Journal: Cell reports

Article Title: Rbf Activates the Myogenic Transcriptional Program to Promote Skeletal Muscle Differentiation

doi: 10.1016/j.celrep.2018.12.080

Figure Lengend Snippet: (A) Developing DLMs labeled using vgAME-lacZ reporter (yellow bar) and anti-Rbf antibodies from 14 to 33 h APF. Magnified images of the yellow, dashed boxes indicated on the overlay image on the left. (B) Developing DLMs at 29 h APF stained with anti-Rbf antibody, phalloidin, and DAPI in Mef2>mCherry-RNAi and Mef2>Rbf-RNAi as negative control. (C and D) Developing DLMs staged at 24 h APF (C) and 21 h APF (D) stained with phalloidin, DAPI, anti-Dp (C) and anti-E2f2 (D) antibodies. Mef2>Dp-RNAi was used as negative control. (E) Developing DLMs labeled using Mhc>tauGFP, anti-Rbf antibody, phalloidin, and DAPI from 52 to 96 h APF. Genotypes are (A) w-;vgAME-lacZ, (B) w-, UAS-Dicer2; +; Mef2-GAL4/UAS-mCherry-RNAi , and w-, UAS-Dicer2; +; Mef2-GAL4/UAS-Rbf-RNAi , (C) w-;vgAME-lacZ as WT and w-; UAS-Dp-RNAi; Mef2-GAL4, (D) y-w-, and (E) w-; P{Mhc-tauGFP}2 . Scale bars, 20 μm.

Article Snippet: 6G11,1:50, DSHB), mouse 22c10 (1:30, DSHB), rabbit anti-Mef2 (1:1,000) , mouse RBF (DX2, 1:20, , rabbit polyclonal anti-Dp (#212, 1:300) , rabbit anti-E2f2 antibodies (#79), rabbit anti-Cleaved Drosophila Dcp-1 (Asp216, 1:500, Cell Signaling), rat anti-Kettin (MAC155,1:1000, Babraham Institute) and rabbit anti-pH3 (Millipore, 1:200), Rhodamine–Phalloidin or fluorescein isothiocyanate–Phalloidin were used to counterstain and stain thin filaments, and 4,6-diamidino-2-phenylindole (DAPI) for nucleus staining.

Techniques: Labeling, Staining, Negative Control

Journal: Oncotarget

Article Title: Increased metastasis with loss of E2F2 in Myc -driven tumors

doi:

Figure Lengend Snippet: MMTV- Myc transgenic mice were interbred with E2F2 −/− mice and tumor latency was examined. Myc tumors developing in the absence of E2F2 had a significantly increased time to tumor onset ( A. p = 0.0057). Metastasis is rarely observed in MMTV- Myc mice with only 13% of tumor bearing mice having lung metastasis ( B. n = 2/15). Metastatic incidence is increased to 67% when Myc tumors develop in the E2F2 knockout background ( n = 6/9; p -value = 0.0361). Histology of a MMTV- Myc mouse lung showing the absence of lung metastasis at 4X C. compared with the metastases observed in the MMTV- Myc E2F2 null strain D. Increased magnification (20X) of these sections revealed secondary structure within the metastatic lesion E and F. To ensure that the metastatic effect of E2F2 loss was not a strain specific effect, MMTV- Myc WT21 mice were interbred with E2F2 −/− mice. Loss of E2F2 in the MMTV- Myc WT21 background resulted in decreased latency G. and trend towards increased percentage of metastasis bearing mice H. Transplantation of MMTV- Myc WT21 E2F2 −/− tumors into MMTV- Myc WT21 or MMTV- Myc WT21 E2F2 −/− backgrounds produced striking metastases, suggesting that loss of E2F2 affected metastasis in a cell autonomous manner I.

Article Snippet: Primary antibodies for immunoblotting were rabbit anti- E2F2 (clone E-19 Santa Cruz, Dallas, TX, 1:300) or rabbit anti- PTPRD (Abcam, Cambridge, MA, 1:100).

Techniques: Transgenic Assay, Knock-Out, Transplantation Assay, Produced

Journal: Oncotarget

Article Title: Increased metastasis with loss of E2F2 in Myc -driven tumors

doi:

Figure Lengend Snippet: Gene expression analysis of MMTV- Myc tumors in E2F the WT, E2F1 −/− , E2F2 −/− and E2F3 +/− backgrounds and 6 sample of lung metastasis were analyzed by microarray. Unsupervised hierarchical clustering revealed that samples were clustered based on their histological type rather than their genotype with lung metastasis samples being clustered together A. The clustering of the metastatic samples in one group, closely related to the papillary subtype was noted. We examined the indicated sub-clusters of genes for predicted transcription factor binding demonstrating that genes upregulated in metastasis, represented in cluster D, were enriched for E2F binding motifs B. Comparison of EMT and lung metastasis samples by GSEA demonstrated enrichment of genes regulated by SMAD2, SMAD3 , and SMAD4 was elevated in EMT relative to lung metastases ( C. p = 0.03). Enrichment of genes up-regulated in invasive ovarian epithelial cancer was noted in the metastasis samples ( D. p = 0.016).

Article Snippet: Primary antibodies for immunoblotting were rabbit anti- E2F2 (clone E-19 Santa Cruz, Dallas, TX, 1:300) or rabbit anti- PTPRD (Abcam, Cambridge, MA, 1:100).

Techniques: Gene Expression, Microarray, Binding Assay, Comparison

Journal: Oncotarget

Article Title: Increased metastasis with loss of E2F2 in Myc -driven tumors

doi:

Figure Lengend Snippet: To examine the regulatory mechanisms involved in E2F2 -mediated human breast cancer metastasis, we established a pipeline for analysis. Genes that were differentially expressed by MMTV- Myc , MMTV- Myc x E2F2 −/− and lung metastasis samples A–C. were identified. Mouse gene expression data for significantly expressed genes was clustered together with human datasets, revealing 451 candidate genes that are homologous to human genes. We ranked these potential target genes based on their correlation with human distant metastasis free survival, their cox-hazard ratio, and the existence of E2F motifs proximal to the transcriptional start site. Comparison between MMTV- Myc tumors and lung metastases revealed elevated PTPRD expression in the lung metastases samples D. Comparison of MMTV- Myc WT21 non-metastatic tumors and MMTV- Myc WT21 E2F2 −/− metastatic tumors passaged in their respective genotype revealed increased PTPRD expression in the MMTV- Myc WT21 E2F2 −/− tumors ( E. D = donor, R = recipient). Elevated levels of PTPRD were found to be correlated with human distant metastasis free survival ( F. p = 0.0153) and is associated with basal breast cancer subtype ( G. p = 0.0085).

Article Snippet: Primary antibodies for immunoblotting were rabbit anti- E2F2 (clone E-19 Santa Cruz, Dallas, TX, 1:300) or rabbit anti- PTPRD (Abcam, Cambridge, MA, 1:100).

Techniques: Gene Expression, Comparison, Expressing

Journal: Oncotarget

Article Title: Increased metastasis with loss of E2F2 in Myc -driven tumors

doi:

Figure Lengend Snippet: E2F2 knockdown in human breast cancer was achieved by transfection of MDA-MB-231 cells with sh E2F2 . Efficacy of E2F2 knockdown was assayed by western blotting A. with untransfected MDA-MB-231 (Con.), MDA-MB-231 transfected with shScramble (Scr.), sh E2F2 construct #3 (C3), sh E2F2 construct #4 (C4). Migration of MDA-MB-231 control cells B. and with E2F2 knockdown C. in transwell migration assays revealed that the percentage of cells that migrated across the membrane increased when the level of E2F2 was decreased ( D. p < 0.0001). In colonization assays with and without the knockdown, lesions were found in the lungs of mice injected with MDA-MB-231 E. and greatly increased with transfection of sh E2F2 F. Quantification of the numbers of metastatic lesions revealed an increased number of metastatic lesions in mice injected with MDA-MB-231 transfected with sh E2F2 ( G. p = 0.0184).

Article Snippet: Primary antibodies for immunoblotting were rabbit anti- E2F2 (clone E-19 Santa Cruz, Dallas, TX, 1:300) or rabbit anti- PTPRD (Abcam, Cambridge, MA, 1:100).

Techniques: Knockdown, Transfection, Western Blot, Construct, Migration, Control, Membrane, Injection

Journal: Oncotarget

Article Title: Increased metastasis with loss of E2F2 in Myc -driven tumors

doi:

Figure Lengend Snippet: We examined a protein-protein interaction network and found that E2F2 and PTPRD were connected through MYC and STAT3 A. Unsupervised clustering of human breast tumor datasets and mouse tumor dataset revealed a cluster in which lung metastasis samples and human tumors were clustered B. Stratification of human Distant Metastasis Free Survival data by E2F2 pathway probability values showed differential effect of E2F2 loss in human tumors C. p -value < 0.0001).

Article Snippet: Primary antibodies for immunoblotting were rabbit anti- E2F2 (clone E-19 Santa Cruz, Dallas, TX, 1:300) or rabbit anti- PTPRD (Abcam, Cambridge, MA, 1:100).

Techniques: