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

Journal: Current Issues in Molecular Biology

Article Title: The Androgen Hormone-Induced Increase in Androgen Receptor Protein Expression Is Caused by the Autoinduction of the Androgen Receptor Translational Activity

doi: 10.3390/cimb44020041

Figure Lengend Snippet: Antibodies used in the study.

Article Snippet: For antibody incubation, cells were incubated for 24 h with primary antibodies at 4 °C (Androgen Receptor (D6F11) XP Rabbit mAb, Cell Signaling Technology, Danvers, MA, USA, Dilution: 1:500).

Techniques:

Journal: Oncotarget

Article Title: Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer.

doi: 10.18632/oncotarget.28059

Figure Lengend Snippet: Figure 2: Overexpression of UGDH in the AD LNCaP background desensitizes AR-mediated gene expression and reduces glucuronidation precursors. Two vector control cell lines (VC1 and VC2) and two UGDH-overexpressing lines (OE1 and OE2) were selected in the LNCaP AD background. Equal cell counts were seeded 48 hours in androgen depleted media, followed by removal and replacement with media containing DMSO (vehicle, 0 nM) or the indicated concentration of DHT. After an additional 48 hours, cells were harvested for analysis. AR-dependent genes PSA (A) and UGT2B17 (B) were analyzed by WB in whole cell lysates; (C) functional synthetic output of each cell line was assessed by Notch1 expression in cell lysates; (D) UDP-sugar pools were measured in cell lysates by LC-MS. In panels A–C, mean ± SEM is plotted for triplicate measurements. In panel D, mean ± SD is plotted for quadruplicate measurements. Statistical significance is indicated as: (a) p < 0.05 relative to VC1 at 0 nM DHT. (b) p < 0.05 relative to VC2 at 0 nM DHT. (c) p < 0.05 comparing OE1 to both VC1 and VC2 at the indicated [DHT]. (d) p < 0.05 comparing OE2 to both VC1 and VC2 at the indicated [DHT].

Article Snippet: Antibodies were purchased and used as follows: polyclonal rabbit anti-human PSA Oncotarget1898www.oncotarget.com (DakoCytomation, Glostrup, Denmark, 1:1500 dilution); rabbit polyclonal anti-human MHC class II [EPR11227] (Abcam, Cambridge, MA, USA, 1:1000); mouse monoclonal anti-human β-tubulin (Sigma, 1:750,000); IRDye 800 conjugated anti-rabbit IgG (Rockland, Gilberstville, PA, USA, 1:5000); IRDye 680 conjugated goat anti-mouse IgG (LI-COR Biosciences, Lincoln, NE, USA, 1:5000); rabbit polyclonal anti-human UGT2B17 (GeneTex, Irvine, CA, USA, 1:100), mouse monoclonal anti-human AR (Santa Cruz Biotechnology, Inc, Dallas, TX, USA, 1:500); rabbit monoclonal anti-human Notch1 (Cell Signaling Technology, Danvers, MA, USA, 1:1000); rabbit monoclonal anti-human FoxA1 (Abcam, Cambridge, MA, USA 1:1000).

Techniques: Over Expression, Gene Expression, Plasmid Preparation, Control, Concentration Assay, Functional Assay, Expressing, Liquid Chromatography with Mass Spectroscopy

Journal: Oncotarget

Article Title: Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer.

doi: 10.18632/oncotarget.28059

Figure Lengend Snippet: Figure 3: Overexpression of UGDH in the CR LNCaP background further suppresses AR-mediated expression of glucuronidation genes and reduces nucleotide sugar pools without impacting proteoglycan production. Two vector control cell lines (VC1 and VC2) and two UGDH-overexpressing lines (OE1 and OE2) were selected in the LNCaP CR background. Equal cell numbers were seeded 48 hours in androgen replete media followed by harvest of cells for analysis of gene expression (A and B) and UDP-sugars (D). For panel (C), equal cell counts were seeded 48 hours in androgen depleted media, followed by removal and replacement with media containing DMSO (vehicle, 0 nM) or the indicated concentration of DHT. After an additional 48 hours, cells and media were harvested for analysis. AR-dependent genes UGT2B17 (A) and FoxA1 (B) were analyzed by WB in whole cell lysates; (C) functional synthetic output of each cell line was assessed by Notch1 expression in cell lysates; (D) UDP-sugar pools were measured in cell lysates by mass spectrometry. Mean ± SEM is plotted for triplicate technical measurements; *p < 0.05 for OE1 and OE2 relative to VC1 and VC2.

Article Snippet: Antibodies were purchased and used as follows: polyclonal rabbit anti-human PSA Oncotarget1898www.oncotarget.com (DakoCytomation, Glostrup, Denmark, 1:1500 dilution); rabbit polyclonal anti-human MHC class II [EPR11227] (Abcam, Cambridge, MA, USA, 1:1000); mouse monoclonal anti-human β-tubulin (Sigma, 1:750,000); IRDye 800 conjugated anti-rabbit IgG (Rockland, Gilberstville, PA, USA, 1:5000); IRDye 680 conjugated goat anti-mouse IgG (LI-COR Biosciences, Lincoln, NE, USA, 1:5000); rabbit polyclonal anti-human UGT2B17 (GeneTex, Irvine, CA, USA, 1:100), mouse monoclonal anti-human AR (Santa Cruz Biotechnology, Inc, Dallas, TX, USA, 1:500); rabbit monoclonal anti-human Notch1 (Cell Signaling Technology, Danvers, MA, USA, 1:1000); rabbit monoclonal anti-human FoxA1 (Abcam, Cambridge, MA, USA 1:1000).

Techniques: Over Expression, Expressing, Plasmid Preparation, Control, Gene Expression, Concentration Assay, Functional Assay, Mass Spectrometry

Journal: Oncotarget

Article Title: Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer.

doi: 10.18632/oncotarget.28059

Figure Lengend Snippet: Figure 5: Loss of UGDH promotes AR-dependent gene expression and reduces proteoglycan production while sustaining glucuronide output. LNCaP AD and CR cells were selected for stable expression of a non-targeting vector control (VC1 and VC2) or a UGDH shRNA knockdown construct (KD1 and KD2). Equal cell counts were seeded 48 hours in androgen depleted media, followed by removal and replacement with media containing 10 nM DHT or DMSO (vehicle, 0 nM). After an additional 48 hours, cells were harvested for analysis by western blot and mass spectrometry. (A) AR-dependent genes PSA (panels a and d) and UGT2B17 (panels b and e) were analyzed by WB in whole cell lysates; functional synthetic output of each cell line was assessed by Notch1 expression in cell lysates (panels c and f). Panels (a–c) depict expression data in the AD background and panels (d–f) illustrate data from the CR background. Mean ± SEM is plotted. Statistical significance is indicated on the plots as: (a) p < 0.05 for that clone, comparing 0 vs 10 nM DHT; (b) p < 0.05 relative to VC1 and VC2, 10 nM DHT; (c) p < 0.05 relative to VC1 and VC2, 0 nM DHT. (B) UDP-sugar pools were measured in cell lysates by LC-MS for both the AD (upper) and CR (lower) backgrounds as indicated. Mean ± SD is plotted. Statistical significance is indicated as: (a) p < 0.05 for both KD1 and KD2 relative to VC1 or VC2, 0 nM DHT. (b) p < 0.05 for both KD1 and KD2 relative to VC1 or VC2, 10 nM DHT. (c) p < 0.05 for that clone, comparing 0 and 10 nM DHT.

Article Snippet: Antibodies were purchased and used as follows: polyclonal rabbit anti-human PSA Oncotarget1898www.oncotarget.com (DakoCytomation, Glostrup, Denmark, 1:1500 dilution); rabbit polyclonal anti-human MHC class II [EPR11227] (Abcam, Cambridge, MA, USA, 1:1000); mouse monoclonal anti-human β-tubulin (Sigma, 1:750,000); IRDye 800 conjugated anti-rabbit IgG (Rockland, Gilberstville, PA, USA, 1:5000); IRDye 680 conjugated goat anti-mouse IgG (LI-COR Biosciences, Lincoln, NE, USA, 1:5000); rabbit polyclonal anti-human UGT2B17 (GeneTex, Irvine, CA, USA, 1:100), mouse monoclonal anti-human AR (Santa Cruz Biotechnology, Inc, Dallas, TX, USA, 1:500); rabbit monoclonal anti-human Notch1 (Cell Signaling Technology, Danvers, MA, USA, 1:1000); rabbit monoclonal anti-human FoxA1 (Abcam, Cambridge, MA, USA 1:1000).

Techniques: Gene Expression, Expressing, Plasmid Preparation, Control, shRNA, Knockdown, Construct, Western Blot, Mass Spectrometry, Functional Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Fully human monoclonal antibody targeting activated ADAM10 on colorectal cancer cells

doi: 10.1016/j.biopha.2023.114494

Figure Lengend Snippet: 1H5 inhibits Notch cleavage. Sandwich ELISA was used to measure the levels of total (A) and cleaved (B) Notch1 in COLO205 cells upon treatment with 1H5. The data represent mean of triplicate experiments, and the bar plots show the effect of treatment with 1H5 relative to untreated control, mean ± SEM. Comparison of notch levels between treated and untreated groups was performed using independent t test. Total Notch1 levels did not significantly differ between the two groups (A), p = 0.162. On the other hand, the mAb-treated group showed significant decrease of the cleaved Notch1 levels when compared with the untreated control (B), p < 0.001.

Article Snippet: To detect NICD1, we used PathScan ® cleaved Notch1 (Val1744) sandwich ELISA Kit (Cell Signaling Technologies).

Techniques: Sandwich ELISA, Control, Comparison

Journal: iScience

Article Title: Molecular docking-aided identification of small molecule inhibitors targeting β-catenin-TCF4 interaction

doi: 10.1016/j.isci.2021.102544

Figure Lengend Snippet:

Article Snippet: Notch CSL reporter , BPS Bioscience , Cat# 60652.

Techniques: Recombinant, Luciferase, Cell Viability Assay, CCK-8 Assay, RNA Sequencing Assay, Negative Control, Mutagenesis, Plasmid Preparation, Software, High Content Screening