anti e2f5  (Santa Cruz Biotechnology)

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