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: Physiological reports

Article Title: Long noncoding RNA TINCR is a novel regulator of human bronchial epithelial cell differentiation state.

doi: 10.14814/phy2.14727

Figure Lengend Snippet: FIGURE 4 (a) Selected gene expressions regarding epithelial mesenchymal markers, differentiation makers and regulators from RNA- sequencing data. (b and c) qRT–PCR and western blot analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression at day4 after siRNA. *p < 0.05. Data presented are from one of two independent experiments with similar results. (d and e) qRT–PCR and western blot analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression for NHBECs transfected with pcDNA3.1-EGFP-Blank or pcDNA3.1-EGFP-TINCR. *p < 0.05. Data presented are from one of two independent experiments with similar results. (f) qRT–PCR analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression at day3 and day14 after airlift with siRNA and 3D organoid matrigel. *p < 0.05. Data presented are from one of two independent experiments with similar results. (g) qRT–PCR analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression at day3 and day14 after airlift with transfection of pcDNA3.1-EGFP-Blank or pcDNA3.1-EGFP-TINCR

Article Snippet: Rabbit anti-SOX2 antibody (cat. No 3579T), rabbit anti-HES1 antibody (cat. No 11988S), rabbit anti-JAG1 antibody (cat. No 2620T), rabbit anti-NOTCH1 antibody (cat. No 3608S), and rabbit anti-NOTCH2 antibody (cat. No 5732T) were from Cell Signaling Technology.

Techniques: RNA Sequencing, Quantitative RT-PCR, Western Blot, Expressing, Transfection

Journal: Physiological reports

Article Title: Long noncoding RNA TINCR is a novel regulator of human bronchial epithelial cell differentiation state.

doi: 10.14814/phy2.14727

Figure Lengend Snippet: FIGURE 5 TINCR binds to STAU1 protein and controls critical regulators of differentiation. (a) Bar graphs show percentage of TINCR in the cytoplasm (black) and nucleus (white). NEAT1 serves as a positive control for nucleus enriched RNA and GAPDH serves as a positive control for cytoplasmic RNA. Data presented are from two independent experiments. (b) RIP experiments were performed using isotype IgG and STAU1 antibody to immunoprecipitated STAU1 protein/mRNAs complexes in total-cell extracts of NHBECs, and relative enrichment was determined as RNA associated with STAU1 IP relative to an input control. Relative ARF1 enrichment served as a positive control and NEAT1 as a negative control as NEAT1 does not interact with STAU1. Data presented are from two independent experiments. (c) Relative mRNA enrichment of STAU1 antibody in total-cell extracts of NHBECs transfected with siSCR or siTICNR. Data presented are from two independent experiments. (d and e) qRT–PCR and western blot analysis of TINCR, TP63, SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression for NHBECs transfected with siSCR or siSTAU1. NHBECs were seeded on 6 well plate at 2 × 105 density and analyzed at day4 after transfection of siRNA reagents. *p < 0.05. (f and g) qRT–PCR and western blot analysis of TINCR, TP63, SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression for NHBECs transfected with siSCR or siSTAU after transfection of pcDNA3.1-EGFP-Blank or pcDNA3.1-EGFP-TINCR for 4 h. NHBECs were seeded on 24 well plate at 1 × 105 density and analyzed at day4 after transfection of siRNA reagents

Article Snippet: Rabbit anti-SOX2 antibody (cat. No 3579T), rabbit anti-HES1 antibody (cat. No 11988S), rabbit anti-JAG1 antibody (cat. No 2620T), rabbit anti-NOTCH1 antibody (cat. No 3608S), and rabbit anti-NOTCH2 antibody (cat. No 5732T) were from Cell Signaling Technology.

Techniques: Positive Control, Immunoprecipitation, Control, Negative Control, Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: Theranostics

Article Title: Rbm24/Notch1 signaling regulates adult neurogenesis in the subventricular zone and mediates Parkinson-associated olfactory dysfunction.

doi: 10.7150/thno.96045

Figure Lengend Snippet: Figure 6. Rbm24 directly regulates Notch1 mRNA stability in the adult NSPCs. (A-B) Representative images of GFAP/Rbm24/Notch1 (A), DCX/Rbm24/Notch1 (B) co-staining in the SVZ from 2-month-old CTL mice. Scale bar, 20 μm, zoom scale bar, 10 μm. (C-D) Immunoblot (C) and quantification (D) of Notch1 protein in the SVZ from 2-month-old CTL and UKO mice (n = 6 mice for each group). (E) Quantification of Notch1 mRNA in the neurospheres from 2-month-old CTL and UKO mice (n = 3 mice for each group). (F) RT-PCR analysis of Notch1 mRNA in input and Rbm24-antibody IP in the SVZ from 2-month-old CTL mice. (G) Quantification of Notch1 mRNA in adult NSPCs from 2-month-old CTL and UKO mice with actinomycin D administration (n = 3 mice for each group). (H) Quantification of luciferase activities for 3’-UTR fragments of Notch1 in 293T cells between pcRbm24 and pcDNA3.1 condition (n = 4 for each group). (I) Schematic diagram showing the effects of Rbm24 deficiency on Notch1. Data are presented as mean ± SEM. *p < 0.05; ***p < 0.001; n.s., not significant. LV: lateral ventricle; NSPCs: neural stem/progenitor cells.

Article Snippet: Slices (20 μm thickness) from the SVZ, RMS, and OB were collected and incubated overnight at 4 °C with primary antibodies: Rbm24 (1:200, ab94567, Abcam, UK), GFAP (1:500, 3670S, Cell Signaling Technology, USA), DCX (1:300, sc271390, Santa Cruz, USA), BrdU (1:250, ab6326, Abcam, UK), Caspase-3 (1:100, T40044, Abmart, China), NeuN (1:500, ab177487, Abcam, UK), TH (1:200, ab1542, Sigma, USA), CalR (1:100, sc365956, Santa Cruz, USA), CalB (1:200, 13176S, Cell Signaling Technology, USA), Notch1 (1:100, 3447S, Cell Signaling Technology, USA), and GFP (1:300, ab290, Abcam, UK).

Techniques: Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Luciferase

Journal: Theranostics

Article Title: Rbm24/Notch1 signaling regulates adult neurogenesis in the subventricular zone and mediates Parkinson-associated olfactory dysfunction.

doi: 10.7150/thno.96045

Figure Lengend Snippet: Figure 7. Rbm24 overexpression ameliorates adult neurogenesis in the SVZ of PD mice. (A-C) Immunoblot (A) and quantification of Rbm24 (B) and Notch1 (C) protein in the SVZ from 7-month-old WT and PD mice (n = 6 mice for each group). (D) Schematic diagram showing the lentivirus injection into the lateral ventricle. (E) Timeline of BrdU (short-term) and lentivirus injection in 7-month-old WT and PD mice. (F-G) Immunoblot (F) and quantification (G) of Rbm24 and Notch1 protein in the SVZ from 7-month-old OEC-PD and OER-PD mice (n = 6 mice for each group). (H) Quantification of Rbm24, Notch1, Heyl mRNA in the SVZ from 7-month-old OEC-PD and OER-PD mice (n = 6 mice for each group). (I-K) Representative images (I) and quantification of GFAP+/BrdU+ (J), GFAP+ (K) cells in the SVZ from 7-month-old WT, PD, OEC-PD, and OER-PD mice (n = 4 mice for each group). Scale bar, 50 μm. (L-N) Representative images (L) and quantification of DCX+/BrdU+ (M), DCX+ (N) cells in the SVZ from 7-month-old WT, PD, OEC-PD, and OER-PD mice (n = 4 mice for each group). Scale bar, 50 μm. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant. LV: lateral ventricle.

Article Snippet: Slices (20 μm thickness) from the SVZ, RMS, and OB were collected and incubated overnight at 4 °C with primary antibodies: Rbm24 (1:200, ab94567, Abcam, UK), GFAP (1:500, 3670S, Cell Signaling Technology, USA), DCX (1:300, sc271390, Santa Cruz, USA), BrdU (1:250, ab6326, Abcam, UK), Caspase-3 (1:100, T40044, Abmart, China), NeuN (1:500, ab177487, Abcam, UK), TH (1:200, ab1542, Sigma, USA), CalR (1:100, sc365956, Santa Cruz, USA), CalB (1:200, 13176S, Cell Signaling Technology, USA), Notch1 (1:100, 3447S, Cell Signaling Technology, USA), and GFP (1:300, ab290, Abcam, UK).

Techniques: Over Expression, Western Blot, Injection

Journal: Theranostics

Article Title: Rbm24/Notch1 signaling regulates adult neurogenesis in the subventricular zone and mediates Parkinson-associated olfactory dysfunction.

doi: 10.7150/thno.96045

Figure Lengend Snippet: Figure 9. Notch1 inhibition blocks the effects of Rbm24 overexpression on SVZ-OB pathway neurogenesis and olfactory performance in PD mice. (A) Timeline of DAPT, BrdU and lentivirus injection in 7-month-old PD mice. (B-C) Immunoblot (B) and quantification (C) of NICD protein levels in the SVZ from 7-month-old OER-PD mice treated with vehicle or DAPT (n = 6 mice for each group). (D-F) Representative images (D) and quantification of GFAP+/BrdU+ (E), GFAP+ (F) cells in the SVZ

Article Snippet: Slices (20 μm thickness) from the SVZ, RMS, and OB were collected and incubated overnight at 4 °C with primary antibodies: Rbm24 (1:200, ab94567, Abcam, UK), GFAP (1:500, 3670S, Cell Signaling Technology, USA), DCX (1:300, sc271390, Santa Cruz, USA), BrdU (1:250, ab6326, Abcam, UK), Caspase-3 (1:100, T40044, Abmart, China), NeuN (1:500, ab177487, Abcam, UK), TH (1:200, ab1542, Sigma, USA), CalR (1:100, sc365956, Santa Cruz, USA), CalB (1:200, 13176S, Cell Signaling Technology, USA), Notch1 (1:100, 3447S, Cell Signaling Technology, USA), and GFP (1:300, ab290, Abcam, UK).

Techniques: Inhibition, Over Expression, Injection, 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 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