Journal: Neoplasia (New York, N.Y.)

Article Title: Lunatic Fringe and p53 Cooperatively Suppress Mesenchymal Stem-Like Breast Cancer.

doi: 10.1016/j.neo.2017.08.006

Figure Lengend Snippet: Figure 6. Human claudin-low breast cancer exhibits low levels of LFNG and HES1 gene expression. (A) Boxplots for expression values of LFNG, NOTCH3, JAG1, HES1 and HEY1 in 6 subtypes of human breast cancer (dataset GSE18229). (B) Heat map for expressions of selected Notch pathways genes in 6 subtypes of human breast cancer (METABRIC dataset). (C) Boxplots for expression values of LFNG, NOTCH3, JAG1, HES1 and HEY1 in 6 subtypes of human breast cancer (METABRIC dataset). Basal: Basal-like. Claudin: Claudin-low. Her2: HER2-positive. LumA: Luminal A. LumB: Luminal B. Normal: Normal breast-like. p values were calculated by comparing expression means across all subtypes.

Article Snippet: Primary antibodies used for immunostaining were: Notch1 (Cell Signaling, No. 3608, 1:100), Notch2 (DSHB, University of Iowa, C651.6DbHN, 1:200), Notch3 (ProteinTech, 55,114–1-AP, 1:100), E-cadherin (Cell Signaling, No. 3195, 1:100), Vimentin (Cell Signaling, No. 5741, 1:100), Twist (Santa Cruz, sc-6070, 1:100), PDGFRα (Santa Cruz, sc-338, 1:100), Cytokeratin 8 (Santa Cruz, sc-101,459, 1:200), Cytokeratin 14 (Santa Cruz, sc-53,253, 1:200), Ki67 (Abcam, ab16667, 1:200), and Lfng (Abcam, ab192788, 1:100).

Techniques: Gene Expression, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Pharmacogenomics of intracellular methotrexate polyglutamates in patients’ leukemia cells in vivo

doi: 10.1172/JCI140797

Figure Lengend Snippet: (A) Relation between NOTCH2 and FPGS mRNA expression in ALL cells. Each ALL subtype is represented by a different symbol, as depicted in Figure 3. (B) ALL subtype differences in NOTCH2 mRNA expression, ordered from left to right for those with the lowest to the highest MTXPG levels (order, blue and gold as defined in Figure 1B). (C) Relation between NOTCH2 and SLC19A1 mRNA expression in ALL cells. Each ALL subtype is represented by a different symbol, as depicted in the symbol key. (D) Relation of NOTCH2 and PBX1 mRNA expression in primary ALL cells. Each ALL subtype is represented by a different symbol, as depicted in the symbol key. (E) ALL subtype differences in PBX1 mRNA expression. Lines depict the best-fit linear model, Pearson’s correlation, and P values. *P < 0.05, **P < 1 × 10–5, and ***P < 1 × 10–10, by t test.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti-E2A (TCF3) (Cell Signaling Technology; D2B1, 12258), which binds to both endogenous TCF3 and the TCF3-PBX1b fusion protein, diluted 1:1000; rabbit polyclonal anti-FPGS (LifeSpan Biosciences; LS-C680494), diluted 1:1000; rabbit polyclonal anti-SLC19A1 (MilliporeSigma; HPA024802), diluted 1:1000; rabbit monoclonal anti-NOTCH2 (Cell Signaling Technology; D76A6, 5732), diluted 1:1000; and mouse monoclonal anti–β-actin (MilliporeSigma; A5441), diluted 1:100,000.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: Pharmacogenomics of intracellular methotrexate polyglutamates in patients’ leukemia cells in vivo

doi: 10.1172/JCI140797

Figure Lengend Snippet: (A and B) Overexpression of the DOX-inducible TCF3-PBX1 protein in the REH human ALL cell line compared with expression in the control LacZ vector. Overexpression of TCF3-PBX1b was associated with a significant increase in expression of NOTCH2-NICD, a significant decrease in expression of SLC19A1 and FPGS, and lower intracellular accumulation of MTXPGs (MTXPG1–7, MTXPG4–7, MTXPG1–3 after incubation with 1 ng/mL DOX and 1 μM MTX for 24 hours, compared with cells that were not treated with DOX. Each value is the average of 12 samples, compared with the MTXPG concentrations in control cells (value set at 1), expressed as the Δ increase (values >1) or decrease (values <1) compared with controls. P values were calculated by t tests. iLacZ, DOX-inducible LacZ; iTCF3-PBX1B, DOX-inducible TCF3-PBX1B. FL-NOTCH2, full-length NOTCH2. (C) Schema of the hypothetical relation between TCF3-PBX1 expression and NOTCH2, SLC19A1, and FPGS expression.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti-E2A (TCF3) (Cell Signaling Technology; D2B1, 12258), which binds to both endogenous TCF3 and the TCF3-PBX1b fusion protein, diluted 1:1000; rabbit polyclonal anti-FPGS (LifeSpan Biosciences; LS-C680494), diluted 1:1000; rabbit polyclonal anti-SLC19A1 (MilliporeSigma; HPA024802), diluted 1:1000; rabbit monoclonal anti-NOTCH2 (Cell Signaling Technology; D76A6, 5732), diluted 1:1000; and mouse monoclonal anti–β-actin (MilliporeSigma; A5441), diluted 1:100,000.

Techniques: Over Expression, Expressing, Plasmid Preparation, Incubation

Journal: Haematologica

Article Title: FGFR1OP2-FGFR1 induced myeloid leukemia and T-cell lymphoma in a mouse model

doi: 10.3324/haematol.2015.137695

Figure Lengend Snippet: Dysregulation of multiple signaling pathways in myeloid leukemia and T-lymphoma induced by FGFR1OP2-FGFR1. (A) RT-PCR shows that Notch1 and several direct target genes are up-regulated in T-lymphomas from FGFR1OP2-FGFR1 mice. (B) Western blot analysis, using activated Notch1 antibodies (Val 1774), shows that activated Notch1 is present in lymph nodes (LN) isolated from diseased mice. (C) Schematic of the Notch1 gene showing the relative locations of the primers used to analyze the deletion mutants (above). A 5 Kb deletion (brackets) creates a 500-base pair fragment using the P1/P2 primers, as shown in LN from diseased mice (below left). In normal LN and thymus (Thy) cells, the wild-type 11.5-kb fragment cannot be amplified. RT-PCR shows that the alternative Notch1 transcript (exon 1a-exon 4) was only found in tumor samples (above), whereas real-time RT-PCR showed that Notch1 transcription levels for exons 30–31 was higher than that for exons 1–3 (below) in the majority of lymphomas, compared with normal cells (below right). (D) Quantitative RT-PCR analysis shows the comparison of gene expression levels in bone marrow cells (BM) from FGFR1OP2-FGFR1 mice (OP2, n=3) compared with normal BALB/c mice (Nor, n=3). (E) Western blot analysis shows Spi1 protein levels in leukemic mouse spleens compared with normal BALB/c mouse spleens. (F) Schematic summary of differentially expressed genes related to stages of myeloid development. * =P<0.05; ** =P<0.01.

Article Snippet: We further identified activated Notch1 (containing the intracellular domain) using the cleaved Notch1 (Val1744) antibody (Cell Signaling) in the T-lymphomas from serially transplanted mice , indicating progressive accumulation of immature T-lymphoma cells during disease development. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2. caption a7 Dysregulation of multiple signaling pathways in myeloid leukemia and T-lymphoma induced by FGFR1OP2-FGFR1. (A) RT-PCR shows that Notch1 and several direct target genes are up-regulated in T-lymphomas from FGFR1OP2-FGFR1 mice. (B) Western blot analysis, using activated Notch1 antibodies (Val 1774), shows that activated Notch1 is present in lymph nodes (LN) isolated from diseased mice. (C) Schematic of the Notch1 gene showing the relative locations of the primers used to analyze the deletion mutants (above).

Techniques: Protein-Protein interactions, Reverse Transcription Polymerase Chain Reaction, Western Blot, Isolation, Amplification, Quantitative RT-PCR, Comparison, Gene Expression