Journal: Molecular cancer therapeutics

Article Title: ASR490, a Small Molecule, Overrides Aberrant Expression of Notch1 in Colorectal Cancer

doi: 10.1158/1535-7163.MCT-19-0949

Figure Lengend Snippet: (A) Structure of ASR490. (B & C) ASR490 or vehicle were used at indicated concentrations to treat HCT 116 and SW-620 cells for 24 h and 48 h followed by the MTT assay for cell viability. (D) Immunoblot analysis of cell lysates from HCT 116 and SW620 cells treated with the IC50 concentration of ASR490 or vehicle (DMSO) for 12 and 24 h. (E) Docking study with Medusa Dock was performed with NRR domain and ASR490, the blue dotted lines (H-bond), Notch1-NRR domain with α-helices (cyan), β-sheets (magenta), loops (salmon) and ASR490 (green licorice) are represented with water molecules (red spheres). (F) Protein run melt profile with temperature plotted against first derivative of fluorescence curve (–df/dt) and the lowest curve points taken at respective melting temperature (Tm) for NRR+ASR490 and NRR+ Vehicle samples. (G) The CETSA assay was performed on ASR490-treated HCT 116 cells at the indicated temperatures followed by ELISA with NRR antibody and temperature was plotted against the absorbance (450nm) changes. (H) Analysis of ASR490-treated cells for p65/Bcl-2 expression in a time-dependent manner. Data are presented as the mean ± standard deviation (SEM/SD) of three independent experiments. Statistical significance between vehicle and treatment at each concentration was calculated with the Student’s t-test. *p < 0.05, **p < 0.01 and ***p < 0.001

Article Snippet: HCT116, T24, RT4 were maintained in McCoy’s medium, TCCSUP, UMUC3, HT1376 in EMEM, SW620 in DMEM and 5637 in RPMI medium, respectively, and supplemented with 10% FBS and penicillin (100 units/mL) and streptomycin (100 units/mL; Millipore Sigma, St Louis, MI, USA) in the presence of 5% CO 2 at 37°C. pCMV6-NOTCH1, vector pCMV6-Entry (NOTCH1 (NM_017617) Human ORF Clone; Origene) and NOTCH1 Human siRNA Oligo Duplex were obtained from Origene Technologies Inc. (Rockville, MD, USA).

Techniques: MTT Assay, Western Blot, Concentration Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Expressing, Standard Deviation

Journal: Molecular cancer therapeutics

Article Title: ASR490, a Small Molecule, Overrides Aberrant Expression of Notch1 in Colorectal Cancer

doi: 10.1158/1535-7163.MCT-19-0949

Figure Lengend Snippet: (A) A trans-well invasion assay was performed for pCMV/HCT and Notch1 overexpressing HCT116 transfectants (C4 and C5) that were treated with either ASR490 or vehicle (DMSO). (B) Migration assays were performed in vector-transfected and Notch1-overexpressing cells (C4 and C5) that were treated with ASR490 or vehicle. Analysis was carried out with Image J software and values are presented as mean ± SEM. *P < 0.05; **P < 0.01 (Student t test). (C) Cell lysates from ASR490-treated and vehicle-treated pCMV/HCT, C4, and C5 cells were subjected to western blot analysis using E-cadherin, N-cadherin, Snail, β-catenin, MMP-9, and Snail antibodies. (D) Densitometry analysis was performed with ImageJ software for the immunoblots. Values plotted are mean ± SEM. Statistical significance between vehicle and treatment at each concentration was calculated by the Student’s t-test. *p < 0.05 and **p < 0.01

Article Snippet: HCT116, T24, RT4 were maintained in McCoy’s medium, TCCSUP, UMUC3, HT1376 in EMEM, SW620 in DMEM and 5637 in RPMI medium, respectively, and supplemented with 10% FBS and penicillin (100 units/mL) and streptomycin (100 units/mL; Millipore Sigma, St Louis, MI, USA) in the presence of 5% CO 2 at 37°C. pCMV6-NOTCH1, vector pCMV6-Entry (NOTCH1 (NM_017617) Human ORF Clone; Origene) and NOTCH1 Human siRNA Oligo Duplex were obtained from Origene Technologies Inc. (Rockville, MD, USA).

Techniques: Invasion Assay, Migration, Plasmid Preparation, Transfection, Software, Western Blot, Concentration Assay

Journal: Molecular cancer therapeutics

Article Title: ASR490, a Small Molecule, Overrides Aberrant Expression of Notch1 in Colorectal Cancer

doi: 10.1158/1535-7163.MCT-19-0949

Figure Lengend Snippet: (A) Western blot analysis of Notch1 basal expression in HCT116 (pCMV-transfected and Notch1-transfected) cells. (B) For assessment of cell viability with MTT assay, IC50 concentration of ASR490 or Vehicle (DMSO) was used to treat HCT116 stable transfectants C4 and C5 for 24h. One way ANOVA with multiple comparison test was used to calculate the statistical significance between different experimental groups. *p < 0.05 and **p < 0.01. (C) Colony-forming assay on pCMV/HCT116 (vector transfected) and HCT116 stable transfectants C4 and C5 treated with ASR490 or vehicle (DMSO) were performed. All experiments were performed in triplicate. One way ANOVA with multiple comparison test was used to calculate the statistical significance between different experimental groups. *p < 0.05 and **p < 0.01. (D) Immunoblot analysis of ASR490-treated pCMV/HCT, C4, and C5 cells for Notch1 and HES1 expression in a time-dependent manner (12 and 24h). (E) Densitometry analysis was performed with ImageJ software for the immunoblots. Values, mean ± SEM. *P < 0.05; **P < 0.01 (Student t test).

Article Snippet: HCT116, T24, RT4 were maintained in McCoy’s medium, TCCSUP, UMUC3, HT1376 in EMEM, SW620 in DMEM and 5637 in RPMI medium, respectively, and supplemented with 10% FBS and penicillin (100 units/mL) and streptomycin (100 units/mL; Millipore Sigma, St Louis, MI, USA) in the presence of 5% CO 2 at 37°C. pCMV6-NOTCH1, vector pCMV6-Entry (NOTCH1 (NM_017617) Human ORF Clone; Origene) and NOTCH1 Human siRNA Oligo Duplex were obtained from Origene Technologies Inc. (Rockville, MD, USA).

Techniques: Western Blot, Expressing, Transfection, MTT Assay, Concentration Assay, Comparison, Plasmid Preparation, Software

Journal: Molecular cancer therapeutics

Article Title: ASR490, a Small Molecule, Overrides Aberrant Expression of Notch1 in Colorectal Cancer

doi: 10.1158/1535-7163.MCT-19-0949

Figure Lengend Snippet: (A) pCMV/HCT116 and stable clones (C4 and C5) were treated with the indicated concentration of ASR490 or vehicle (DMSO) for the indicated time points and total protein lysates were analyzed for the pro-survival markers NF-κB (p65) and Bcl-2. (B) Densitometry analysis was performed with ImageJ software for the immunoblots. Values, mean ± SEM. Statistical significance between vehicle and treatment at each concentration was calculated with the Student’s t-test. *p < 0.05. (C) Total protein lysates from ASR490-treated pCMV/HCT 116, C4, and C5 cells were analyzed for expression of the pro-apoptotic markers cleaved PARP and Bax. (D) FACS analysis was performed (Annexin V-FITC and PI staining) in non-transfected and Notch1-overexpressing HCT116 transfectants that were treated with the IC50 concentration of ASR490 or vehicle (DMSO). Values, mean ± SEM. *P < 0.05; **P < 0.01 (Student t test).

Article Snippet: HCT116, T24, RT4 were maintained in McCoy’s medium, TCCSUP, UMUC3, HT1376 in EMEM, SW620 in DMEM and 5637 in RPMI medium, respectively, and supplemented with 10% FBS and penicillin (100 units/mL) and streptomycin (100 units/mL; Millipore Sigma, St Louis, MI, USA) in the presence of 5% CO 2 at 37°C. pCMV6-NOTCH1, vector pCMV6-Entry (NOTCH1 (NM_017617) Human ORF Clone; Origene) and NOTCH1 Human siRNA Oligo Duplex were obtained from Origene Technologies Inc. (Rockville, MD, USA).

Techniques: Clone Assay, Concentration Assay, Software, Western Blot, Expressing, Staining, Transfection

Journal: Molecular cancer therapeutics

Article Title: ASR490, a Small Molecule, Overrides Aberrant Expression of Notch1 in Colorectal Cancer

doi: 10.1158/1535-7163.MCT-19-0949

Figure Lengend Snippet: pCMV/HCT116 and C4 (1×106) were injected subcutaneously into separate flanks of the mice (n=6–8). ASR490 (5mg/kg) or 1% DMSO (Vehicle) (100μl volume) was administered intraperitoneally thrice a week. (A) Weekly thrice the tumor volume (mm3) was measured in both ASR490 and vehicle treated mice.. Mean tumor volumes ± SEM are shown. *p < 0.05 and **p < 0.01 by two-tailed Student t test. (B) IHC analysis of Ki-67, Notch1, HES1, and NFκB (p65) (C) Protein isolated from tissue samples taken from HCT/Notch1 xenografts was subjected to immunoblot analysis with Notch1 and HES1 antibodies.

Article Snippet: HCT116, T24, RT4 were maintained in McCoy’s medium, TCCSUP, UMUC3, HT1376 in EMEM, SW620 in DMEM and 5637 in RPMI medium, respectively, and supplemented with 10% FBS and penicillin (100 units/mL) and streptomycin (100 units/mL; Millipore Sigma, St Louis, MI, USA) in the presence of 5% CO 2 at 37°C. pCMV6-NOTCH1, vector pCMV6-Entry (NOTCH1 (NM_017617) Human ORF Clone; Origene) and NOTCH1 Human siRNA Oligo Duplex were obtained from Origene Technologies Inc. (Rockville, MD, USA).

Techniques: Injection, Two Tailed Test, Isolation, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: Inhibition of Dll4/Notch1 pathway promotes angiogenesis of Masquelet’s induced membrane in rats

doi: 10.1038/s12276-018-0062-9

Figure Lengend Snippet: a – b The protein expression and quantitative data for Notch1, PCNA, ICAM-1 and SDF-1 in EPCs treated with control-siRNA or Notch1-siRNA. c – d The protein expression and quantitative data for Notch1, PCNA, ICAM-1 and SDF-1 in EPCs treated with the protein extracted from the induced membrane derived from the subcutaneous site and femoral defect site with or without DAPT intervention. The data in the figures represent averages ± S.D. Significant differences between the different groups are indicated as * P < 0.05, ** P < 0.01; for each group, n = 5

Article Snippet: siRNA for rat Notch1 gene was designed and synthesized (RiboBio, Guangzhou, China) with the following sequence: 5′-AAGTGGGACCTGCCTGAATGG-3′.

Techniques: Expressing, Control, Membrane, Derivative Assay