Journal: Cancer research

Article Title: NR2F1 Is a Barrier to Dissemination of Early-Stage Breast Cancer Cells.

doi: 10.1158/0008-5472.CAN-21-4145

Figure Lengend Snippet: Figure 1. NR2F1 is downstream of p38 and HER2 signaling in ECCs. A. QPCR and percentage of cells/duct with nuclear NR2F1 shown by IHC from mammary glands of MMTV-HER2 females treated with SB203580. N=2 mice/condition. B. Percentage of HER2 positive+ cells per field of view (FOV) that has either NR2F1HIGH

Article Snippet: Acini were also transduced with shRNA against mouse Nr2f1 gene (Origene #TL516691C and #TL516691D), or controlshRNA (Origene #TR30021, non-effective scrambled shRNA).

Techniques:

Journal: Cancer research

Article Title: NR2F1 Is a Barrier to Dissemination of Early-Stage Breast Cancer Cells.

doi: 10.1158/0008-5472.CAN-21-4145

Figure Lengend Snippet: Figure 2. NR2F1 inhibits MMTV-HER2 ECCs invasion. A. Percentage of invading cells with high/low NR2F1 reporter activity in acini by IF. Arrows show invading cells. B. Schematic of invasive protrusions and length-over-width (L/W) ratio. C&D. Time lapse imaging of siControl (C) or siNR2F1 (D) acini. E. L/W ratio of protrusions in siControl (black) or siNr2f1 (blue) acini. F. Percentage of invasive and non-invasive protrusions in siControl and siNr2f1 in acini. N=2. Fisher’s exact test. G. Migration assay for NR2F1-overexpressing or empty vector control MMTV-HER2 ECCs. N=2. H. Schematic of intravital imaging using mammary gland windows (Adapted[19]). I&J. Percentage of acinar structures with outward invasion (J) by intravital imaging (I) in mice orthotopically injected with shControl or shNr2f1 MMTV-HER2 ECCs. N=3-4mice/

Article Snippet: Acini were also transduced with shRNA against mouse Nr2f1 gene (Origene #TL516691C and #TL516691D), or controlshRNA (Origene #TR30021, non-effective scrambled shRNA).

Techniques: Activity Assay, Imaging, Migration, Plasmid Preparation, Control, Injection

Journal: Cancer research

Article Title: NR2F1 Is a Barrier to Dissemination of Early-Stage Breast Cancer Cells.

doi: 10.1158/0008-5472.CAN-21-4145

Figure Lengend Snippet: Figure 3. NR2F1 depletion allows a partial EMT and hybrid luminal/basal program in MMTV- HER2 ECC acini. A. QPCR for the indicated genes in siControl vs. siNr2f1 MMTV-HER2 ECC acini. N=3-9. B. Percentage of E-cadherin+ acini by IF. N=3. C. Percentage of TWIST1+ cells per acinus. N=2. D. Percentage of PRRX1+ acini by IF. N=2. E. IF staining for NR2F1 (green), PRRX1 (red) and panCK (gray) in HER2− (n=6), HER2+ (n=7) and benign adjacent (n=6) DCIS samples. Representative images for PRRX1 levels are shown (left). Representative cell with NR2F1LOW/PRRX1HIGH signature is shown (middle). Fisher’s exact test. F. Percentage of β-catenin+ MMTV-HER2 ECC acini by IF after transfection with siRNA. N=2. G. Percentage of β-catenin+ acini after transfection with siNr2f1#2 and treatment with rDKK1.

Article Snippet: Acini were also transduced with shRNA against mouse Nr2f1 gene (Origene #TL516691C and #TL516691D), or controlshRNA (Origene #TR30021, non-effective scrambled shRNA).

Techniques: Staining, Transfection

Journal: Cancer research

Article Title: NR2F1 Is a Barrier to Dissemination of Early-Stage Breast Cancer Cells.

doi: 10.1158/0008-5472.CAN-21-4145

Figure Lengend Snippet: Figure 4. NR2F1 inhibits systemic dissemination of MMTV-HER2 ECCs. A. Tumor volume measurements in nude mice orthotopically injected with shControl and shNr2f1 MMTV-HER2 ECCs or MMTV-HER2 tumor-derived (PT) cells. B. Percentage of PH3+ cells/acinus in mammary glands orthotopically injected with shControl or shNr2f1 MMTV-HER2 ECCs 10-16 day post-injections. N=2 mice/group. C. Experimental design for detection of eDCCs in nude mice 10-16 days after ortothopic injection with shControl and shNr2f1 MMTV-HER2 ECCs. N=4mice/group. D. Number of eDCCs (HER2+) per lung area as described in C. Number of DCCs= 4,399 shControl; 4,305 shNr2f1. Representative image shown, scale bar=5 μm. E. Percentage of PH3+/HER2+ lung eDCCs as described in C. Number of eDCCs= 4,399 shControl; 4,305 shNr2f1. Image depicts a

Article Snippet: Acini were also transduced with shRNA against mouse Nr2f1 gene (Origene #TL516691C and #TL516691D), or controlshRNA (Origene #TR30021, non-effective scrambled shRNA).

Techniques: Injection, Derivative Assay

Journal: Cancer research

Article Title: NR2F1 Is a Barrier to Dissemination of Early-Stage Breast Cancer Cells.

doi: 10.1158/0008-5472.CAN-21-4145

Figure Lengend Snippet: Figure 5. Graphical summary. Our data suggest that p38 inactivity reduces NR2F1 levels in normal mammary epithelial cells (left) and MMTV-HER2 ECCs (right). In the latter scenario, NR2F1 downregulation led to a partial EMT, a shift β-catenin localization from membrane to nuclei and a hybrid luminal/basal phenotype allowing dissemination of ECCs to distant organs. Thus, NR2F1 acts as a barrier for early dissemination and its loss in early lesions could indicate the presence of early disseminated cancer cells (eDCCs). Image by Jill Gregory. Used with permission of ©Mount Sinai Health System.

Article Snippet: Acini were also transduced with shRNA against mouse Nr2f1 gene (Origene #TL516691C and #TL516691D), or controlshRNA (Origene #TR30021, non-effective scrambled shRNA).

Techniques: Membrane

Journal: Biochemistry

Article Title: An open library of human kinase domain constructs for automated bacterial expression

doi: 10.1021/acs.biochem.7b01081

Figure Lengend Snippet: Kinase domain constructs with yields >2 μ g/mL culture for 96-kinase expression screen. Kinases are listed by Uniprot designation and whether they were co-expressed with Lambda or truncated YopH164 phosphatase. Yield (determined by Caliper GX II quantitation of the expected size band) reported in μ g/mL culture, where total eluate volume was 120 μ L from 900 μ L bacterial culture. Yields are shaded green (yield > 12 μ g/mL), yellow (12 > yield > 7 μ g/mL) and orange (yield <7 μ g/mL); kinase domain constructs with yields that were undetectable or < 2 μ g/mL are not listed. ‡ denotes that the second kinase domain of KS6A1_HUMAN was expressed; all other kinases were the first or only kinase domain occurring in the ORF. Construct boundaries are listed in UniProt residue numbering for the UniProt canonical isoform. An interactive table of expression yields and corresponding constructs is available at http://choderalab.org/kinome-expression

Article Snippet: MK03_HUMAN , 1–379 , Addgene 23509 , Lambda , 36.4.

Techniques: Construct, Expressing, Quantitation Assay, Residue, Plasmid Preparation

Journal: PLoS Genetics

Article Title: The Gene Desert Mammary Carcinoma Susceptibility Locus Mcs1a Regulates Nr2f1 Modifying Mammary Epithelial Cell Differentiation and Proliferation

doi: 10.1371/journal.pgen.1003549

Figure Lengend Snippet: A) Schematic representation of the rat Mcs1a locus and mouse orthologous locus that was deleted using mutagenic insertion and chromosome engineering resource (MICER) vector-assisted targeting. The horizontal bar, genetic markers and chromosomal base pair positions in purple represent the location of the Mcs1a critical interval on rat chromosome 2 (UCSC Genome Browser, rn4). The marker and base pair position in light purple delineate the location of the distal end of the Mcs1a critical interval at the time of design of the MICER project. The MD on mouse chromosome 13 is depicted as a red horizontal bar. The coordinates (in bp) are from the 2007 version of the mouse genome (UCSC Genome Browser, mm9). At the indicated base pair positions, a compatible pair of MICER vectors was placed, such that upon Cre-recombinase-driven excision in the embryonic stem-cell stage, a functional Hypoxanthine-guanine phosphoribosyltransferase ( HPRT ) gene was formed, which allowed for selection of a correctly targeted ES cell clone. Construct MHPP256h04 contained Agouti ( A ), HPRT exons 3–9, loxP (grey triangle) and a Puromycin resistance gene ( puro ); Construct MHPN5k06 contained a Neomycin resistance gene ( neo ), loxP (grey triangle), HPRT exons 1 and 2, and Tyrosinase ( TYR ). B) Graph showing the delayed eye-opening phenotype in MD mice (n = 83; FVB) as compared with WT littermates (n = 88; FVB). Plotted are on the vertical axis the percentage of mice in each genotype group with both eyes completely open and on the horizontal axis age in days. C) RNA-seq analysis of mammary gland gene expression from MD and WT control mice (n = 4 each; FVB). The transcript levels are shown as the average (+/− sem) counts from the RSEM algorithm output (used to estimate transcript levels ), normalized to the average of the WT group. Shown are transcripts within 2.5 Mb surrounding the gene desert. Only the transcript level of the orphan nuclear receptor gene Nr2f1 was significantly different between MD and WT mice (P<0.05, indicated by an asterisk). D) Quantitative real-time PCR (Q-PCR) analysis verifying the differential transcript level of Nr2f1 in mammary gland from MD and WT control mice (n = 12 each; FVB). The Nr2f1 transcript level is shown relative to the transcript level of the ActB endogenous control gene.

Article Snippet: TaqMan quantitative PCR primers and probes were ordered as premade assays (ABI/Applied Biosystems): rat Nr2f1 Rn01489978_m1 (FAM), mouse Nr2f1 Mm01354342_m1 (FAM), human NR2F1 Hs00818842_m1 (FAM), rat ActB Rn00667869_m1 (VIC, endogenous control), mouse ActB Mm00607939_s1 (VIC, endogenous control) and human GAPDH Hs03929097_g1(VIC, endogenous control).

Techniques: Plasmid Preparation, Marker, Functional Assay, Selection, Construct, RNA Sequencing, Gene Expression, Control, Real-time Polymerase Chain Reaction

Journal: PLoS Genetics

Article Title: The Gene Desert Mammary Carcinoma Susceptibility Locus Mcs1a Regulates Nr2f1 Modifying Mammary Epithelial Cell Differentiation and Proliferation

doi: 10.1371/journal.pgen.1003549

Figure Lengend Snippet: A–C) Q-PCR analysis of Nr2f1 transcript levels in mammary gland (MG; panel A), rat mammary epithelial cell (RMEC, panel B) and DMBA- or MNU-induced mammary carcinoma (carc.; panel C) samples from resistant congenic (res.; n = 54 panel A, n = 18 panel B, n = 12 each panel C) and WF.Cop susceptible congenic control (susc.; n = 19 panel A, n = 11 panel B, n = 6 each panel C) rat lines. Data derived from both the W4 and W5 congenic lines are used in the Mcs1a resistant congenic data points. Nr2f1 transcript levels are shown relative to the transcript level of the ActB endogenous control gene. D) Chromosome conformation capture (3C) assay for the Nr2f1 promoter and the Mcs1a critical interval. The region is shown as a UCSC Genome Browser view (version rn4 of rat genome) and the location of the Mcs1a critical interval in indicated as a horizontal black line. The evolutionary sequence conservation track is also shown. The locations of the 3C assay primers are shown as vertical purple lines. The fixed primer in the Nr2f1 promoter is shown with respect to Bgl II restriction sites in the Nr2f1 gene span. Graphed is the average relative interaction frequency (+/− sem) of the Bgl II fragment in the Nr2f1 promoter containing the fixed primer with each of the Bgl II fragments in Mcs1a containing the 3C assay primers (n = 4 or more templates). Significantly increased relative interaction frequency is indicated with 1 asterisk for a background cut-off interaction frequency of 0.05 and 2 asterisks for a background cut-off interaction frequency of 0.1. The horizontal axis indicates the genomic distance (in Kb) from the 3C assay primers in Mcs1a to the fixed primer in the Nr2f1 promoter. The main peak in the interaction profile coincides with blocks of strong evolutionary sequence conservation (to zebrafish and frog, X. tropicalis ). Sequence variation within the interacting Mcs1a region is outlined in . E) Schematic drawing of the higher-order chromatin interaction of Mcs1a with the Nr2f1 promoter. The Mcs1a critical interval is indicated as a thick area in the black line that represents the DNA. The green, orange and red shapes represent the putative DNA-binding proteins involved in the structure.

Article Snippet: TaqMan quantitative PCR primers and probes were ordered as premade assays (ABI/Applied Biosystems): rat Nr2f1 Rn01489978_m1 (FAM), mouse Nr2f1 Mm01354342_m1 (FAM), human NR2F1 Hs00818842_m1 (FAM), rat ActB Rn00667869_m1 (VIC, endogenous control), mouse ActB Mm00607939_s1 (VIC, endogenous control) and human GAPDH Hs03929097_g1(VIC, endogenous control).

Techniques: Control, Derivative Assay, Sequencing, DNA Binding Assay

Journal: PLoS Genetics

Article Title: The Gene Desert Mammary Carcinoma Susceptibility Locus Mcs1a Regulates Nr2f1 Modifying Mammary Epithelial Cell Differentiation and Proliferation

doi: 10.1371/journal.pgen.1003549

Figure Lengend Snippet: A) Heatmap of expression correlation clustering analysis of 412 genes (which have 1-1-1 mouse-rat-human orthologues) that are differentially expressed between mammary gland samples from megadeletion (MD) and wild type mice (WT), both FVB. B) Heatmap of expression correlation clustering analysis of the same 412 genes in 243 human breast cancers from GSE3494, downloaded from the Gene Expression Omnibus. For both panels, strong correlation is indicated in blue, strong anti-correlation is indicated in yellow. The position of Nr2f1/NR2F1 in group 1 is indicated by a dark blue vertical line. Below the panels, a summary of the gene ontology enrichment analysis is given. Smaller print indicates weak enrichment, larger print indicates strong enrichment ( , ).

Article Snippet: TaqMan quantitative PCR primers and probes were ordered as premade assays (ABI/Applied Biosystems): rat Nr2f1 Rn01489978_m1 (FAM), mouse Nr2f1 Mm01354342_m1 (FAM), human NR2F1 Hs00818842_m1 (FAM), rat ActB Rn00667869_m1 (VIC, endogenous control), mouse ActB Mm00607939_s1 (VIC, endogenous control) and human GAPDH Hs03929097_g1(VIC, endogenous control).

Techniques: Expressing, Gene Expression

Journal: PLoS Genetics

Article Title: The Gene Desert Mammary Carcinoma Susceptibility Locus Mcs1a Regulates Nr2f1 Modifying Mammary Epithelial Cell Differentiation and Proliferation

doi: 10.1371/journal.pgen.1003549

Figure Lengend Snippet: A) Average (+/− sem) of the normalized median NR2F1 probe intensities, obtained from a total of 12 breast cancer expression studies (with 120+ samples each study) available through Oncomine. G = Histological grade, ER = Estrogen receptor, PR = Progestrone receptor, HER2 = Human epidermal growth factor receptor 2, TN = Triple-negative. B) Average (+/− sem) of the normalized NR2F1 probe intensity in sample groups organized by grade and ER status (from GSE3494; left panel) or by grade and HER2 status (from GSE5460; right panel). Left panel: ER+G1 n = 62, ER+G2 n = 116, ER+G3 n = 33, ER-G2 n = 11, ER-G3 n = 21. Right panel: HER2+G2 n = 7, HER2+G3 n = 23, HER2-G1 n = 27, HER2-G2 n = 24, HER2-G3 n = 46. Significantly different NR2F1 transcript level (P<0.05) is indicated by an asterisk.

Article Snippet: TaqMan quantitative PCR primers and probes were ordered as premade assays (ABI/Applied Biosystems): rat Nr2f1 Rn01489978_m1 (FAM), mouse Nr2f1 Mm01354342_m1 (FAM), human NR2F1 Hs00818842_m1 (FAM), rat ActB Rn00667869_m1 (VIC, endogenous control), mouse ActB Mm00607939_s1 (VIC, endogenous control) and human GAPDH Hs03929097_g1(VIC, endogenous control).

Techniques: Expressing

Journal: PLoS Genetics

Article Title: The Gene Desert Mammary Carcinoma Susceptibility Locus Mcs1a Regulates Nr2f1 Modifying Mammary Epithelial Cell Differentiation and Proliferation

doi: 10.1371/journal.pgen.1003549

Figure Lengend Snippet: The Mcs1a resistance allele displays increased mammary Nr2f1 transcript levels as compared with the susceptible allele. Lower Nr2f1 transcript levels in the mammary gland are associated with susceptibility, a lower percentage of luminal rat mammary epithelial cells (RMEC), a higher percentage of basal RMECs and increased colony-forming ability of the clonogenic luminal RMEC population, indicative of increased proliferation.

Article Snippet: TaqMan quantitative PCR primers and probes were ordered as premade assays (ABI/Applied Biosystems): rat Nr2f1 Rn01489978_m1 (FAM), mouse Nr2f1 Mm01354342_m1 (FAM), human NR2F1 Hs00818842_m1 (FAM), rat ActB Rn00667869_m1 (VIC, endogenous control), mouse ActB Mm00607939_s1 (VIC, endogenous control) and human GAPDH Hs03929097_g1(VIC, endogenous control).

Techniques: