Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A: Histology of tumor invasive front. H&E staining. Scale bar, 100μm. B: Distribution of NOTCH3-positive fibroblastic cells. C: Distribution of α-SMA-positive fibroblastic cells. Cells stained brown in B and C represent positive cells for each antibody. Arrows in A , B and C indicate blood vessel layer, which is positive internal control of each antibody. D , E , F: Dual immunohistochemical analysis for α-SMA (red) and NOTCH3 (Green). Co-localization of α-SMA and NOTCH3 in fibroblasts was observed in cancer stroma. Scale bar, 50μm. Ca, cancer cells. Dotted lines in A - F indicate the interface of cancer nests and stroma. G: Kaplan-Meier curve for overall survival in relation to NOTCH3 expression in CAFs using 93 human OSCC cases. Log-rank test was used to calculate significance.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Staining, Control, Immunohistochemical staining, Expressing

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: α-SMA and NOTCH3 expression in fibroblasts in the OSCC stroma.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: Clinico-pathological Significance of NOTCH3 expression in CAFs.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A: NHDFs were cultured alone (ctl; control) or cocultured with represented oral and maxillary SCC cell lines. 3 days after coculture, NHDFs were isolated from this coculture by using anti-Fibroblast microbeads for Western blot analysis. B and C: Immunofluorostainig using coculture with HO1-N-1 and NHDFs. Culture of NHDFs alone was used as a control. The bundles of NOTCH3-positive NHDFs (green) were observed around the AE1/AE3-positve HO1-N-1 cancer nests (red) ( B ). Double NOTCH3 and α-SMA-positive NHDFs were intervened between HO1-N-1 cancer nests. Ca, HO1-N-1 cancer nests. Dotted lines demonstrate the interface of HO1-N-1 cancer nests and NHDFs. ( C ). Scale bar, 100μm. The nuclei were counterstained by DAPI. D-G: NHDFs transfected with negative control siRNA (siCtrl) or siRNA for NOTCH3 (siN3) was cultured alone or cocultured with HO1-N-1 cells. 3 days after coculture, NHDFs were isolated from this coculture and subjected to western blot ( D ) and qPCR analyses to measure NOTCH3 ( E ), HEY1 ( F ), α-SMA ( G ). H: NHDFs were cultured alone (control), or directly cocultured with HO1-N-1 cells, or cocultured with transwell-separated HO1-N-1 cells. 3 days after culture, NHDFs were isolated and subjected to qPCR analysis. I: qPCR to measure each NOTCH mRNA expression in NHDFs isolated from coculture with HO1-N-1 cells. ND, not detected. * P < 0.05, ** P < 0.01.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Cell Culture, Control, Isolation, Western Blot, Transfection, Negative Control, Expressing

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A: JAGGED1 expression in various oral cancer cell lines was analyzed by Western blot. B: The effect of NOTCH3 on cell proliferation. The proliferation of HO1-N-1 cells with or without recombinant human NOTCH3-Fc chimera treatment was monitored for 72hr.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Recombinant

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A and B : Comparison of the microvessel density (MVD) between NOTCH3(-) CAFs and NOTCH3(+)CAFs cases. Immunofluorostaining for α-SMA (green), NOTCH3 (green) and CD34 (red) using human tongue OSCC samples. Ca, cancer nests. Dotted lines showed the interface of the cancer nests and stroma. Arrows demonstrated the CD34-positive microvessel. Scale bar, 50μm. The nuclei were counterstained by DAPI ( A ). Quantitative evaluation of MVD between these two groups ( B ). C-F : In vitro angiogenesis assay cocultured with HO1-N-1 cells, HUVECs and siRNA transfected NHDFs. Immunofluorostaining for NOTCH3 (green) and CD31 (red). Ca, HO1-N-1 cancer nests. Dotted lines indicate the margin of the HO1-N-1 cancer nests. Scale bar, 100μm. The nuclei were counterstained by DAPI ( C ). NHDFs isolated from this coculture were subjected to Western blot ( D ). Representative image of tube formation by HUVECs in each condition. Scale bar, 100μm ( E ). Quantitative evaluation of tube formation area (CD31-positive area) in each condition ( F ). siCtrl, negative control siRNA. siN3, siRNA for NOTCH3. ** P < 0.01.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Comparison, In Vitro, Angiogenesis Assay, Transfection, Isolation, Western Blot, Negative Control

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A: NHDFs were cocultured with various cancer cell lines derived from non-oral lesions. 3 days after coculture, NHDFS were isolated from this coculture and subjected to Western blot analysis to detect NOTCH3 and α-SMA. B-D: In vitro angiogenesis assay cocultured with A549, HUVECs and siRNA transfected NHDFs. Representative images of tube formation ( B ) and its quantitative evaluation ( C ) in each condition. Scale bar, 100μm. NHDFs isolated from this angiogenesis assay were subjected to Western blot analysis ( D ). siCtrl, negative control siRNA. siN3, siRNA for NOTCH3. ** P < 0.01.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Derivative Assay, Isolation, Western Blot, In Vitro, Angiogenesis Assay, Transfection, Negative Control

Journal: PLoS ONE

Article Title: NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0154112

Figure Lengend Snippet: A: NHDFs were cocultured with HO1-N-1 for 3 days. Prior to coculture, NHDFs were transfected with negative control siRNA or siRNA for NOTCH3. After 3 days, NHDFs were isolated from this coculture by using anti-fibroblast microbeads. NHDFs were subjected to real-time qPCR analyses to measure Notch3 expression. B-C: These isolated NHDFs were seeded on 24-well plates (2.5×10 5 cells/well) and cocultured with HUVECs (3.0×10 4 cells/well) for 4 days. Representative images of tube formation ( B ) and its quantitative evaluation ( C ) in each condition. Scale bar, 100μm. ctlNHDF(siCtl), negative control siRNA transfected NHDF isolated from culture alone. co-NHDF(siCtl), negative control siRNA transfected NHDF isolated from coculture with HO1-N-1. co-NHDF(siN3), siRNA for NOTCH3 transfected NHDF isolated from coculutre with HO1-N-1. ** P < 0.01.

Article Snippet: To examine the effect of NOTCH3 on OSCC cell line proliferation, HO1-N-1 were seeded (2.0×10 3 cells/well) onto a 96-well plate coated with a recombinant human NOTCH3 Fc chimera (R&D Systems, Minneapolis, MN, USA).

Techniques: Transfection, Negative Control, Isolation, Expressing

Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A) C2C12 cells were engineered to expressed Notch1 receptors lacking the extracellular domain (N1DECD, green). This receptor is inactive in the presence of the γ-secretase inhibitor DAPT (red), but constitutively active when DAPT concentration is reduced in the culture medium. (B) Comparison of transcript levels in C2C12-N1ΔECD cells at 1 hr or 6 hr after DAPT removal. The blue line represents equal expression at 1 hr and 6 hr, and the gray lines represent 5-fold changes in either direction. Circled genes are putative direct Notch targets. The blue circle highlights target genes that are upregulated >5-fold at 1 hr but not 6 hr, while red circles indicate target genes that are upregulated >5-fold only after 6 hr. See also and . (C) qPCR time course measurement of Hes1 (blue), Hey1 (orange), and HeyL (yellow) mRNA levels following complete DAPT removal at t = 0 hr. (D) Duration dependence of Hes1 (blue) and Hey1 (orange) response to DAPT removal for 5 min, 15 min, or 30 min followed by replenishment (“Pulse”), or no replenishment until the 1 hr or 4 hr measurement (“Sustained”). Error bars represent SEM calculated from duplicate experiments (n = 2). See also Figure S4 .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Concentration Assay, Comparison, Expressing

Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A) Both Dll1 (blue) and Dll4 (red) activate the Notch1 receptor (green) to induce proteolytic release of the Notch intracellular domain (NICD), but are used in different biological contexts (blue and red boxes, bottom). The released NICD translocates to the nucleus and, in complex with CSL/RBPjκ (yellow), activates Notch target genes (white). (B) Left: Engineered CHO-K1 “sender” cell lines contain stably integrated constructs expressing Dll1 (blue) or Dll4 (red), each with a co-translational (T2A, brown) H2B-mCh readout (purple), from a 4epi-Tetracycline (4epi-Tc) inducible promoter. Right: “Receiver” cells stably express a chimeric receptor combining the Notch1 extracellular domain (Notch1ECD) with a Gal4 transcription factor (orange), which can activate a stably integrated fluorescent H2B-3xCitrine reporter gene (chartreuse). (C) Left (schematics): A minority of receiver cells (green) are co-cultured with an excess of either Dll1 (blue) or Dll4 (red) sender cells. Right: Filmstrips showing representative sustained (top, Dll4 senders) or pulsatile (bottom, Dll1 senders) response of a single receiver cell (center, automatically segmented nucleus outlined in white). Grey channel shows DIC images of cells, while the rate of increase in Citrine fluorescence, scaled to 25%–75% of its total range, is indicated using green pseudo-coloring. See also and . (D) Left: Representative traces showing total nuclear Citrine fluorescence levels (top) or corresponding derivatives of the total Citrine ( d Citrine/ d t), i.e., promoter activity (bottom), in individual receiver cells activated by Dll4. Right: Average values of total fluorescence (top) and promoter activity (bottom) in receiver cells activated by Dll4. Solid traces represent medians, lighter shades indicate SEM, and gray shading indicates SD. n, number of traces included in the alignment. See for alignment and normalization procedure. (E) Left: Corresponding plots (as in D) showing total nuclear Citrine fluorescence levels (top) and promoter activity (bottom) in individual receiver cells in co-culture with Dll1. Right: Average values of total fluorescence (top) and promoter activity (bottom) in receiver cells activated by Dll1. The percentage value (60%) in the plots on right indicates the fraction of receiver traces included in the alignment ( STAR Methods , see also ). (F) 95 th percentile of (absolute, non-normalized) promoter activity values between 0 and 7.5 hr (after alignment) in the traces included in (D) and (E). This time window is chosen to simultaneously estimate the promoter activity at the peak of Dll1 pulses and at steady-state levels of Dll4 signaling. Solid horizontal lines represent medians, while the boxes delineate 25 th –75 th percentile values. p value calculated by two-sided Kolmogorov-Smirnov (K-S) test. See also Figures S1 and S2 .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Stable Transfection, Construct, Expressing, Cell Culture, Fluorescence, Activity Assay, Co-Culture Assay

Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A) Developing chick embryo (dorsal view schematic). Dll1 (blue cells in 3) is expressed in a fraction of neural crest cells (gray, see 2, 3). These cells activate Notch1-expressing Pax7 + progenitor cells in the dorsomedial lip (DML, magenta) of the somite. When activated, these progenitor cells (green, 3) upregulate Hes1 and the muscle regulatory gene MyoD1. (B–D) Representative images showing effects of Dll1 or Dll4 electroporation into the neural crest, on Hes1, Hey1, and MyoD1 expression in the DML. White arrows indicate the somites on the electroporated side. The dotted lines indicate the DMLs of somites or the central line of the neural tube. (B) Top: Dll1-T2A-EGFP (i, blue), electroporated into the left side of the neural tube, is expressed in the neural tube and neural crest, resulting in upregulation of Hes1 (ii, red) and MyoD1 (iii, green) in the somites on the electroporated (left) side compared to the right side, which serves as negative control. Bottom: When Dll4-T2A-EGFP (iv, blue) is electroporated, Hey1 (v, red) is upregulated on the electroporated side, and MyoD1 (vi, green) expression is decreased. (C) Dll1-T2A-EGFP (blue, left) electroporation does not affect expression of Hey1 (red, right) in adjacent somites. (D) Dll4-T2A-EGFP (blue, left) electroporation increases expression of Hes1 (red, right) in adjacent somites. See also and .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Expressing, Electroporation, Negative Control

Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A and B) Dll4 ECD -Dll1 ICD and Dll1 ECD -Dll4 ICD were constructed by exchanging the intracellular domain (ICD) of Dll4 with that of Dll1. (A) Median response profiles in activated receiver cells co-cultured with Dll4 sender cells (red, top left) or Dll4 ECD -Dll1 ICD sender cells (magenta, right) under excess receiver conditions (as in ). Solid traces represent medians, lighter colored regions represent SEM, and gray shading represents SD. n, number of cell traces included in the alignment. See for alignment and normalization procedures. Bottom left: 95 th percentile of (absolute, non-normalized) promoter activity values between 0 and 7.5 hr (after alignment) in individual traces included in the averaging. Solid horizontal lines represent medians, while the boxes delineate 25 th –75 th percentile values. p value calculated by two-sided K-S test. (B) Corresponding response profiles (right, top left) and amplitudes (bottom left) in activated receiver cells co-cultured with Dll1 sender cells (blue) or Dll1 ECD- Dll4 ICD sender cells (purple) under excess sender conditions. (C) Representative images of “excess sender” co-cultures of receiver cells (R) expressing full-length Notch1 and sender cells (S) expressing either Dll4 ECD -Dll1 ICD (left) or Dll4 (Dll4 ECD -Dll4 ICD , right), immunostained for Notch1ECD. Examples of dispersed, low intensity staining or higher-intensity puncta are indicated by the white circles. (D) Left: Median values of number of puncta detected (see ) in Dll1 ICD (blue) or Dll4 ICD (red) sender cells neighboring receiver cells. Right: Median values of the (background subtracted) mean pixel intensity of dispersed signal (see ) within Dll1 ICD (blue) or Dll4 ICD (red) sender cells that neighbor receiver cells. Error bars represent SEM. p value calculated using the two-sided K-S test. (E) Schematic: Proposed differences in the abilities of ligands containing the Dll1 (blue) and Dll4 (red) ICDs to initiate transendocytosis in different clustering states. See also Figure S6 .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Construct, Cell Culture, Activity Assay, Expressing, Staining