Journal: Oncology reports

Article Title: Notch1 is a potential therapeutic target for the treatment of human hepatitis B virus X protein-associated hepatocellular carcinoma.

doi: 10.3892/or.2013.2917

Figure Lengend Snippet: Figure 1. Notch1-shRNA suppresses L02/HBx cell proliferation in vitro. (A) Identification of the effective shRNA targeting the Notch1 gene. The relative mRNA and protein levels of Notch1 were assessed by qRT-PCR and western blotting in L02/HBx cells 48 h after transient transfection with Notch1-shRNA1, Notch1-shRNA2 or negative control-shRNA (NC), respectively. (B) The components of the Notch1 signaling pathway were downregulated in the L02/ HBx-Notch1 shRNA2 cells. The mRNA and protein expression levels of Notch1 and Hes1 were assessed by qRT-PCR and western blotting, respectively. Actin was used as a loading control for both quantitative RT-PCR and western blotting. (C) CCK-8 assay and (D) colony formation assay of L02/HBx cells stably transfected with control or Notch1 shRNA2. Data are shown as the mean ± SEM from at least three independent experiments. Statistically significant differences are indicated as *P<0.05, **P<0.01 vs. L02/HBx cells.

Article Snippet: For immunohistochemical analysis, staining for Notch1 was carried out using rabbit polyclonal antibodies against Notch1 (Proteintech Group) at a dilution of 1:50.

Techniques: shRNA, In Vitro, Quantitative RT-PCR, Western Blot, Transfection, Negative Control, Expressing, Control, CCK-8 Assay, Colony Assay, Stable Transfection

Journal: Oncology reports

Article Title: Notch1 is a potential therapeutic target for the treatment of human hepatitis B virus X protein-associated hepatocellular carcinoma.

doi: 10.3892/or.2013.2917

Figure Lengend Snippet: Figure 2. Notch1-shRNAs suppresses L02/HBx cell proliferation in vivo. (A) The growth curve of the tumors derived from L02/HBx cells pretreated with Notch1-shRNA or control-shRNA in nude mice. Data represent means ± SEM of six samples. *P<0.05, **P<0.01. (B) Images of representative mice and dis sected tumors from nude mice. (C) Tumor tissues from nude mice inoculated with NC or Notch1-shRNA cells were stained with hematoxylin and eosin (H&E). Original magnification, x200. (D) Immunohistochemistry of Notch1 expression in tumor tissues from nude mice. Original magnification, x400.

Article Snippet: For immunohistochemical analysis, staining for Notch1 was carried out using rabbit polyclonal antibodies against Notch1 (Proteintech Group) at a dilution of 1:50.

Techniques: In Vivo, Derivative Assay, shRNA, Control, Staining, Immunohistochemistry, Expressing

Journal: Oncology reports

Article Title: Notch1 is a potential therapeutic target for the treatment of human hepatitis B virus X protein-associated hepatocellular carcinoma.

doi: 10.3892/or.2013.2917

Figure Lengend Snippet: Figure 3. Notch1-shRNA induces cell cycle arrest via the CyclinD1/CDK4 pathway in L02/HBx cells. (A) Notch1-shRNA induced cell cycle arrest in L02/ HBx cells. Cell cycle distribution was examined by flow cytometry after staining with PI. Results are visualized as a representative experiment or means ± SEM of three experiments. (B) Notch1-shRNA downregulates the CyclinD1/CDK4 pathway in L02/HBx cells. The expression levels of cell cycle regulatory genes CyclinD1, CDK4, E2F1, Rb, p21 and CyclinE1 were analyzed by qRT-PCR and western blotting. Data represent the mean ± SEM from three independent experiments. Statistically significant differences are indicated as *P<0.05, **P<0.01 vs. L02/HBx cells.

Article Snippet: For immunohistochemical analysis, staining for Notch1 was carried out using rabbit polyclonal antibodies against Notch1 (Proteintech Group) at a dilution of 1:50.

Techniques: shRNA, Cytometry, Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Oncology reports

Article Title: Notch1 is a potential therapeutic target for the treatment of human hepatitis B virus X protein-associated hepatocellular carcinoma.

doi: 10.3892/or.2013.2917

Figure Lengend Snippet: Figure 4. Notch1-shRNA increases cell apoptosis via the caspase-9-caspase-3 pathway in L02/HBx cells. (A) Notch1-shRNA increased cell apoptosis in L02/ HBx cells. Analysis of apoptosis was performed using PE Annexin V and 7-AAD staining by FACS analysis. Results are visualized as a representative experi ment or means ± SEM of three experiments. (B) Notch1-shRNA upregulated the caspase-9-caspase-3 pathway in L02/HBx cells. The expression of caspase-9, -8 and -3 was analyzed by qRT-PCR and western blotting. Data are shown as the mean ± SEM of three independent experiments. Statistically significant differences are indicated as *P<0.05, **P<0.01 vs. L02/HBx cells.

Article Snippet: For immunohistochemical analysis, staining for Notch1 was carried out using rabbit polyclonal antibodies against Notch1 (Proteintech Group) at a dilution of 1:50.

Techniques: shRNA, Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Biochimica et biophysica acta

Article Title: The proteins DLK1 and DLK2 modulate NOTCH1-dependent proliferation and oncogenic potential of human SK-MEL-2 melanoma cells.

doi: 10.1016/j.bbamcr.2014.07.015

Figure Lengend Snippet: Fig. 4. Overexpression of human DLK1 or DLK2 proteins inhibits NOTCH1 activation and signaling in SK-MEL-2 cells. (A) Representative Western blot analysis (left) for active NOTCH1 (active NICD1 ~ 110 kDa) in SK-MEL-2 cells stably transfected with empty vector or plasmids HDLK1S, HDLK2S or HDLK2aS. SK-MEL-2 cells treated with 10 μM DAPT for 24 h were used as a control. NICD1 expression was normalized to total NOTCH1 expression (~120 kDa) and data were finally normalized to those of cells transfected with the empty vector. These data were represented in the graph (right) as the mean ± SD of two different transfectants for each construct, in at least three independent experiments. The empty vector transfectants are the reference control for cells transfected both with DLK1 and with DLK2 expressing plasmids. (B) Analysis of NOTCH transcriptional activity, as measured by luciferase assays, in SK-MEL-2 stably transfected with empty vector or plasmids HDLK1S, HDLK2S or HDLK2aS, and transiently transfected with plasmid pGLucWT, which expresses a NOTCH- dependent luciferase reporter gene. The relative luciferase activity was calculated by normalizing data to those obtained from cells transfected with the empty vector and it is represented as the mean ± SD of two different transfectants for each construct, in at least three independent experiments. (C) Level of expression of the NOTCH target genes HES1, HEY1 and HEY2, in HDLK1S, HDLK2S or HDLK2aS stably transfected SK-MEL-2 cells. Data were normalized to GADPH mRNA levels in quantitative RT-PCR assays. Student's t-test results relative to vector cells: *(P b 0.05), **(P b 0.01), ***(P b 0.001).

Article Snippet: Western blot was performed as described previously [34] by using the following antibodies: anti-DLK1 [16], diluted 1:2000; anti-DLK2 (Abnova, Heidelberg, Germany), diluted 1:500; anti-cleaved NOTCH1 (Val 1744, Cell Signaling, Beverly, MA, USA), diluted 1:500; anti-NOTCH1 (C-20; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), diluted 1:1000: and antitubulin (Sigma), diluted 1:5000.

Techniques: Over Expression, Activation Assay, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Control, Expressing, Construct, Activity Assay, Luciferase, Quantitative RT-PCR

Journal: Biochimica et biophysica acta

Article Title: The proteins DLK1 and DLK2 modulate NOTCH1-dependent proliferation and oncogenic potential of human SK-MEL-2 melanoma cells.

doi: 10.1016/j.bbamcr.2014.07.015

Figure Lengend Snippet: Fig. 6. NOTCH activation and signaling in SK-MEL-2 cells treated with the γ-secretase inhibitor DAPT and/or DLK proteins. (A) Analysis of active NOTCH1 protein (active NICD1 ~ 110 kDa) in the presence of the γ-secretase inhibitor DAPT at the indicated concentrations. A representative Western blot assay is shown (left). NICD1 expression was normalized to total NOTCH1 expression (~120 kDa) and data were finally normalized to those obtained from cells non-treated with DAPT. These data were represented in the graph (right) as the mean ± SD of three independent experiments. (B) Analysis of NOTCH transcriptional activity, as measured by luciferase assays, in SK-MEL-2 cells transiently co-transfected with pGLucWT and empty vector or HDLK1S, HDLK2S or HDLK2aS plasmids. Empty vector transfectants were treated with DAPT at the indicated concentrations. These data were represented in the graph as the mean ± SD of three independent experiments. Student's t-test results relative to vector cells without DAPT treatment. (C) Representative Western blot analysis (left) of active NICD1 protein in stable SK-MEL-2 transfectants overexpressing DLK1 or DLK2 and treated, or not, with DAPT at the indicated concentrations. NICD1 expression (~110 kDa) was normalized to total NOTCH1 expression (~120 kDa) and data were finally normalized to those obtained from cells transfected with the empty vector. These data were represented in the graph as the mean ± SD of two different transfectants for each construct, in at least three independent experiments. Student's t-test results relative to cell samples are indicated in the figure. (D) Analysis of NOTCH transcriptional activity, as measured by luciferase assays, in SK-MEL-2 cells transiently co-transfected with pGLucWT and empty vector or plasmids HDLK1S or HDLK2S, and treat- ed, or not, with the γ-secretase inhibitor DAPT at the indicated concentrations. The relative luciferase activities were calculated by normalizing the data to those obtained from cells transfected with the empty vector and treated, or not, with DAPT, and they were represented as the mean ± SD of two different transfectants for each construct, in at least three indepen- dent experiments. Student's t-test results relative to cell samples are indicated in the figure: *(P b 0.05), **(P b 0.01), ***(P b 0.001).

Article Snippet: Western blot was performed as described previously [34] by using the following antibodies: anti-DLK1 [16], diluted 1:2000; anti-DLK2 (Abnova, Heidelberg, Germany), diluted 1:500; anti-cleaved NOTCH1 (Val 1744, Cell Signaling, Beverly, MA, USA), diluted 1:500; anti-NOTCH1 (C-20; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), diluted 1:1000: and antitubulin (Sigma), diluted 1:5000.

Techniques: Activation Assay, Western Blot, Expressing, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Construct

Journal: Oncogene

Article Title: Lunatic Fringe is a potent tumor suppressor in Kras-initiated pancreatic cancer.

doi: 10.1038/onc.2015.306

Figure Lengend Snippet: Figure 1. Lfng is expressed in a subset of acinar cells in the adult pancreas. (a–f) Expression of Lfng in the adult pancreas. Shown are representative photomicrographs of whole-mount (a–c) and sectioned. (d–f) X-Gal staining of the pancreas from wild-type, LfnglacZ/+ and LfnglacZ/lacZ mice at 6 months of age. (g–j) Expression of individual Notch receptors in the adult pancreas. Shown are representative photomicrographs of anti-Notch1, anti-Notch2, anti-Notch3 and anti-Notch4 immunostaining in the wild pancreas at 6 months of age. Arrow in g inset points to Notch1 expression in the blood vessel. Arrows in i and j indicate Notch3 expression in ductal cells and Notch4 expression in islet, respectively. Scale bars: 50 μm.

Article Snippet: Primary antibodies used for immunostaining were: Notch1 (Cell Signaling, Danvers, MA, USA; No. 3608, 1:100), Notch2 (DSHB, University of Iowa, C651.6DbHN, 1:200), Notch3 (ProteinTech, Chicago, IL, USA; 55114-1-AP, 1:100), Notch4 (Millipore, Billerica, MA, USA; 09-089, 1:100), Ki67 (Abcam, Cambridge, MA, USA; ab16667, 1:100), Aldh1 (Abcam, ab52492, 1:200), CK19 (DSHB, University of Iowa, TROMA-III, 1:400), E-cadherin (Cell Signaling, No. 3195, 1:100), Vimentin (Cell Signaling, No. 5741, 1:100), Clusterin (Santa Cruz, Dallas, TX, USA; sc-6420, 1:50), Phospho-Smad2 (Ser465/467) (Cell Signaling, No. 3101, 1:100), and GFP (Invitrogen, Grand Island, NY, USA; A11122, 1:200).

Techniques: Expressing, Staining, Immunostaining

Journal: Oncogene

Article Title: Lunatic Fringe is a potent tumor suppressor in Kras-initiated pancreatic cancer.

doi: 10.1038/onc.2015.306

Figure Lengend Snippet: Figure 2. Deletion of Lfng enhanced Notch signaling in the KrasLSL-G12D/+;Pdx1-Cre pancreas. (a) Quantitative RT–PCR for Lfng and Notch downstream target gene Hes1 in the pancreas from Pdx1-Cre (C), Lfngflox/flox;Pdx1-Cre (LC), KrasLSL-G12D/+;Pdx1-Cre (KC) and Lfngflox/flox;KrasLSL-G12D/+; Pdx1-Cre (LKC) mice of indicated age. *Po0.05; **Po0.005; ***Po0.0005. (b) Western blot analysis of individual Notch receptors in the pancreas from C and KC mice at 3 months of age, with quantification of band densities relative to β-actin. (c) Western blot analysis of Notch receptors in the pancreas from KC and LKC mice of indicated age, and quantification of band densities relative to β-actin. (d) Representative photomicrographs of anti-Notch1 and anti-Notch3 immunostaining in the pancreas from 3 and 5-month-old KC and LKC mice. Arrows point to ADM lesions in 3-month-old mice and ductal cells in 5-month-old mice, respectively. Scale bars: 50 μm.

Article Snippet: Primary antibodies used for immunostaining were: Notch1 (Cell Signaling, Danvers, MA, USA; No. 3608, 1:100), Notch2 (DSHB, University of Iowa, C651.6DbHN, 1:200), Notch3 (ProteinTech, Chicago, IL, USA; 55114-1-AP, 1:100), Notch4 (Millipore, Billerica, MA, USA; 09-089, 1:100), Ki67 (Abcam, Cambridge, MA, USA; ab16667, 1:100), Aldh1 (Abcam, ab52492, 1:200), CK19 (DSHB, University of Iowa, TROMA-III, 1:400), E-cadherin (Cell Signaling, No. 3195, 1:100), Vimentin (Cell Signaling, No. 5741, 1:100), Clusterin (Santa Cruz, Dallas, TX, USA; sc-6420, 1:50), Phospho-Smad2 (Ser465/467) (Cell Signaling, No. 3101, 1:100), and GFP (Invitrogen, Grand Island, NY, USA; A11122, 1:200).

Techniques: Quantitative RT-PCR, Western Blot, Immunostaining