Journal: OncoTargets and therapy

Article Title: Epigallocatechin gallate inhibits the proliferation of colorectal cancer cells by regulating Notch signaling

doi: 10.2147/OTT.S40914

Figure Lengend Snippet: The effect of EGCG on expressions of Notch1, Notch2, HES1 , and JAG1 in the colorectal cancer cells: ( A ) the expression of HES1 ; ( B ) the expression of JAG1 ; ( C ) the expression of Notch1 ; and ( D ) the expression of Notch2 . Notes: The x-axis shows the Lovo, SW480, HT-29, and HCT-8 colorectal cell lines. The y-axis shows the mRNA of the colorectal cell lines after treatment with EGCG. An independent-sample t -test was used to analyze the mRNA levels of the colorectal cell lines. Abbreviation: EGCG, epigallocatechin gallate.

Article Snippet: The primary antibodies used were Abcam Hes1 antibody (ab71559), CST Jagged1 (28H8Rabbit mAb), CST Notch1 (D1E11 XP™ Rabbit mAb), and CST Notch2 (8A1Rabbit mAb) (Sigma-Aldrich, St Louis, MO, USA).

Techniques: Expressing

Journal: The Journal of cell biology

Article Title: Aberrant phosphorylation inactivates Numb in breast cancer causing expansion of the stem cell pool.

doi: 10.1083/jcb.202112001

Figure Lengend Snippet: Figure 3. Characterization of Numb phosphomutants. (A) Scheme showing the behavior of a SC (A) and daughter progenitor cell (B) in WT vs. Numb-KO (Tosoni et al., 2015). (B) Scheme of the growth of MS from WT and Numb-KO MECs (Tosoni et al., 2015). (C and D) WT and Numb-KO cells, transduced with the indicated constructs (DsRed fusion proteins; EV, empty vector), were assessed for SFE (C, by counting only red cells or MS) and size (D, N = number of epifluorescent MS analyzed). Results are expressed relative to WT cells (see also Table S2). Significance was calculated vs. EV cells. Representative images of the MS are in D, top panel. Bar, 100 µm. (E) WT and Numb-KO MS, transduced with the indicated constructs (Flag-tagged), were analyzed by IB. Arrows, endogenous (black) or overexpressed (red) Numb (also in G). Activated Notch (Act. Notch) was detected with the anti Val1744 Ab (in this and all subsequent figures). Right: Quantitation of three independent experiments. (F) HEK-293 cells, transfected as indicated (all Numb constructs were Flag-tagged and also codify for an sh-RNA sequence against endogenous Numb; EV, empty vector), were IP and IB as shown. (G) HEK-293 cells were stably transduced with Notch- NΔE (Notch-TFX; NT, not transfected) and transfected with the indicated Numb-Flag constructs (as in F). IP and IB were as shown. (H and I) MCF-7 or Cal51 cells were either transduced with Notch-NΔE (Notch-TFX; I) or not (H). Cells were treated with BIS (or mock-treated) and IP and IB as shown. In H, IP-Ctr is anti-Flag; in I, IP-Ctr is goat IgG. Data are reported ± SD (C and E) or ± SE (D). Statistical analysis was with the Student’s t test two-tailed (C and D) or with the one-sample t test (E). Source data are available for this figure: SourceData F3.

Article Snippet: Ab for immunoblot (IB) were directed against Numb (AB21, a mouse monoclonal Ab against amino acids 537–551 of hNumb [Colaluca et al., 2008]), and for the experiment in Fig. S3 B, the anti-Numb C29G11, rabbit monoclonal from Cell Signaling Technologies (Cat. 4140) was used; Vinculin (mouse monoclonal; Sigma-Aldrich, Cat. V9131); GRP94 (9G10, rat monoclonal, Cat. ADI-SPA-851; Enzo Life Sciences); Tubulin (11H10, rabbit monoclonal, Cat. 2125; Cell Signaling Technologies); p53 (1C12, mouse monoclonal, Cat. 2524; Cell Signaling Technologies, Figs. 3 E, 5, A–C, and 9 D; FL393, Santa Cruz Biotechnology, goat polyclonal, Cat. sc-6243-G, Fig. 3 F; goat polyclonal, Bio-techne, Cat. AF1355 Fig. 3 H); PKCζ (C24E6, rabbit monoclonal, Cat. 9368; Cell Signaling Technologies); p-PKCζ (H-2, mouse monoclonal, Cat. sc-271962; Santa Cruz Biotechnology); PAN-PKC (A-9, mouse monoclonal, Cat. sc-17804; Santa Cruz Biotechnology); pPAN PKC (rabbit polyclonal, Cat. 9371; Cell Signaling Technologies); Notch (5B5, rat monoclonal, Cat. 3447; Cell Signaling Technologies); Cleaved Notch1 Val1744 (D3B8, rabbit monoclonal, Cat. 4147; Cell Signaling Technologies); Flag (D6W5B, rabbit monoclonal, Cat. 2368; Cell Signaling Technologies); Par3 (Cat. 07-330; Millipore); Mdm2 (Mouse Monoclonal, Cat. Op46; Calbiochem); anti GAPDH (rabbit monoclonal, Cat. 5174; Cell Signaling Technologies); and pNumb-Ser276 (rabbit monoclonal, Cat. 4140; Cell Signaling Technologies).

Techniques: Transduction, Construct, Plasmid Preparation, Quantitation Assay, Transfection, Sequencing, Stable Transfection, Two Tailed Test

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: Molecular medicine reports

Article Title: Disorder of the mevalonate pathway inhibits calcium-induced differentiation of keratinocytes.

doi: 10.3892/mmr.2017.7128

Figure Lengend Snippet: Figure 2. Treatment of KCs with inhibitors of the mevalonate pathway reduced the expression levels of Notch1 and p53. Representative western blot images and quantification of protein expression levels of Notch1 and p53 in KCs treated with PRA, ALD, FTI‑277 and GGTI‑298 inhibitors (A) alone, (B) in combination with two inhibitors or (C) in combination with three or four inhibitors for 48 h, in the presence or absence of Ca2+. Data are expressed as mean ± standard error of three independent experiments. *P<0.01 and #P<0.05 vs. untreated control in the presence of Ca2+. PRA, pravastatin; ALD, alendronate; FTI‑277, farnesyl transferase inhibitor; GGTI‑298, geranylgeranyl transferase inhibitor; KCs, keratinocytes; NEXT, Notch1 extracellular truncation; NCID, Notch1 intracellular domain.

Article Snippet: Anti-K1(mouse monoclonal IgG2a; sc-376224), involucrin (INV; rabbit polyclonal IgG; sc-28557), Notch1 (goat polyclonal IgG; sc-6014), p53 (rabbit polyclonal IgG; sc-6243), phosphorylated (p)-extracellular signal-regulated kinase (ERK; rabbit polyclonal IgG; sc-101761), p-protein kinase B (AKT; rabbit polyclonal IgG; sc-33437) and β-actin (mouse monoclonal IgG1; sc-376421) primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Expressing, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: α-Synuclein Induces Alterations in Adult Neurogenesis in Parkinson Disease Models via p53-mediated Repression of Notch1

doi: 10.1074/jbc.m112.354522

Figure Lengend Snippet: FIGURE 2. Alterations in the expression of Notch1 signaling components in NPC-derived neural progeny expressing -syn. A, absolute quantification of Notch1 mRNA levels throughout neuronal differentiation, showing decay of signal as neuronal differentiation progress. Notch1 levels were significantly lower in -syn-overexpressing cells by day 4. B, quantitative real time PCR analysis of the mRNA levels of Notch1, Hes1, and Hes5 in NPC-derived neuronal progeny of cells infected with LV--syn in comparison with LV-control-infected cells at day 4. C, detection of -syn, Notch1, Hes1, and Hes5 levels by Western blotting at day 4 of neuronal differentiation. D, image analysis showing integrated pixel intensity of the immunoreactivity of Notch1 signaling components. ***, p 0.001 by paired Student’s t test. *, p 0.05 compared with LV-control-infected cells by one-way ANOVA with post hoc Tukey’s test.

Article Snippet: Double Immunocytochemical Analysis and Confocal Microscopy—Neuronal differentiated ARH-NPCs grown on coverslips were double labeled with polyclonal antibodies against -syn and Notch1 (1:1000) and detected with the Tyramide Signal AmplificationTM-Direct (Red) system (1:100; PerkinElmer Life Sciences), and the mouse monoclonal antibodies against tubulin III (Tuj1 clone; 1:100) or p53 (1:500), detected with FITC-conjugated secondary antibodies (1:75; Vector Laboratories, Burlingame, CA) (24).

Techniques: Expressing, Derivative Assay, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Infection, Comparison, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: α-Synuclein Induces Alterations in Adult Neurogenesis in Parkinson Disease Models via p53-mediated Repression of Notch1

doi: 10.1074/jbc.m112.354522

Figure Lengend Snippet: FIGURE 3. Decreased endogenous -synuclein levels alter neuronal maturation in adult rat hippocampus neural progenitor cells. A, ARH-NPCs were infected with a lentiviral vector expressing a shRNA directed to the rat -syn (LV-shRNA--syn::GFP) or an shRNA specific for luciferase as control (LV-shRNA-Luc::GFP) at a MOI of 30. 48 h post-infection, when endogenous expression of -syn reached the lowest level, neuronal differentiation was started (day 0). Immunohistochemical analysis of the expression of tubulin III (as a marker of neuronal maturation), PCNA (as a marker of proliferation), active caspase 3 (apoptosis marker), and Notch1 was performed at days 0, 2, 4, and 8 of neuronal differentiation. Compared with LV-shRNA-Luc, the NPC-derived neural progeny from LV-shRNA--syn-infected cells showed an earlier appearance of more mature neuronal phenotypes as evidenced by longer -tubulin-immu- noreactive neurites, whereas no significant changes were detected in proliferation, apoptosis, or Notch1 expression. Scale bar, 10 m. B–E, quantitative image analysis showing levels of tubulin III, PCNA, active caspase 3, and Notch1. *, p 0.05 compared with LV-shRNA-Luc-infected cells by one-way ANOVA with post hoc Tukey’s test.

Article Snippet: Double Immunocytochemical Analysis and Confocal Microscopy—Neuronal differentiated ARH-NPCs grown on coverslips were double labeled with polyclonal antibodies against -syn and Notch1 (1:1000) and detected with the Tyramide Signal AmplificationTM-Direct (Red) system (1:100; PerkinElmer Life Sciences), and the mouse monoclonal antibodies against tubulin III (Tuj1 clone; 1:100) or p53 (1:500), detected with FITC-conjugated secondary antibodies (1:75; Vector Laboratories, Burlingame, CA) (24).

Techniques: Infection, Plasmid Preparation, Expressing, shRNA, Luciferase, Control, Immunohistochemical staining, Marker, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: α-Synuclein Induces Alterations in Adult Neurogenesis in Parkinson Disease Models via p53-mediated Repression of Notch1

doi: 10.1074/jbc.m112.354522

Figure Lengend Snippet: FIGURE 4. p53 and -syn binding to Notch1 promoter. A, schematic representation of a proximal regulatory region in the rat Notch1 promoter depicting two potential p53 response elements (RE900 and RE500). Boxes show the sequence of the RE. The bold bigger font represents the core element, and bold smaller fonts show regulatory flanking sequences. Capital letters indicate conserved residues from the consensus site, and lowercase letters show deviation from consensus. Arrows indicate the approximate position of the primers used in PCR amplification. B, binding of endogenous p53 to the Notch1 promoter was assessed in vivo by ChIP assays in B103 cells infected with LV-control (LV-CT) or LV--syn. ChIP was performed using specific antibodies against p53 or control IgG. Input chromatin represents the portion of the enzymatic-sheared chromatin prior to immunoprecipitation. The immunoprecipitated chromatin was analyzed by PCR amplification with primers flanking each RE tested. C, quantification of the immunoprecipitated DNA was analyzed by quantitative real time PCR and is shown as a percentage of the total DNA input. D, EMSAs show shifts in mobility when increasing concentrations of human recombinant -syn (2 and 5 g) were incubated with both RE900 and RE500, suggesting direct binding to DNA. -Synuclein did not alter the mobility of a control double-stranded randomized oligonucleotide.

Article Snippet: Double Immunocytochemical Analysis and Confocal Microscopy—Neuronal differentiated ARH-NPCs grown on coverslips were double labeled with polyclonal antibodies against -syn and Notch1 (1:1000) and detected with the Tyramide Signal AmplificationTM-Direct (Red) system (1:100; PerkinElmer Life Sciences), and the mouse monoclonal antibodies against tubulin III (Tuj1 clone; 1:100) or p53 (1:500), detected with FITC-conjugated secondary antibodies (1:75; Vector Laboratories, Burlingame, CA) (24).

Techniques: Binding Assay, Sequencing, Amplification, In Vivo, Infection, Control, Immunoprecipitation, Real-time Polymerase Chain Reaction, Recombinant, Incubation

Journal: Journal of Biological Chemistry

Article Title: α-Synuclein Induces Alterations in Adult Neurogenesis in Parkinson Disease Models via p53-mediated Repression of Notch1

doi: 10.1074/jbc.m112.354522

Figure Lengend Snippet: FIGURE 5. p53 is a negative regulator of Notch1 promoter-driven transcription and -syn further represses its expression. A, schematic representation of Notch1 promoter fragments fused to luciferase-reporter vectors to test the effects of p53 on the transcription driven by Notch1 promoter. NP1002 expands from 1002 to 81 and contains both RE900 and RE500. NP580 contains the fragment 580 to 81 and only RE500. Promoter-less vector pGLuc Basic was used as control of basal luciferase expression. B, transfection of 293T cells with NP1002 or NP580 resulted in a more than 4-fold increase of luciferase expression in comparison with pGluc control plasmid. ***, p 0.001 by paired Student’s t test. C and D, p53 represses the transcription from NP1002 (C) and NP580 (D) as observed by a decrease in luciferase expression in cells transfected with 2 g of a plasmid expressing rat p53. Overexpression of -syn by infection of the cells with LV--syn aggravates the repression of transcription mediated by p53 only for NP1002. ***, p 0.001; **, p 0.01 compared with control cells by one-way ANOVA with post hoc Tukey’s test. E, transfection of 293T cells with increasing amounts of the p53-expression plasmid resulted in a 10% increase of cell death relative to mock-transfected cells.

Article Snippet: Double Immunocytochemical Analysis and Confocal Microscopy—Neuronal differentiated ARH-NPCs grown on coverslips were double labeled with polyclonal antibodies against -syn and Notch1 (1:1000) and detected with the Tyramide Signal AmplificationTM-Direct (Red) system (1:100; PerkinElmer Life Sciences), and the mouse monoclonal antibodies against tubulin III (Tuj1 clone; 1:100) or p53 (1:500), detected with FITC-conjugated secondary antibodies (1:75; Vector Laboratories, Burlingame, CA) (24).

Techniques: Expressing, Luciferase, Plasmid Preparation, Control, Transfection, Comparison, Over Expression, Infection

Journal: Journal of Biological Chemistry

Article Title: α-Synuclein Induces Alterations in Adult Neurogenesis in Parkinson Disease Models via p53-mediated Repression of Notch1

doi: 10.1074/jbc.m112.354522

Figure Lengend Snippet: FIGURE 7. Knockdown of p53 expression partially rescues the repression of Notch1 transcription induced by -syn. A, delivery of specific siRNA directed toward rat p53 by lentiviral vectors (LV-si p53) to ARH-NPCs resulted in 65% reduction of p53 protein levels 48 h post-infection. B, quantitative real time PCR analysis of the mRNA levels of Notch1 in NPC-derived neuronal progeny of cells infected with LV--syn in comparison with LV-control-infected cells and co-infected with LV-si p53 to knock down p53 expression, or LV-si Luc as control for double viral infection. **, p 0.01 by paired Student’s t test. C, immuno- histochemical analysis of p53 and Notch1 expression in the NPC-derived neuronal cells infected with LV--syn or control and with or without knocking down of p53 expression (si p53 or si Luc). Compared with LV--syn/si Luc cells, the cells that received si p53 show higher expression of Notch1, suggesting release of transcriptional repression. Scale bar, 20 m. D and E, quantitative image analysis showing levels of p53 and Notch1. F, expression of human -syn in cells infected with the different lentiviral constructs assayed, showing robust expression also in cells co-infected with si p53 and si Luc. * and #, p 0.05 compared with control cells by one-way ANOVA with post hoc Tukey’s test.

Article Snippet: Double Immunocytochemical Analysis and Confocal Microscopy—Neuronal differentiated ARH-NPCs grown on coverslips were double labeled with polyclonal antibodies against -syn and Notch1 (1:1000) and detected with the Tyramide Signal AmplificationTM-Direct (Red) system (1:100; PerkinElmer Life Sciences), and the mouse monoclonal antibodies against tubulin III (Tuj1 clone; 1:100) or p53 (1:500), detected with FITC-conjugated secondary antibodies (1:75; Vector Laboratories, Burlingame, CA) (24).

Techniques: Knockdown, Expressing, Infection, Real-time Polymerase Chain Reaction, Derivative Assay, Comparison, Control, Construct