997-9

Figure Lengend Snippet: miR-1-3p inhibits proliferation and migration of ESCC cells in vitro by down-regulating E2F5. The miR-1-3p levels ( A ), E2F5 mRNA levels ( B ), and E2F5 protein levels in KYSE150 and Eca109 cells in vitro co-transfected with miR-1-3p mimics and E2F5 expression plasmids. The proliferation ( D ) and migration ( E and F ) of transfected cells. ns, no significance; * P < 0.05, *** P < 0.001

Article Snippet: Antibodies information used in this study is provided below: anti-TSG101 (1:2000, 14497-1-AP, Proteintech), anti-CD81 (66866-1-Ig), anti-CD9 (60232-1-Ig), anti-E2F5 (1:2000, bs-1734R, Bioss), anti-GAPDH (1:5000, 60004-1-Ig, Proteintech), anti-p-p38 (1:1000, 4511, CST), anti-p38 (1:5000, 66234-1-lg, Proteintech), anti-p-ERK (1:1000, AP0974, ABclonal), anti-ERK (1:700, A4782, ABclonal), anti-p-Akt (1:2000, 28731-1-AP, Proteintech), anti-p-mTOR (1:500, AF3308, Affinity), HRP Goat anti-Rabbit IgG (1: 5000, AS014, ABclonal), HRP Goat anti-Mouse IgG (1: 5000, AS003, ABclonal).

Techniques: Migration, In Vitro, Transfection, Expressing

Journal: Journal of Translational Medicine

Article Title: Exosomes loaded with the anti-cancer molecule mir-1-3p inhibit intrapulmonary colonization and growth of human esophageal squamous carcinoma cells

doi: 10.1186/s12967-024-05997-9

Figure Lengend Snippet: miR-1-3p inhibits invasion of ESCC cells in vitro by down-regulating E2F5, which may involve the MAPK/ERK signaling pathway. ( A and B ) The invasion of KYSE150 and Eca109 cells in vitro co-transfected with miR-1-3p mimics and E2F5 expression plasmids. ( C ) The protein levels of p-p38, p38, p-ERK, ERK in transfected cells, detected with western blot. ns, no significance; *** P < 0.001

Article Snippet: Antibodies information used in this study is provided below: anti-TSG101 (1:2000, 14497-1-AP, Proteintech), anti-CD81 (66866-1-Ig), anti-CD9 (60232-1-Ig), anti-E2F5 (1:2000, bs-1734R, Bioss), anti-GAPDH (1:5000, 60004-1-Ig, Proteintech), anti-p-p38 (1:1000, 4511, CST), anti-p38 (1:5000, 66234-1-lg, Proteintech), anti-p-ERK (1:1000, AP0974, ABclonal), anti-ERK (1:700, A4782, ABclonal), anti-p-Akt (1:2000, 28731-1-AP, Proteintech), anti-p-mTOR (1:500, AF3308, Affinity), HRP Goat anti-Rabbit IgG (1: 5000, AS014, ABclonal), HRP Goat anti-Mouse IgG (1: 5000, AS003, ABclonal).

Techniques: In Vitro, Transfection, Expressing, Western Blot

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: Translational Andrology and Urology

Article Title: Fu Zheng Xiao Yu San Jie Decoction affects the proliferation of renal cell carcinoma via regulating E2F5 gene

doi: 10.21037/tau-2025-aw-739

Figure Lengend Snippet: Potential target genes of FZXYJD in E2F family were predicted. (A) E2F5 has low expression in ccRCC. (B) Patients had poorer survival with low expression of E2F5 in ccRCC. (C-F) FZXYJD can increase the expression level of E2F5 in ccRCC cell line. *, P<0.05; **, P<0.01; ***, P<0.001. Control, cultured in SD rat serum. LD, cultured in FZXYJD lower dose group serum. MD, cultured in FZXYJD medium dose group serum. HD, cultured in FZXYJD high-dose group serum. ccRCC, clear cell renal cell carcinoma; FZXYSJD, Fu Zheng Xiao Yu San Jie Decoction; HD, high-dose; HR, hazard ratio; KIRC, kidney renal clear cell carcinoma; LD, low-dose; MD, medium-dose; N, normal; SD, Sprague-Dawley; T, tumor; TPM, transcripts per million.

Article Snippet: It was then incubated overnight at 4 °C with the primary antibodies: E2F5 (1:1,000, Catalog: D197169, BBI, Shanghai, China) and β-Actin (1:1,000, Catalog: ab8227, Abcam, Shanghai, China).

Techniques: Expressing, Control, Cell Culture

Journal: Translational Andrology and Urology

Article Title: Fu Zheng Xiao Yu San Jie Decoction affects the proliferation of renal cell carcinoma via regulating E2F5 gene

doi: 10.21037/tau-2025-aw-739

Figure Lengend Snippet: E2F5 was involved in FZXYJD suppression of ccRCC cells proliferation. (A) Western blot and RT-qPCR were performed to analyze E2F5 expression in si E2F5 -transfected ccRCC cell line. β-actin was used as an internal quantitative control. (B) CCK-8 assay. (C) Cell colony formation assay [IPHONE X PRO MAX] (magnification: 1×; crystal violet stained). *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; ccRCC, clear cell renal cell carcinoma; FZXYSJD, Fu Zheng Xiao Yu San Jie Decoction; NC, negative control; OD, optical density; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; si E2F5 , small interfering RNA targeting E2F5 .

Article Snippet: It was then incubated overnight at 4 °C with the primary antibodies: E2F5 (1:1,000, Catalog: D197169, BBI, Shanghai, China) and β-Actin (1:1,000, Catalog: ab8227, Abcam, Shanghai, China).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Transfection, Control, CCK-8 Assay, Colony Assay, Staining, Cell Counting, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Translational Andrology and Urology

Article Title: Fu Zheng Xiao Yu San Jie Decoction affects the proliferation of renal cell carcinoma via regulating E2F5 gene

doi: 10.21037/tau-2025-aw-739

Figure Lengend Snippet: Three-dimensional and two-dimensional docking patterns and interactions of FZXYSJD identified components with the E2F5 protein. FZXYSJD, Fu Zheng Xiao Yu San Jie Decoction.

Article Snippet: It was then incubated overnight at 4 °C with the primary antibodies: E2F5 (1:1,000, Catalog: D197169, BBI, Shanghai, China) and β-Actin (1:1,000, Catalog: ab8227, Abcam, Shanghai, China).

Techniques:

Journal: bioRxiv

Article Title: Transcriptional Regulation of Mammary Alveolar Proliferation and Differentiation during Early Pregnancy

doi: 10.1101/2024.11.27.625731

Figure Lengend Snippet: (A) Normalized expression of E2F5 in whole mammary glands at different stages of development: virgin, pregnancy day 6 (P6), pregnancy day 18 (P18), lactation day 1 (L1), and lactation day 10 (L10). (B) Normalized expression of E2F5 in basal and luminal cells isolated from mammary glands at different stages: virgin, pregnancy day 18.5 (Preg day 18.5), and lactation day 2 (Lac day 2). (C) Venn diagram showing overlap between E2f5 CUT&RUN target genes and differentially expressed genes in early pregnancy (virgin vs. P6). (D) Normalized expression of several up-regulated E2f5 target genes known to be associated with proliferation and differentiation during pregnancy. (E) Volcano plot of differentially expressed genes between virgin and P6, with E2f5 targets labeled.

Article Snippet: For CUT&RUN, rabbit IgG (Epicypher #13-0042; RRID:AB_2923178), rabbit monoclonal anti-H3K27me3 (Invitrogen #MA5-11198; RRID:AB_1100074), rabbit monoclonal anti-H3K4me3 (Epicypher #13-0041; RRID:AB_3076423), and rabbit polyclonal anti-E2F5 (Invitrogen #PA585578; RRID:AB_2792718) were used at 0.5 ug per sample replicate in 50 uL of volume.

Techniques: Expressing, Isolation, Labeling

Journal: bioRxiv

Article Title: Transcriptional Regulation of Mammary Alveolar Proliferation and Differentiation during Early Pregnancy

doi: 10.1101/2024.11.27.625731

Figure Lengend Snippet: Paired mammary wholemounts (A) and H&E staining (B) from 12 weeks virgin adult E2F5 CKO, day 5 pregnant E2F5 f/f and day 5 pregnant E2F5 CKO mice shown an impairment on alveoli formation during pregnancy on mice lacking E2F5 in mammary epithelium. (C) PCNA expression in pregnant mice shows reduced proliferative activity in E2F5 CKO mice. (D) Alveoli quantification shown reduced proliferative activity in mice lacking E2F5 in mammary epithelium (p=0.0025).

Article Snippet: For CUT&RUN, rabbit IgG (Epicypher #13-0042; RRID:AB_2923178), rabbit monoclonal anti-H3K27me3 (Invitrogen #MA5-11198; RRID:AB_1100074), rabbit monoclonal anti-H3K4me3 (Epicypher #13-0041; RRID:AB_3076423), and rabbit polyclonal anti-E2F5 (Invitrogen #PA585578; RRID:AB_2792718) were used at 0.5 ug per sample replicate in 50 uL of volume.

Techniques: Staining, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Transcriptional Regulation of Mammary Alveolar Proliferation and Differentiation during Early Pregnancy

doi: 10.1101/2024.11.27.625731

Figure Lengend Snippet: (A) Early pregnancy in E2F5 CKO females undergoing a second gestation shows reduced number of alveoli in comparison with E2F5 f/f group. Upon day 14 of pregnancy, E2F5 CKO females shows non phenotypic differences in comparison with control groups. (B) Matched histological sections shown evidence of early but no late pregnancy defects in E2F5 CKO females.

Article Snippet: For CUT&RUN, rabbit IgG (Epicypher #13-0042; RRID:AB_2923178), rabbit monoclonal anti-H3K27me3 (Invitrogen #MA5-11198; RRID:AB_1100074), rabbit monoclonal anti-H3K4me3 (Epicypher #13-0041; RRID:AB_3076423), and rabbit polyclonal anti-E2F5 (Invitrogen #PA585578; RRID:AB_2792718) were used at 0.5 ug per sample replicate in 50 uL of volume.

Techniques: Comparison, Control

Journal: bioRxiv

Article Title: Transcriptional Regulation of Mammary Alveolar Proliferation and Differentiation during Early Pregnancy

doi: 10.1101/2024.11.27.625731

Figure Lengend Snippet: (A) Principal component analysis (PCA) plot showing clustering of CUT&RUN data based on chromatin occupancy for E2F5, H3K4me3, H3K27me3, and IgG across two replicates each. (B) De novo motif discovery using MEME-ChIP revealed a motif that is distinct from the known E2F consensus motif. (C) Motif similarity analysis using Tomtom identified overlap between the discovered E2F5 motif and the known E2F consensus motif. (D) Integrative Genomics Viewer (IGV) tracks showing E2F5 and H3K4me3 occupancy at the promoters of known cell cycle regulatory genes, including CDKN2A, CDKN2B, CDKN2C, CDK4, and CCND1, supporting the role of E2F5 in regulating genes involved in cellular proliferation.

Article Snippet: For CUT&RUN, rabbit IgG (Epicypher #13-0042; RRID:AB_2923178), rabbit monoclonal anti-H3K27me3 (Invitrogen #MA5-11198; RRID:AB_1100074), rabbit monoclonal anti-H3K4me3 (Epicypher #13-0041; RRID:AB_3076423), and rabbit polyclonal anti-E2F5 (Invitrogen #PA585578; RRID:AB_2792718) were used at 0.5 ug per sample replicate in 50 uL of volume.

Techniques:

Journal: bioRxiv

Article Title: Transcriptional Regulation of Mammary Alveolar Proliferation and Differentiation during Early Pregnancy

doi: 10.1101/2024.11.27.625731

Figure Lengend Snippet: (A) Integrative Genomics Viewer (IGV) tracks showing E2F5, H3K4me3, and IgG CUT&RUN signal at the STAT6 promoter in HC11 mouse mammary epithelial cells. (B) Luciferase reporter assay in 293T cells showing that E2F5 represses STAT6 promoter activity. (C) STAT6 promoter-luciferase reporter assay of MCF7 cells treated with vehicle or R5020 and co-transfected with CMV-HA-E2F5 or CMV-Neo-Bam (empty vector).

Article Snippet: For CUT&RUN, rabbit IgG (Epicypher #13-0042; RRID:AB_2923178), rabbit monoclonal anti-H3K27me3 (Invitrogen #MA5-11198; RRID:AB_1100074), rabbit monoclonal anti-H3K4me3 (Epicypher #13-0041; RRID:AB_3076423), and rabbit polyclonal anti-E2F5 (Invitrogen #PA585578; RRID:AB_2792718) were used at 0.5 ug per sample replicate in 50 uL of volume.

Techniques: Luciferase, Reporter Assay, Activity Assay, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Transcriptional Regulation of Mammary Alveolar Proliferation and Differentiation during Early Pregnancy

doi: 10.1101/2024.11.27.625731

Figure Lengend Snippet: In the absence of progesterone signaling, E2F5 binds to the STAT6 promoter and represses its transcription (left panel). Upon activation of PR, E2F5-mediated repression is relieved, leading to transcriptional activation of STAT6. This could occur either by displacement of E2F5 from the STAT6 promoter or through cooperation between E2F5 and activated PR at the STAT6 promoter (right panel).

Article Snippet: For CUT&RUN, rabbit IgG (Epicypher #13-0042; RRID:AB_2923178), rabbit monoclonal anti-H3K27me3 (Invitrogen #MA5-11198; RRID:AB_1100074), rabbit monoclonal anti-H3K4me3 (Epicypher #13-0041; RRID:AB_3076423), and rabbit polyclonal anti-E2F5 (Invitrogen #PA585578; RRID:AB_2792718) were used at 0.5 ug per sample replicate in 50 uL of volume.

Techniques: Activation Assay

Journal: Oncogene

Article Title: Insight into mammary gland development and tumor progression in an E2F5 conditional knockout mouse model

doi: 10.1038/s41388-024-03172-4

Figure Lengend Snippet: Using a mouse mammary scRNAseq dataset that was split to various functional stages ( A ) including nulliparous (Null P) pregnancy day 14.5 (Preg D14.5), lactation day 6 (Lact D6) and involution day 11 (Inv D11). For each stage, the level of E2F1 ( B ) and E2F5 ( C ) expression was plotted. Using a separate scRNAseq dataset that was not sorted for epithelial cells ( D ), expression of E2F5 was examined across cell populations in the involuting mammary gland, revealing expression in endothelial cells, fibroblasts and alveoli with elevated signal in red and lower signaling in green ( E ). Examining lineage commitment ( F ), we overlaid E2F1 and E2F5 expression with elevated expression in green. To generate a signature for E2F5 activity, HMECs were infected with increasing multiplicity of infection (MOI) for an adenovirus expressing GFP or E2F5. A western blot demonstrated increasing levels of E2F5 with increasing MOI ( G ). Generation of a signature for E2F5 activation revealed genes up (orange/red) and down (blue) regulated. The activity of the signature was predicted in several human breast cancer cell lines and was plotted against the observed levels in a western blot for E2F5 revealing correlation between the two ( H ). A mammary gland developmental dataset (Stein et al., 2004) was limited to the E2F5 signature genes and was clustered, revealing that genes regulated by E2F5 stratified mammary developmental stages ( I ).

Article Snippet: The following antibodies were used: 1:100 E2F5 (Santa Cruz Biotech sc-374268), 1:1000 Grb2 (Cell Signaling Technology 3976), 1:4000 Vinculin (Cell Signaling Technology 13901), 1:2000 Cyclin D1 (Thermo Scientific 516356.), 1:2000 Cyclin D3 (Thermo Scientific PA5-97551) and 1:1000 Cyclin D2 (Proteintech 10934-1-AP).

Techniques: Functional Assay, Expressing, Activity Assay, Infection, Western Blot, Activation Assay

Journal: Oncogene

Article Title: Insight into mammary gland development and tumor progression in an E2F5 conditional knockout mouse model

doi: 10.1038/s41388-024-03172-4

Figure Lengend Snippet: A gene targeting strategy to flank exons 2 and 3 of E2F5 with loxP sites was employed with genotyping primers shown ( A ). With the introduction of Cre Recombinase under the mammary epithelial specific control of the MMTV promoter/enhancer, exons 2 and 3 are lost resulting in a tissue specific knockout. Examining ductal extension through wholemounts at 4 weeks we examined 20 control mice (E2F5 flox/flox ) ( B ) and 14 E2F5 CKO mice ( C ), revealing a delay in outgrowth. This delay was quantified revealing a consistent outgrowth delay, 0 = 0.0019 ( D ). After mice aged to 12 months, virgin mammary glands were assessed by both wholemount and histology. Relative to the MMTV-Cre controls ( E ) with their somewhat spiked ductal appearance, the E2F5 CKO mammary glands resembled a lactating mammary gland with alveoli engulfing the entire fat pad ( F ).

Article Snippet: The following antibodies were used: 1:100 E2F5 (Santa Cruz Biotech sc-374268), 1:1000 Grb2 (Cell Signaling Technology 3976), 1:4000 Vinculin (Cell Signaling Technology 13901), 1:2000 Cyclin D1 (Thermo Scientific 516356.), 1:2000 Cyclin D3 (Thermo Scientific PA5-97551) and 1:1000 Cyclin D2 (Proteintech 10934-1-AP).

Techniques: Control, Knock-Out

Journal: Oncogene

Article Title: Insight into mammary gland development and tumor progression in an E2F5 conditional knockout mouse model

doi: 10.1038/s41388-024-03172-4

Figure Lengend Snippet: Regular palpation of the mammary glands revealed the onset of tumor formation in E2F5 CKO virgin and multiparous mice, but not in the control MMTV-Cre line ( A ). The resulting tumors were histologically diverse with numerous patterns noted, including squamous ( B ), solid with collagen tracks ( C ), papillary ( D ) and microacinar ( E ). 20-micron scale bars are included. The tumors were also metastatic, a pulmonary section reveals numerous metastatic lesions at both low and high power ( F ), this includes adenosquamous (left panel), normal lung (center panel) and EMT (right panel). Applying the E2F5 signature to human breast cancer stratified the patients to high/low quartiles. These results were consistent with the mouse model as low E2F5 activity was associated with worse survival ( G ).

Article Snippet: The following antibodies were used: 1:100 E2F5 (Santa Cruz Biotech sc-374268), 1:1000 Grb2 (Cell Signaling Technology 3976), 1:4000 Vinculin (Cell Signaling Technology 13901), 1:2000 Cyclin D1 (Thermo Scientific 516356.), 1:2000 Cyclin D3 (Thermo Scientific PA5-97551) and 1:1000 Cyclin D2 (Proteintech 10934-1-AP).

Techniques: Control, Activity Assay

Journal: Oncogene

Article Title: Insight into mammary gland development and tumor progression in an E2F5 conditional knockout mouse model

doi: 10.1038/s41388-024-03172-4

Figure Lengend Snippet: Whole genome sequencing on five E2F5 CKO tumors were generated. Analysis of WGS data revealed CNVs, SNVs and translocation for each tumor. An example of a Circos plot generated for a single tumor provides a bird eye view of the genetic alteration identified ( A ). Starting from the outermost region of each plot, an ideogram with labelled mouse chromosomes is first, followed by SNVs at the next innermost ring. SNVs were marked as low (yellow), moderate (orange), and high (red) predicted impact as defined by Mutect2 annotation. The next innermost ring contains all predicted CNVs as defined by the consensus of Delly and Lumpy; deletions are colored blue and duplications are colored red. Within each tumor, the height of CNVs are scaled relative to the CNV with the largest amount of evidence supporting it as determined by Lumpy annotation (e.g. a duplication with 40 pieces of evidence will only be half the height of a deletion with 80 pieces of evidence). The width of each CNV is determined by the start and stop positions determined by the consensus of Delly and Lumpy on the length of the genome. Duplications point outward while deletions point inward starting from a shared midpoint. The innermost ring contains predicted high impact translocations and high to moderate impact inversions by the consensus of Delly and Lumpy calls. Inversions are colored black. Translocation color matches the ideogram color of one of the two chromosomes involved in the event. For all CNV and SNVs found in at least 3 tumors an oncoprint style plot was generated with SNV/CNV per tumor shown above and the SNV/CNV per gene shown at right. For each tumor sample in each column, only the high confidence calls are presented ( B ). Copy number alterations found in the tumors are illustrated with each column representing a chromosome location ( C ). Red indicates amplification while blue indicates deletion. A closer examination at the CNVs located on Chromosome 6 revealed some shared events between the tumors ( D ). The boxed region in ( D ) is expanded for the three tumors containing an amplification at that point in panel E. The COSMIC SBS mutation signatures enriched among the tumors were identified and are presented in panel F.

Article Snippet: The following antibodies were used: 1:100 E2F5 (Santa Cruz Biotech sc-374268), 1:1000 Grb2 (Cell Signaling Technology 3976), 1:4000 Vinculin (Cell Signaling Technology 13901), 1:2000 Cyclin D1 (Thermo Scientific 516356.), 1:2000 Cyclin D3 (Thermo Scientific PA5-97551) and 1:1000 Cyclin D2 (Proteintech 10934-1-AP).

Techniques: Sequencing, Generated, Translocation Assay, Amplification, Mutagenesis

Journal: Oncogene

Article Title: Insight into mammary gland development and tumor progression in an E2F5 conditional knockout mouse model

doi: 10.1038/s41388-024-03172-4

Figure Lengend Snippet: Analysis of differentially upregulated genes in E2F5KO using EnRichR on whole mammary glands lacking E2F5 relative to E2F5 CKO tumors revealed putative targets ( A ) and biological pathways ( B ). Further analysis with StringDb revealed the predicted interactions between E2F5 and the differentially upregulated genes ( C ). Using a filtering strategy to determine genes involved with E2F5 CKO tumor formation, we identified Cyclin D1. Comparison of mammary gland expression of cyclin D1 through RNAseq revealed an upregulation in whole mammary glands, tumors and more extensively in the cell lines derived from the tumors ( D ). Validation of these results through qRT-PCR for cyclin D1 in 3 MMTV-Cre and 3 E2F5 CKO whole mammary glands revealed a 15 fold upregulation of cyclin D1 in the pre-tumor mammary glands ( t -test P -value 0.077) ( E ). Examination of Wnt1, Neu and E2F5 CKO tumors revealed that each strain had elevated Cyclin D1 but only Wnt1 tumors had upregulation of Cyclin D2 ( F ). Indeed, the E2F5 CKO tumors closely resembled the MMTV-Neu tumors with elevated Cyclin D1 and lower levels of both Cyclin D2 and D3 (quantification in Supplemental Table ).

Article Snippet: The following antibodies were used: 1:100 E2F5 (Santa Cruz Biotech sc-374268), 1:1000 Grb2 (Cell Signaling Technology 3976), 1:4000 Vinculin (Cell Signaling Technology 13901), 1:2000 Cyclin D1 (Thermo Scientific 516356.), 1:2000 Cyclin D3 (Thermo Scientific PA5-97551) and 1:1000 Cyclin D2 (Proteintech 10934-1-AP).

Techniques: Comparison, Expressing, Derivative Assay, Biomarker Discovery, Quantitative RT-PCR

Journal: Oncogene

Article Title: Insight into mammary gland development and tumor progression in an E2F5 conditional knockout mouse model

doi: 10.1038/s41388-024-03172-4

Figure Lengend Snippet: Implantation of E2F5 CKO tumors into FVB MMTV-Cre recipients resulted in mammary and axillary tumor formation. The strategy for serial transplantation of E2F5 CKO axillary tumors into the abdominal mammary gland to enrichlymphatic metastasis is shown ( A ). In an example of an enrichment necropsy, the primary tumor has been excised (abdominal gland, bottom of panel) but a tumor has also formed in the region of the axial lymph node (top) ( B ) Labels include ALNT (axial lymph node tumor), LV (enlarged lymphatic vessel) and the remnants of the excised primary tumor (PT) in the abdominal gland. Cross section of the lymph node reveals both lymph (left side) tissue and metastatic (right side) cells ( C ). Staining with a pan-cytokeratin antibody reveals nests of metastatic cells throughout the lymph node ( D ). Cross section and staining of the enlarged vessel from ( B ) for podoplanin reveals a positively staining lymphatic vessel containing counterstained tumor cells ( E ). A summary for four transplanted lines shows the number of rounds of transplantation and where the optimal lymph node enrichment point was observed with the percent of tumor bearing mice with lymph node metastasis included for each ( F ). Examining human breast cancer with known lymph node status for the E2F5 activation gene signature revealed that metastatic tumors had lower E2F5 activity ( G ). Splitting the samples into quartiles, the samples with the lowest levels of E2F5 were most likely to have lymph node metastasis ( P < 0.0001, Fisher test) ( H ).

Article Snippet: The following antibodies were used: 1:100 E2F5 (Santa Cruz Biotech sc-374268), 1:1000 Grb2 (Cell Signaling Technology 3976), 1:4000 Vinculin (Cell Signaling Technology 13901), 1:2000 Cyclin D1 (Thermo Scientific 516356.), 1:2000 Cyclin D3 (Thermo Scientific PA5-97551) and 1:1000 Cyclin D2 (Proteintech 10934-1-AP).

Techniques: Transplantation Assay, Staining, Activation Assay, Activity Assay