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: 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: Journal of Cerebral Blood Flow & Metabolism

Article Title: Sonic hedgehog is expressed in human brain arteriovenous malformations and induces arteriovenous malformations in vivo

doi: 10.1177/0271678x20912405

Figure Lengend Snippet: Figure 1. Immunohistochemical analysis of Shh pathway expression in human brain AVMs. Representative images of human brain AVMs and control brain vessels. Cells in the intimal layer of AVMs are strongly immunopositive for Shh and Gli1. COUP-TFII immunopositive cells are detectable in the intimal and medial layers of AVMs. Control brain vessels are negative in terms of Shh, Gli1, and COUP-TFII expression. Shh: Sonic hedgehog.

Article Snippet: For the detection of COUP-TFII expression in brain vasculature, sections were incubated with lectin from Lycopersicon esculentum (tomato) biotin conjugate (1:500) (SigmaAldrich, St. Louis, MO, USA) together with a rabbit polyclonal anti-COUP-TFII antibody (Origene, Rockville, MD, USA).

Techniques: Immunohistochemical staining, Expressing, Control

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Sonic hedgehog is expressed in human brain arteriovenous malformations and induces arteriovenous malformations in vivo

doi: 10.1177/0271678x20912405

Figure Lengend Snippet: Figure 7. Expression of COUP-TFII in phShh-induced brain neovessels. Sections of brain hemispheres injected with phShh and empty plasmid were stained for DAPI (blue staining) to identify cell nuclei, lectin (red staining) to identify blood vessels, and COUP- TFII (green staining) to identify cells expressing this vascular differentiation marker. (a) COUP-TFII-positive cells were found in the neovessels grown in the brain in response to phShh injection, both at the level of the intimal layer and the vascular wall. (b) COUP- TFII-positive cells were virtually absent in brain hemispheres injected with the empty plasmid.

Article Snippet: For the detection of COUP-TFII expression in brain vasculature, sections were incubated with lectin from Lycopersicon esculentum (tomato) biotin conjugate (1:500) (SigmaAldrich, St. Louis, MO, USA) together with a rabbit polyclonal anti-COUP-TFII antibody (Origene, Rockville, MD, USA).

Techniques: Expressing, Injection, Plasmid Preparation, Staining, Marker