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: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The Hippo pathway effector TAZ induces intrahepatic cholangiocarcinoma in mice and is ubiquitously activated in the human disease

doi: 10.1186/s13046-022-02394-2

Figure Lengend Snippet: Primary antibodies used for immunohistochemistry (IHC) and Western blot analysis (WB)

Article Snippet: Notch2 , IHC , 1:50 , Cell Signaling Technology , 5732.

Techniques: Immunohistochemistry, Western Blot, Concentration Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The Hippo pathway effector TAZ induces intrahepatic cholangiocarcinoma in mice and is ubiquitously activated in the human disease

doi: 10.1186/s13046-022-02394-2

Figure Lengend Snippet: Validated Gene Expression Assays used for real-time qRT-PCR experiments

Article Snippet: Notch2 , IHC , 1:50 , Cell Signaling Technology , 5732.

Techniques: Gene Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The Hippo pathway effector TAZ induces intrahepatic cholangiocarcinoma in mice and is ubiquitously activated in the human disease

doi: 10.1186/s13046-022-02394-2

Figure Lengend Snippet: Liver lesions developed in AKT/TAZ mice display activation of Hippo and AKT/mTOR pathways. ( A ) Representative immunohistochemical patterns of a cholangiocellular tumor exhibiting immunoreactivity for HA-Tag(AKT) and nuclear TAZ, as well as for downstream effectors of the Hippo (NOTCH1, NOTCH2, and JAG1) and AKT/mTOR (phosphorylated/activated AKT or p-AKT; phosphorylated/inactivated GSK-3β or p-GSK-3β; and phosphorylated/activated RPS6 or p-RPS6) pathways. Original magnification: 200x; scale bar: 100 μm. ( B ) Upregulation of the Hippo pathway targets CCN1 , CCN2 , and NOTCH 2 in AKT/TAZ livers (10 weeks post-injection) compared with livers injected with the empty vector (Vector), as assessed by quantitative real-time RT-PCR. Abbreviations: H&E, hematoxylin and eosin staining; ST, non-tumorous surrounding tissue; T, tumor. Data are expressed as means ± SD. *** p < 0.0001 and ** p < 0.01 vs. empty vector-injected mice

Article Snippet: Notch2 , IHC , 1:50 , Cell Signaling Technology , 5732.

Techniques: Activation Assay, Immunohistochemical staining, Injection, Plasmid Preparation, Quantitative RT-PCR, Staining

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: Cell Death & Disease

Article Title: The bHLH transcription factor DEC1 promotes thyroid cancer aggressiveness by the interplay with NOTCH1

doi: 10.1038/s41419-018-0933-y

Figure Lengend Snippet: a , b qRT-PCR analysis ( a ) and Western blot analysis ( b ) of Notch1 expression in DEC1 silenced or control TPC1 cells. c Western blot analysis of NOTCH1 in TPC1 cells transfected in transient with a DEC1 overexpressing plasmid. Western blot for beta-actin was used as loading control. d , e qRT-PCR and western blot analysis of NOTCH target-genes in DEC1 silenced or control cells. f Analysis of cell proliferation in DEC1 overexpressing clones upon DAPT treatment. The histograms represent the percentage of cells in the indicated conditions relatively to NI samples 72 h after DAPT treatment. g Analysis of cell invasion in DEC1 overexpressing clones upon DAPT treatment. The histogram represents the percentage of invading cells in the indicated conditions relatively to NI samples h , i qRT-PCR ( h ) and western blot analysis ( i ) of DEC1 expression in TPC1 cells treated or not with DAPT. j qRT-PCR of DEC1 expression in TPC1 cells transfected with NOTCH1 specific siRNA or with scramble oligos

Article Snippet: Immunohistochemistry was performed on 4 μm formalin-fixed, paraffin-embedded tissue sections with a 1:300 dilution of rabbit polyclonal anti-human DEC1 antibody (Bethyl Laboratories, Inc. Montgomery, TX) and NOTCH1 antibody (D1E11-Cell Signaling) for 32′ at 37°.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Control, Transfection, Plasmid Preparation, Clone Assay

Journal: Cell Death & Disease

Article Title: The bHLH transcription factor DEC1 promotes thyroid cancer aggressiveness by the interplay with NOTCH1

doi: 10.1038/s41419-018-0933-y

Figure Lengend Snippet: Correlation analysis of DEC1 and NOTCH1 expression in thyroid cancer samples

Article Snippet: Immunohistochemistry was performed on 4 μm formalin-fixed, paraffin-embedded tissue sections with a 1:300 dilution of rabbit polyclonal anti-human DEC1 antibody (Bethyl Laboratories, Inc. Montgomery, TX) and NOTCH1 antibody (D1E11-Cell Signaling) for 32′ at 37°.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: The bHLH transcription factor DEC1 promotes thyroid cancer aggressiveness by the interplay with NOTCH1

doi: 10.1038/s41419-018-0933-y

Figure Lengend Snippet: a Volcano Plot showing the distribution of RNA-Seq data. b , c Histograms representing the distribution of log2 fold change. d , e Protein–protein interaction network showing DEC1 target genes involved in cell cycle regulation and DNA stability. f qRT-PCR validation analysis of a panel of DEC1 target genes within the network shown in ( c ). Histogram represents the relative fold change ± SD of these genes in DEC1 silenced TPC1 cells as compared to control cells (set as 1, black line). g qRT-PCR analysis of selected DEC1 target genes in TPC1 cells transfected with NOTCH1 #1 or NOTCH1#1+2 siRNA or scramble siRNA as control. Histogram represents the relative fold change ± SD of these genes in NOTCH1 silenced TPC1 cells as compared to control cells (set as 1, black line)

Article Snippet: Immunohistochemistry was performed on 4 μm formalin-fixed, paraffin-embedded tissue sections with a 1:300 dilution of rabbit polyclonal anti-human DEC1 antibody (Bethyl Laboratories, Inc. Montgomery, TX) and NOTCH1 antibody (D1E11-Cell Signaling) for 32′ at 37°.

Techniques: RNA Sequencing, Quantitative RT-PCR, Biomarker Discovery, Control, Transfection

Journal: Cell reports

Article Title: LSD1 Inhibition Promotes Epithelial Differentiation through Derepression of Fate-Determining Transcription Factors

doi: 10.1016/j.celrep.2019.07.058

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-Notch3 (D11B8) , Cell Signaling , Cat# 5276 RRID: AB_10560515.

Techniques: Virus, Transfection, Expressing, Recombinant, Isolation, Library Quantification, Control, Software

Journal: The Journal of pathology

Article Title: Alcohol drinking inhibits NOTCH–PAX9 signaling in esophageal squamous epithelial cells

doi: 10.1002/path.5602

Figure Lengend Snippet: IHC staining showed expression of NOTCH1, NOTCH2, NICD1, and HES1 in the ethanol-exposed mouse squamous epithelium as compared with controls (A). Hierarchical clustering analysis of gene microarray data (GSE75373) was performed based on differential expression of Notch components (B). qRT-PCR and Western blotting confirmed the downregulation of Hes1 mRNA (C) and NOTCH components (D) in mouse squamous epithelium due to alcohol drinking (C-E). Broken lines indicate groups of samples and joining of panels. * p < 0.05, ** p < 0.01. Bar represented mean ± SD (n=3/group). Scale bar=50 μm. P values were determined using the Student’s t-test.

Article Snippet: For IHC staining, deparaffinized sections were pretreated to retrieve antigens with a Tris-based Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA, USA), before blocking with 10% normal serum and then applying either a rabbit monoclonal anti-NOTCH1 antibody (1:200, Cat #: D1E11, Cell Signaling Technology), a rabbit polyclonal anti-NOTCH2 antibody (1:100, Cat #: ab52302, Abcam), a rabbit polyclonal anti-activated NOTCH1 antibody (1:100, Cat #: ab8925, Abcam), a rabbit monoclonal anti-PAX9 antibody (1:50, Cell Signaling Technology) or a rabbit monoclonal anti-HES1 antibody (1:50, Cell Signaling Technology) at 4°C for overnight.

Techniques: Immunohistochemistry, Expressing, Microarray, Quantitative Proteomics, Quantitative RT-PCR, Western Blot

Journal: Nature Communications

Article Title: Plasma membrane-derived extracellular microvesicles mediate non-canonical intercellular NOTCH signaling

doi: 10.1038/s41467-017-00767-2

Figure Lengend Snippet: Enrichment of NOTCH pathway components in ARMMs

Article Snippet: Primary antibodies used in the study include rabbit polyclonal ARRDC1 antibody as described previously , mouse monoclonal CD9 antibody (Santa Cruz, catalogue #sc-13118, at 1:1000 dilution), rabbit monoclonal GFP antibody (Cell Signaling, catalogue #2956S, at 1:1000 dilution) and rabbit monoclonal NOTCH2 and ITCH antibodies (Cell Signaling, catalogue #4530S, 12117S, both at 1:2000 dilution).

Techniques:

Journal: Nature Communications

Article Title: Plasma membrane-derived extracellular microvesicles mediate non-canonical intercellular NOTCH signaling

doi: 10.1038/s41467-017-00767-2

Figure Lengend Snippet: NOTCH2 receptor is secreted into ARMMs. a Schematic drawing of domains in NOTCH2 receptor. Relative positions of peptides identified by mass spectrometry were indicated. ECD: extracellular domain; TM: transmembrane domain; NICD: NOTCH intracellular domain. b Western blotting of NOTCH2 protein in ARMMs. HEK293T cells were transfected with GFP or ARRDC1-GFP. Extracellular vesicles were pelleted by ultracentrifugation. Both cell lysates and extracellular vesicle ( EV ) pellets were used for anti-NOTCH2, anti-ARRDC1 and anti-CD9 western blotting. c Effect of ARRDC1 knockout on NOTCH release into ARMMs. CRISPR was used to knock out the ARRDC1 gene in HEK293T cells. EVs from conditioned culture media of ARRDC1-KO and wild-type HEK293T cells were pelleted and subjected to western blotting along with corresponding cell lysates. d Re-expression of ARRDC1 restored NOTCH2 release into ARMMs in ARRDC1-KO cells. ARRDC1-KO HEK293T cells were transfected with the indicated amount of ARRDC1-GFP construct. Two days after transfection, EVs were pelleted from the conditioned media and subjected to western blotting along with the cell lysates

Article Snippet: Primary antibodies used in the study include rabbit polyclonal ARRDC1 antibody as described previously , mouse monoclonal CD9 antibody (Santa Cruz, catalogue #sc-13118, at 1:1000 dilution), rabbit monoclonal GFP antibody (Cell Signaling, catalogue #2956S, at 1:1000 dilution) and rabbit monoclonal NOTCH2 and ITCH antibodies (Cell Signaling, catalogue #4530S, 12117S, both at 1:2000 dilution).

Techniques: Mass Spectrometry, Western Blot, Transfection, Knock-Out, CRISPR, Expressing, Construct

Journal: Nature Communications

Article Title: Plasma membrane-derived extracellular microvesicles mediate non-canonical intercellular NOTCH signaling

doi: 10.1038/s41467-017-00767-2

Figure Lengend Snippet: ITCH interacts with ARRDC1 and mediates NOTCH2 incorporation into ARMMs. a ARMMs contain ITCH. HEK293T cells were transfected with GFP or ARRDC1-GFP. EVs were pelleted by ultracentrifugation. Both cell lysates and EVs were used for anti-ITCH, anti-ARRDC1 and anti-CD9 western blotting. b Effect of ARRDC1 knockout on ITCH release into EVs. EVs from conditioned culture media of ARRDC1-KO and wild-type HEK293T cells were pelleted and subjected to anti-ITCH, anti-ARRDC1 and anti-CD9. c Co-immunoprecipitation ( IP ) showing the interaction between ARRDC1 and ITCH. HEK293T cells were co-transfected with Flag-tagged ITCH and one of the following: control vector, HA-tagged-ARRDC1 or ARRDC1-delPPXY (deletion of two PPXY motifs). Two days after transfection, cell lysates were collected and incubated with anti-HA agarose beads for immunoprecipitation. IP-ed complexes were subjected to anti-HA or anti-Flag western blotting. d Effect of ITCH knockdown on NOTCH2 release into ARMMs. HEK293T cells were co-transfected with ITCH siRNA (or scrambled control) and ARRDC1-GFP (or control GFP). Three days after transfection, EVs from conditioned culture media were pelleted and subjected to western blotting. The amount of NOTCH2 in EV was quantified. Data represent an average of three independent repeats. e Model depicting the role of ITCH on NOTCH2 release into ARMMs

Article Snippet: Primary antibodies used in the study include rabbit polyclonal ARRDC1 antibody as described previously , mouse monoclonal CD9 antibody (Santa Cruz, catalogue #sc-13118, at 1:1000 dilution), rabbit monoclonal GFP antibody (Cell Signaling, catalogue #2956S, at 1:1000 dilution) and rabbit monoclonal NOTCH2 and ITCH antibodies (Cell Signaling, catalogue #4530S, 12117S, both at 1:2000 dilution).

Techniques: Transfection, Western Blot, Knock-Out, Immunoprecipitation, Control, Plasmid Preparation, Incubation, Knockdown

Journal: Nature Communications

Article Title: Plasma membrane-derived extracellular microvesicles mediate non-canonical intercellular NOTCH signaling

doi: 10.1038/s41467-017-00767-2

Figure Lengend Snippet: ARMMs deliver functional NOTCH2 to recipient cells. a Schematic drawing of the transwell assay in which cultured recipient and donor cells were separated by a 0.4 μm membrane. b Western blotting of NOTCH2 (GFP-tagged) in donor and recipient cells. HEK293T cells were transfected with GFP, NOTCH2 delECD -GFP (NOTCH2 used here does not contain the extracellular domain) and ARRDC1-GFP. Twenty-four hours later, the transfected cells were seeded as donor cells in transwells and co-cultured with recipient HEK293T cells. Recipient cells were washed with PBS and lysed for anti-GFP western blotting analysis. GAPDH western blotting was done to ensure equal loading. Asterisk indicates a non-specific protein band. c Expression of NOTCH target genes in recipient cells. Donor HEK293T cells were transfected with GFP, NOTCH2 delECD -GFP or NOTCH2 delECD -GFP plus HA-ARRDC1, and co-cultured with recipient HEK293T cells in transwells for 48 h. RNAs were then extracted from the recipient cells and used for qRT-PCR to measure the expression of HES1 and HES5 genes. d qRT-PCR data showing HES1/HES5 mRNA levels in recipient cells that received ARMMs from control or ARRDC1-KO HEK293T cells. Data represent an average of three independent repeats for each condition. Error bars indicate standard deviation (s.d.). * p < 0.05

Article Snippet: Primary antibodies used in the study include rabbit polyclonal ARRDC1 antibody as described previously , mouse monoclonal CD9 antibody (Santa Cruz, catalogue #sc-13118, at 1:1000 dilution), rabbit monoclonal GFP antibody (Cell Signaling, catalogue #2956S, at 1:1000 dilution) and rabbit monoclonal NOTCH2 and ITCH antibodies (Cell Signaling, catalogue #4530S, 12117S, both at 1:2000 dilution).

Techniques: Functional Assay, Transwell Assay, Cell Culture, Membrane, Western Blot, Transfection, Expressing, Quantitative RT-PCR, Control, Standard Deviation

Journal: Nature Communications

Article Title: Plasma membrane-derived extracellular microvesicles mediate non-canonical intercellular NOTCH signaling

doi: 10.1038/s41467-017-00767-2

Figure Lengend Snippet: Roles of ADAM10 and γ-secretase in NOTCH2 release and activation. a Schematic drawing of NOTCH2 receptor with indicated cleavage sites for ADAM10 and γ-secretase. b Effect of ADAM10 knockdown on NOTCH2 release into ARMMs. Left panel : qRT-PCR data showing siRNA-mediated knockdown of ADAM10 in HEK293T cells. Right panel : Western blot analysis. HEK293T cells were transfected with scrambled control or ADMA10 siRNAs. EVs and cell lysates were collected and subjected to western blotting. c Effect of γ-secretase inhibitor on NOTCH activation in recipient cells. HEK293T donor cells were transfected with NOTCH2 delECD -GFP alone or together with HA-ARRDC1. Donor cells were then co-cultured with recipient HEK293T cells in the absence or presence of 100 nM γ-secretase inhibitor DAPT. qRT-PCR data showed the expression of HES genes in recipient cells. Data represent an average of three independent repeats for each condition. Error bars indicate standard deviation (s.d.). * p < 0.05. d Proposed model for ARMMs-mediated non-canonical NOTCH2 signaling

Article Snippet: Primary antibodies used in the study include rabbit polyclonal ARRDC1 antibody as described previously , mouse monoclonal CD9 antibody (Santa Cruz, catalogue #sc-13118, at 1:1000 dilution), rabbit monoclonal GFP antibody (Cell Signaling, catalogue #2956S, at 1:1000 dilution) and rabbit monoclonal NOTCH2 and ITCH antibodies (Cell Signaling, catalogue #4530S, 12117S, both at 1:2000 dilution).

Techniques: Activation Assay, Knockdown, Quantitative RT-PCR, Western Blot, Transfection, Control, Cell Culture, Expressing, Standard Deviation

Journal: Scientific reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate.

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Figure 1. The stable over-expression of each one of the Notch genes on 3T3-L1 cells enhances adipogenesis. (A) qRT-PCR analysis of the relative mRNA expression levels of the adipocyte markers aP2 and Pparg in differentiated 3T3-L1 cells. (B) qRT-PCR analysis of the relative Notch and Hes1 mRNA expression levels in differentiated 3T3-L1 cells. Representative Western blots (C) and densitometric analysis (D) of each NOTCH receptor expression in 3T3-L1 adipocytes compared to non-differentiated cells. In the case of the Notch1 gene transfectant intracellular NOTCH1 (NICD1) and complete NOTCH1 protein signals are shown. In the case of the Notch2 gene transfectant, the intracellular NOTCH2 (NICD2) protein signal is shown. For the Notch3 gene transfectant, the complete and the intracellular NOTCH3 (NICD3) protein signals are shown. Finally, for the Notch4 gene transfectant, the complete and the intracellular NOTCH4 (NICD4) protein signals are shown. (E) qRT-PCR analysis of the relative aP2 and Pparg mRNA expression levels in differentiated stable Notch1 gene transfectant (L1-N1D), stable Notch2 gene transfectant (L1-N2D), stable Notch3 gene transfectant (L1-N3D), and stable gene Notch4 transfectant (L1-N4D). Data from qRT-PCR assays were previously normalized to P0 mRNA expression levels. The expression of alpha-tubulin was used as a loading control in all Western blots to normalize expression data. Blot signals from empty vector and over-expressing cells were cropped from original blots and delineated with horizontal white spaces (original blots for each protein signal are shown in Supplementary Figure 4). The fold activation or inhibition was calculated relative to the seven-day differentiated non-transfected or empty-vector-transfected cells, which was set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t-tests is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: Protein Dilution of primary and secondary antibodies Company NOTCH1 Rabbit anti-NOTCH1 C20R (1:1000) Santa Cruz Biotechnology Active NICD1 Rabbit anti-Cleaved NOTCH1 (Val 1744) (1:000) Cell signaling NOTCH2 Goat anti-NOTCH2 M20 (1:500) Santa Cruz Biotechnology NOTCH3 Rabbit anti-NOTCH3 (1:1000) Abcam NOTCH4 Rabbit anti-NOTCH4 (1:1000) Upstate Millipore HA Mouse anti-HA 16B12 (1:5000) Covance DLK1 Rabbit anti-DLK1 (1:1000) Nueda et al.49 DLK2 Rabbit anti-DLK2 (1:500) Proteintech α-tubulin Mouse anti-alpha-Tubulin (1:5000) Sigma Table 2.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Transfection, Control, Plasmid Preparation, Activation Assay, Inhibition

Journal: Scientific reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate.

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Figure 2. Effects of stable over-expression of each one of the Notch genes in 3T3-L1 adipocyte browning. (A) Representative microscopy images (400X magnification) of 3T3-L1 adipocytes (L1D) seven days after standard adipogenic induction (48 hours with IBMX and dexamethasone, and 5 days with insulin, see Methods) and non-treated 3T3-L1 cells (L1C). Scale bar (250 μm) is shown. (B) qRT-PCR mRNA expression analysis of the brown adipocyte markers Ucp1, Pgc1a, Gyk, Prdm16, Cidea and Sirt1 in seven-day-differentiated 3T3-L1 cells. qRT-PCR analysis of the relative mRNA expression levels of Ucp1, Pgc1a, Gyk, Prdm16, Cidea and Sirt1 markers in seven-day differentiated Notch1 gene transfectant (L1-N1D) (C), Notch2 gene transfectant (L1-N2D) (D), Notch3 gene transfectant (L1-N3D) (E), Notch4 gene transfectant (L1-N4D) (F). Data from qRT-PCR assays were previously normalized to P0 mRNA expression levels. qPCR analysis of mitochondrial CytB DNA amplification (related to genomic ApoB DNA amplification, see Methods) in seven-day differentiated 3T3-L1 cells over-expressing Notch1 gene (G) and Notch2, Notch3 or Notch4 genes (H). The fold activation or inhibition was calculated relative to the seven-day differentiated non-transfected or empty-vector-transfected cells, which was set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance of the Student’s t-tests performed is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: Protein Dilution of primary and secondary antibodies Company NOTCH1 Rabbit anti-NOTCH1 C20R (1:1000) Santa Cruz Biotechnology Active NICD1 Rabbit anti-Cleaved NOTCH1 (Val 1744) (1:000) Cell signaling NOTCH2 Goat anti-NOTCH2 M20 (1:500) Santa Cruz Biotechnology NOTCH3 Rabbit anti-NOTCH3 (1:1000) Abcam NOTCH4 Rabbit anti-NOTCH4 (1:1000) Upstate Millipore HA Mouse anti-HA 16B12 (1:5000) Covance DLK1 Rabbit anti-DLK1 (1:1000) Nueda et al.49 DLK2 Rabbit anti-DLK2 (1:500) Proteintech α-tubulin Mouse anti-alpha-Tubulin (1:5000) Sigma Table 2.

Techniques: Over Expression, Microscopy, Quantitative RT-PCR, Expressing, Transfection, DNA Amplification, Activation Assay, Inhibition, Plasmid Preparation

Journal: Scientific reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate.

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Figure 4. Release of glycerol and lactate to the extracellular medium in 3T3-L1 adipocytes over-expressing Dlk or Notch genes. Relative levels of glycerol released to the extracellular medium in response to isoproterenol from Dlk1 or Dlk2 genes (A), and Notch1, 2, 3 or 4 genes (B) over-expressing adipocytes. (C) Representative microscopy images (400X magnification) of non-transfected (L1C) and transfected 3T3-L1 adipocytes (Empty vectors V1, V2, V3 and V4, and their corresponding over-expressing transfectant) under study. The size of their lipid droplets is showed. Scale bar (80 μm) is shown. Relative levels of lactate in the culture supernatant of differentiated non-transfected 3T3-L1 cells (D), Dlk1 or Dlk2 genes over-expressing adipocytes (E), and each of the Notch genes over-expressing adipocytes (F). The fold activation or inhibition was calculated relative to the seven-day differentiated non-transfected or empty-vector-transfected cells, which was set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t-tests is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: Protein Dilution of primary and secondary antibodies Company NOTCH1 Rabbit anti-NOTCH1 C20R (1:1000) Santa Cruz Biotechnology Active NICD1 Rabbit anti-Cleaved NOTCH1 (Val 1744) (1:000) Cell signaling NOTCH2 Goat anti-NOTCH2 M20 (1:500) Santa Cruz Biotechnology NOTCH3 Rabbit anti-NOTCH3 (1:1000) Abcam NOTCH4 Rabbit anti-NOTCH4 (1:1000) Upstate Millipore HA Mouse anti-HA 16B12 (1:5000) Covance DLK1 Rabbit anti-DLK1 (1:1000) Nueda et al.49 DLK2 Rabbit anti-DLK2 (1:500) Proteintech α-tubulin Mouse anti-alpha-Tubulin (1:5000) Sigma Table 2.

Techniques: Expressing, Microscopy, Transfection, Activation Assay, Inhibition, Plasmid Preparation

Journal: Scientific reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate.

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Figure 5. Oxygen consumption rate (OCR) in 3T3-L1 adipocytes over-expressing Dlk and Notch genes. Analysis of the relative oxygen consumption rate (OCR) in non-transfected 3T3-L1 cells (A) and 3T3-L1 cells over-expressing Dlk1 or Dlk2 genes (B), and Notch1 (C), Notch2 (D), Notch3 (E) or Notch4 genes (F). The fold activation or inhibition was calculated relative to the time 0 of seven-day differentiated non-transfected or empty-vector-transfected cells, which was set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t-tests is indicated at 120 minutes (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: Protein Dilution of primary and secondary antibodies Company NOTCH1 Rabbit anti-NOTCH1 C20R (1:1000) Santa Cruz Biotechnology Active NICD1 Rabbit anti-Cleaved NOTCH1 (Val 1744) (1:000) Cell signaling NOTCH2 Goat anti-NOTCH2 M20 (1:500) Santa Cruz Biotechnology NOTCH3 Rabbit anti-NOTCH3 (1:1000) Abcam NOTCH4 Rabbit anti-NOTCH4 (1:1000) Upstate Millipore HA Mouse anti-HA 16B12 (1:5000) Covance DLK1 Rabbit anti-DLK1 (1:1000) Nueda et al.49 DLK2 Rabbit anti-DLK2 (1:500) Proteintech α-tubulin Mouse anti-alpha-Tubulin (1:5000) Sigma Table 2.

Techniques: Expressing, Transfection, Activation Assay, Inhibition, Plasmid Preparation

Journal: Scientific reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate.

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Figure 7. NOTCH activation and signaling in 3T3-L1 cells stably over-expressing each one of the four NOTCH receptors. (A) qRT-PCR analysis of the relative Hes1 and Hey1 mRNA expression levels in the stable Notch1 gene transfectant (L1-N1), the stable Notch2 gene transfectant (L1-N2), the stable Notch3 gene transfectant (L1-N3), and the stable Notch4 gene transfectant (L1-N4). (B) NOTCH transcriptional activity, as measured by gene reporter luciferase assays, in these four Notch genes stable transfectants. (C) qRT-PCR analysis of the relative individual Notch mRNA expression levels in stable Notch1 gene transfectant (L1-N1), the stable Notch2 gene transfectant (L1-N2), the stable Notch3 gene transfectant (L1-N3), and the stable Notch4 gene transfectant (L1-N4). The relative luciferase activities were always normalized with renilla values and referred to those of control cells. Data in all qRT-PCR assays were normalized to P0 mRNA expression levels. The fold activation or inhibition in all assays is measured relative to the empty vector control, set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance of Student’s t-tests results is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: Protein Dilution of primary and secondary antibodies Company NOTCH1 Rabbit anti-NOTCH1 C20R (1:1000) Santa Cruz Biotechnology Active NICD1 Rabbit anti-Cleaved NOTCH1 (Val 1744) (1:000) Cell signaling NOTCH2 Goat anti-NOTCH2 M20 (1:500) Santa Cruz Biotechnology NOTCH3 Rabbit anti-NOTCH3 (1:1000) Abcam NOTCH4 Rabbit anti-NOTCH4 (1:1000) Upstate Millipore HA Mouse anti-HA 16B12 (1:5000) Covance DLK1 Rabbit anti-DLK1 (1:1000) Nueda et al.49 DLK2 Rabbit anti-DLK2 (1:500) Proteintech α-tubulin Mouse anti-alpha-Tubulin (1:5000) Sigma Table 2.

Techniques: Activation Assay, Stable Transfection, Expressing, Quantitative RT-PCR, Transfection, Activity Assay, Luciferase, Control, Inhibition, Plasmid Preparation

Journal: Scientific reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate.

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Figure 8. Feedback modulation among Notch and Dlk gene expression in 3T3-L1 preadipocytes. (A) qRT- PCR analysis of the relative individual Dlk (B) mRNA expression levels in the stable Notch1 gene transfectant (L1-N1), the stable Notch2 gene transfectant (L1-N2), the stable Notch3 gene transfectant (L1-N3), and the stable Notch4 gene transfectant (L1-N4). (B) qRT-PCR analysis of the relative Hes1 and Dlk mRNA expression levels in the stable Hes1 gene transfectant (L1-H1). (C) qRT-PCR analysis of the relative Hes1 and Hey1 mRNA expression levels in the stable Dlk1 gene transfectant (L1-DLK1) and the stable Dlk2 gene transfectant (L1- DLK2). qRT-PCR analysis of the relative Notch (D) and Dlk (E) mRNA expression levels in the stable Dlk1 gene transfectant (L1-DLK1), and the stable Dlk2 gene transfectant (L1-DLK2). In all qRT-PCR assays, data were normalized to P0 mRNA expression levels. The fold activation or inhibition in all assays was measured relative to the empty vector, set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance of Student’s t-tests results is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: Protein Dilution of primary and secondary antibodies Company NOTCH1 Rabbit anti-NOTCH1 C20R (1:1000) Santa Cruz Biotechnology Active NICD1 Rabbit anti-Cleaved NOTCH1 (Val 1744) (1:000) Cell signaling NOTCH2 Goat anti-NOTCH2 M20 (1:500) Santa Cruz Biotechnology NOTCH3 Rabbit anti-NOTCH3 (1:1000) Abcam NOTCH4 Rabbit anti-NOTCH4 (1:1000) Upstate Millipore HA Mouse anti-HA 16B12 (1:5000) Covance DLK1 Rabbit anti-DLK1 (1:1000) Nueda et al.49 DLK2 Rabbit anti-DLK2 (1:500) Proteintech α-tubulin Mouse anti-alpha-Tubulin (1:5000) Sigma Table 2.

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Transfection, Activation Assay, Inhibition, Plasmid Preparation

Journal: bioRxiv

Article Title: NOTCH3 signalling controls human trophoblast stem cell expansion and differentiation

doi: 10.1101/2023.07.03.547490

Figure Lengend Snippet: (A-B) Relative mRNA expression in matched CTBs and STBs, purified by a two-step protease digestion protocol from first trimester placentae (n=7, 6 th to 12 th week), was measured by RT-qPCR (duplicates). Data were normalized to transcript levels of TATA box binding protein ( TBP ). Graphs represent mean values±s.e.m. Asterisks denote statistical differences (*P<0.05, **P<0.01, ***P<0.001) as determined by unpaired, two-tailed Student’s t -test. ns, not significant. nd, not detectable. AU, arbitrary units; (C-D) Representative Western blots showing protein expression in the isolated trophoblast samples of early placentae (n=3, 7 th to 9 th week). GAPDH was used as a loading control. (E) In situ localization in first trimester placental tissues. Representative immunofluorescence images of placental sections from 6 th (n=3) and 12 th week (n=3) of gestation are shown. Stippled line indicates the border between villous cytotrophoblast (CTB) and syncytiotrophoblast (STB). Higher magnifications of inset pictures are shown in the bottom right corner (digitally zoomed). Scale bar: 50 µm. TEAD4 and CDH1 (E-cadherin) mark the CTB cell layer. Nuclei are stained with DAPI. Images with 10 µm scale bar depict selected CTB nuclei expressing NOTCH3-ICD. VC, villous core;

Article Snippet: Immunoprecipitations were performed using NOTCH3 or MAML1 antibodies, as well as appropriate rabbit IgG controls (listed in Table S1) according to manufacturer’s instructions (Cell Signaling, no. 73778).

Techniques: Expressing, Purification, Quantitative RT-PCR, Binding Assay, Two Tailed Test, Western Blot, Isolation, Control, In Situ, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: NOTCH3 signalling controls human trophoblast stem cell expansion and differentiation

doi: 10.1101/2023.07.03.547490

Figure Lengend Snippet: (A-B) RT-qPCR analyses measuring transcript levels in proliferating TSCs (-cAMP; n=6) and differentiated TSCs, treated with forskolin for 5 days (+cAMP, n=6). Data were normalized to TBP (AU, arbitrary units). Graphs represent mean values±s.e.m. Asterisks denote statistical differences (*P<0.05, **P<0.01, ***P<0.001) as calculated by unpaired, two-tailed Student’s t -test. ns, not significant. nd, not detectable. (C-D) Representative Western blots showing protein expression in proliferating and cAMP-differentiated TSCs (n=3). TEAD4/YAP1 and ENDOU were selected as markers of stemness and cell fusion, respectively. GAPDH was used as loading control. (E) Immunofluorescence in self-renewing and cAMP-treated TSCs (n=3). Scale bar: 25 µM. CDH1 (E-cadherin) and SDC1 were used as markers of expanding and fused TSCs, respectively. (F) Immunoprecipitation in cell lysates, prepared from proliferating TSCs (n=3), using antibodies binding NOTCH3 and MAML1, respectively. A representative example is shown. IgG was used as negative control. IP, immunoprecipitation;

Article Snippet: Immunoprecipitations were performed using NOTCH3 or MAML1 antibodies, as well as appropriate rabbit IgG controls (listed in Table S1) according to manufacturer’s instructions (Cell Signaling, no. 73778).

Techniques: Quantitative RT-PCR, Two Tailed Test, Western Blot, Expressing, Control, Immunofluorescence, Immunoprecipitation, Binding Assay, Negative Control

Journal: bioRxiv

Article Title: NOTCH3 signalling controls human trophoblast stem cell expansion and differentiation

doi: 10.1101/2023.07.03.547490

Figure Lengend Snippet: Representative Western blots detecting protein expression of trophoblast stemness- and proliferation/mitosis-associated genes upon overexpression of NOTCH3-ICD in (A) primary CTBs (n=3) and (D) TSCs (n=3). GAPDH was used as loading control. (B) Representative immunofluorescence images showing EdU labelling in TSCs that express NOTCH3-ICD. Scale bar: 50 µM. Percentage of EdU + TSCs (n=9) was measured by counting EdU + /DAPI ratio of 10 individual areas per experiment and condition, each containing between 800 and 950 nuclei. Graphs represent mean values±s.e.m. Asterisks indicate statistical differences (***P<0.001) as determined by unpaired, two-tailed Student’s t-test. (C) Immunofluorescence pictures illustrating SDC1 + areas (encircled by stippled lines) in the absence or presence of exogenous NOTCH3-ICD. A representative experiment is shown. Scale bar: 200 µM. To determine the size of SDC1 + areas, 10 different regions of NOTCH3-ICD-expressing TSCs (n=9) and controls, each containing between 650 and 800 nuclei, were evaluated. Graphs represent mean values±s.e.m. Asterisks denote statistical differences (***P<0.001) as determined by unpaired, two-tailed Student’s t-test.

Article Snippet: Immunoprecipitations were performed using NOTCH3 or MAML1 antibodies, as well as appropriate rabbit IgG controls (listed in Table S1) according to manufacturer’s instructions (Cell Signaling, no. 73778).

Techniques: Western Blot, Expressing, Over Expression, Control, Immunofluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: NOTCH3 signalling controls human trophoblast stem cell expansion and differentiation

doi: 10.1101/2023.07.03.547490

Figure Lengend Snippet: Active NOTCH3-ICD, interacting with the transcriptional co-activator MAML1, promotes self-renewal of CTB progenitors and TSCs and maintains its expression in an autocrine manner, whereas downregulation of the NOTCH3 pathway results in STB formation.

Article Snippet: Immunoprecipitations were performed using NOTCH3 or MAML1 antibodies, as well as appropriate rabbit IgG controls (listed in Table S1) according to manufacturer’s instructions (Cell Signaling, no. 73778).

Techniques: Expressing

Journal: Oncogene

Article Title: Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor suppressor activities

doi: 10.1038/onc.2014.169

Figure Lengend Snippet: A frozen vial of primary culture of EPC2 cells (27.5 PD) was thawed and grown in the presence or absence of GSI for a period indicated in ( a ). Cells were harvested at indicated time points to determine population doubling ( a ) as well as doubling time ( b ), and subjected to Western blotting ( c ) and SABG assays ( d and e ). In ( c ), β-actin served as a loading control; ICN1 Val1744 , the activated form of Notch1; p-Rb, phospho-Rb S780 . In densitometry, the signal intensity for molecule of interest was calibrated by that of β-actin at each time point. In ( d ), representative bright-field and phase contrast images demonstrate SABG-positive cells and the corresponding cells with flat and enlarged cell morphology (arrows) as scored in ( e ); *, P <0.05 vs. Day 23 and GSI (−); #, P <0.05 vs. Day 42 and GSI (−); (n=6). Note a reduced cell density at day 42 (43 PD) without GSI. Note that GSI suppressed ICN1 Val174 ( c ), preventing the extension of doubling time ( b ) as well as the induction of SABG positive cells in ( d ) and ( e ).

Article Snippet: Following electrotransfer, Immobilon-P membranes (Millipore) were incubated with primary antibodies for Notch1 (1:1000 rat monoclonal 5B5; Cell Signaling, Danvers, MA), ICN1 Val1744 (1:1000 rabbit monoclonal anticleaved NOTCH1 Val1744 D3B8; Cell Signaling), ICN3 (1:1000 rat monoclonal anti-NOTCH3 8G5; Cell Signaling), JAG1 (1:1000 rabbit monoclonal anti-Jagged1 28H8; Cell Signaling), pRb (1:1000 rabbit polyclonal anti-Phospho-Rb Ser 780 ; Cell Signaling), p16 (1:1000 mouse monoclonal anti-Human p16 G175–1239; BD Biosciences), p15 (1:200 mouse monoclonal anti-p15 15P06; Santa Cruz), p21 (1:1000 mouse monoclonal anti-Human Cip1; BD Biosciences), p53 (1:1000 Rabbit polyclonal anti-p53; Cell Signaling) IVL (1:1000 mouse monoclonal anti-Involucrin clone SY5; Sigma-Aldrich, St Louis, MO), β-actin (1:30,000 mouse monoclonal anti-β-actin AC-74; Sigma Aldrich), Cat# A5316, and then with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). β-actin served as a loading control.

Techniques: Western Blot, Control

Journal: Oncogene

Article Title: Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor suppressor activities

doi: 10.1038/onc.2014.169

Figure Lengend Snippet: EPC2-hTERT carrying ICN1 Tet-On was treated with indicated concentrations of doxycycline (DOX); or 0 µg/ml [DOX (−)] or 1 µg/ml [DOX (+)] of DOX to induce ICN1. In ( a ), ( d ) and ( e )–( f ), cells were exposed to DOX for 7 days. In ( b ) and ( c ), cells were exposed to DOX for indicated time period. Following DOX treatment, cells were analyzed by Western blotting for ICN1, phospho-Rb S780 (p-Rb), p53 and cell-cycle regulators at indicated time points with densitometry in ( a ) and ( b ); WST1 assays for cell proliferation in ( c ); flow cytometry for cell-cycle in ( d ); and SABG assays in ( e ) and ( f ). In ( a ) and ( b ), β-actin served as a loading control. * denotes transmembrane/intracellular region of endogenous Notch1 which was suppressed by RNAi directed against Notch1 (data not shown). Bracket indicates lentivirally expressed ICN1 induced by DOX. A doublet appears consistently and may represent a posttranslational modification. Or note, anit-Notch1 (5B5) antibody was used to detect lentivirally expressed ICN1 which lacks the epitope recognized by anti-ICN1 Val1744 antibody. In ( c ), *, P < 0.05 vs. DOX (−) at day 7 (n=6). In ( d ), representative histogram plots are shown. Proportions of cells in G0/G1, S and G2/M cell-cycle phases were determined. *, P < 0.05 vs. DOX (-)(n=3). In ( f ), representative bright-field and phase contrast images of SABG-positive cells with flat and enlarged cell morphology (arrows) as scored in ( e ); *, P <0.05 vs. 0 µg/ml DOX (n=6).

Article Snippet: Following electrotransfer, Immobilon-P membranes (Millipore) were incubated with primary antibodies for Notch1 (1:1000 rat monoclonal 5B5; Cell Signaling, Danvers, MA), ICN1 Val1744 (1:1000 rabbit monoclonal anticleaved NOTCH1 Val1744 D3B8; Cell Signaling), ICN3 (1:1000 rat monoclonal anti-NOTCH3 8G5; Cell Signaling), JAG1 (1:1000 rabbit monoclonal anti-Jagged1 28H8; Cell Signaling), pRb (1:1000 rabbit polyclonal anti-Phospho-Rb Ser 780 ; Cell Signaling), p16 (1:1000 mouse monoclonal anti-Human p16 G175–1239; BD Biosciences), p15 (1:200 mouse monoclonal anti-p15 15P06; Santa Cruz), p21 (1:1000 mouse monoclonal anti-Human Cip1; BD Biosciences), p53 (1:1000 Rabbit polyclonal anti-p53; Cell Signaling) IVL (1:1000 mouse monoclonal anti-Involucrin clone SY5; Sigma-Aldrich, St Louis, MO), β-actin (1:30,000 mouse monoclonal anti-β-actin AC-74; Sigma Aldrich), Cat# A5316, and then with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). β-actin served as a loading control.

Techniques: Western Blot, Flow Cytometry, Control, Modification

Journal: Oncogene

Article Title: Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor suppressor activities

doi: 10.1038/onc.2014.169

Figure Lengend Snippet: EPC2-hTERT carrying ICN1 Tet-On was stably transduced with lentivirus expressing two independent shRNA sequences directed against CSL (CSL-1 and CSL-2) or a non-silencing control scramble shRNA (Scr.) sequence. In ( b ), cells were transiently transfected with 8x CSL-luc 24 h before DOX treatment. Cells were treated with DOX at a concentration of 0 µg/ml [DOX (−)] or 1 µg/ml [DOX (+)] to induce ICN1 for 48 h in ( b ) and 7 d in ( c )–( f ). Cells were harvested at indicated time points in ( d ). Following DOX treatment, cells were subjected to quantitative RT-PCR for CSL mRNA in ( a ); luciferase assays for 8xCSL-luc reporter activity in ( b ); Western blotting for ICN1, phospho-Rb S780 (p-Rb), p53 and cell-cycle regulators in ( c ); WST1 assays for cell proliferation in ( d ); and SABG assays in ( e ) and ( f ). In ( a ), β-actin served as an internal control. *, P < 0.05 vs. Scr. (n=3). In ( b ), *, P < 0.05 vs. Scr. and DOX (−); #, P < 0.05 vs. Scr. and DOX (+); (n=3). In ( c ), β-actin served as a loading control. * denotes transmembrane/intracellular region of endogenous Notch1. Bracket indicates lentivirally expressed ICN1 induced by DOX. In ( d ), *, P < 0.05 vs. Scr. and DOX (−) at day 7; #, P < 0.05 vs. Scr. and DOX (+) at day 7; (n=6). In ( e ), representative bright-field and phase contrast images of SABG-positive cells with flat and enlarged cell morphology (arrows) as scored in ( e ); *, P < 0.05 vs. Scr. and DOX (−); #, P < 0.05 vs. Scr. and DOX (+); (n=6).

Article Snippet: Following electrotransfer, Immobilon-P membranes (Millipore) were incubated with primary antibodies for Notch1 (1:1000 rat monoclonal 5B5; Cell Signaling, Danvers, MA), ICN1 Val1744 (1:1000 rabbit monoclonal anticleaved NOTCH1 Val1744 D3B8; Cell Signaling), ICN3 (1:1000 rat monoclonal anti-NOTCH3 8G5; Cell Signaling), JAG1 (1:1000 rabbit monoclonal anti-Jagged1 28H8; Cell Signaling), pRb (1:1000 rabbit polyclonal anti-Phospho-Rb Ser 780 ; Cell Signaling), p16 (1:1000 mouse monoclonal anti-Human p16 G175–1239; BD Biosciences), p15 (1:200 mouse monoclonal anti-p15 15P06; Santa Cruz), p21 (1:1000 mouse monoclonal anti-Human Cip1; BD Biosciences), p53 (1:1000 Rabbit polyclonal anti-p53; Cell Signaling) IVL (1:1000 mouse monoclonal anti-Involucrin clone SY5; Sigma-Aldrich, St Louis, MO), β-actin (1:30,000 mouse monoclonal anti-β-actin AC-74; Sigma Aldrich), Cat# A5316, and then with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). β-actin served as a loading control.

Techniques: Stable Transfection, Transduction, Expressing, shRNA, Control, Sequencing, Transfection, Concentration Assay, Quantitative RT-PCR, Luciferase, Activity Assay, Western Blot

Journal: Oncogene

Article Title: Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor suppressor activities

doi: 10.1038/onc.2014.169

Figure Lengend Snippet: In ( a ), siRNA was designed to target p14 ARF (p14 siRNA) and p16 iNK4A (p16 siRNA) on the INK4A locus. p14/p16 siRNA was used to knockdown p14 ARF and p16 iNK4A concurrently as shown in . In ( b )–( e ), EPC2-T carrying ICN1 Tet-On was treated with 0 µg/ml [DOX (−)] or 1 µg/ml [DOX (+)] of DOX to induce ICN1 following transfection with p14 siRNA, p16 siRNA or a non-silencing control scramble short interfering RNA (Scr.). Starting 24 h after transfection, cells were treated with DOX for 7 days in ( b ), ( d ) and ( e ); and indicated time points in ( c ); and subjected to Western blotting for ICN1, phospho-Rb S780 (p-Rb), p53 and cell-cycle regulators in ( b ); WST1 assays for cell proliferation in ( c ); and SABG assays in ( d ) and ( e ). In ( b ), β-actin served as a loading control. * denotes transmembrane/intracellular region of endogenous Notch1. Bracket indicates lentivirally expressed ICN1 induced by DOX. In ( c ), *, P < 0.05 vs. Scr. and DOX (−) at day 7; #, P < 0.05 vs. Scr. and DOX (+) at day 7; ns , not significant vs. Scr. and DOX (+) at day 7 (n=6). In ( d ), representative bright-field and phase contrast images of SABG-positive cells with flat and enlarged cell morphology (arrows) as scored in ( e ); *, P < 0.05 vs. Scr. and DOX (−); #, P < 0.05 vs. Scr. and DOX (+); ns , not significant vs. Scr. and DOX (+); (n=6). Note that densitometry from ( b ) was summarized along with cell proliferation and SABG data in ( c )–( e ) in .

Article Snippet: Following electrotransfer, Immobilon-P membranes (Millipore) were incubated with primary antibodies for Notch1 (1:1000 rat monoclonal 5B5; Cell Signaling, Danvers, MA), ICN1 Val1744 (1:1000 rabbit monoclonal anticleaved NOTCH1 Val1744 D3B8; Cell Signaling), ICN3 (1:1000 rat monoclonal anti-NOTCH3 8G5; Cell Signaling), JAG1 (1:1000 rabbit monoclonal anti-Jagged1 28H8; Cell Signaling), pRb (1:1000 rabbit polyclonal anti-Phospho-Rb Ser 780 ; Cell Signaling), p16 (1:1000 mouse monoclonal anti-Human p16 G175–1239; BD Biosciences), p15 (1:200 mouse monoclonal anti-p15 15P06; Santa Cruz), p21 (1:1000 mouse monoclonal anti-Human Cip1; BD Biosciences), p53 (1:1000 Rabbit polyclonal anti-p53; Cell Signaling) IVL (1:1000 mouse monoclonal anti-Involucrin clone SY5; Sigma-Aldrich, St Louis, MO), β-actin (1:30,000 mouse monoclonal anti-β-actin AC-74; Sigma Aldrich), Cat# A5316, and then with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). β-actin served as a loading control.

Techniques: Knockdown, Transfection, Control, Small Interfering RNA, Western Blot

Journal: Oncogene

Article Title: Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor suppressor activities

doi: 10.1038/onc.2014.169

Figure Lengend Snippet: EN60 cells expressing HPV E6/E7 were transiently transfected with two independent siRNA sequences directed against either HPV E7 (E7-A and E7-B) or a non-silencing control scramble short interfering RNA (Scr.) along with or without siRNA directed against Notch1 (N1-A and N1-B), p16 iNK4A (p16) or p14 ARF (p14). In ( h ), cells were concurrently transfected with indicated reporter constructs. Cells were analyzed 7 days after transfection by RT-PCR for HPV E6 and E7 transcripts in ( a ); Western blotting for indicated molecules in ( b ); WST1 assays for cell proliferation in ( c ); flow cytometry for cell-cycle in ( d ); and SABG assays in ( e ) and ( f ); quantitative RT-PCR for indicated mRNA in ( g ); and luciferase assays for activation of the TGF-β (3TP-Lux) and Notch (8xCSL-luc) reporters in ( h ). In ( a ), β-actin served as an internal control. In ( b ), β-actin served as a loading control. In ( d ), representative histogram plots are shown. Proportions of cells in G0/G1, S and G2/M cell-cycle phases were determined. *, P < 0.05 vs. Scr.; (n=3). In ( e ) and ( f ), SABG-positive cells were scored (see for representative photomicrographs). *, P < 0.05 vs. Scr. only; #, P < 0.05 vs. Scr. + either E7-A or E7-B; ns , not significant vs. Scr. + either E7-A or E7-B; (n=6) in ( e ) and ( f ). In ( g ), β-actin served as an internal control. *, P < 0.05 vs. Scr.; (n=3). In ( h ), *, P < 0.05 vs. Scr.; (n=4).

Article Snippet: Following electrotransfer, Immobilon-P membranes (Millipore) were incubated with primary antibodies for Notch1 (1:1000 rat monoclonal 5B5; Cell Signaling, Danvers, MA), ICN1 Val1744 (1:1000 rabbit monoclonal anticleaved NOTCH1 Val1744 D3B8; Cell Signaling), ICN3 (1:1000 rat monoclonal anti-NOTCH3 8G5; Cell Signaling), JAG1 (1:1000 rabbit monoclonal anti-Jagged1 28H8; Cell Signaling), pRb (1:1000 rabbit polyclonal anti-Phospho-Rb Ser 780 ; Cell Signaling), p16 (1:1000 mouse monoclonal anti-Human p16 G175–1239; BD Biosciences), p15 (1:200 mouse monoclonal anti-p15 15P06; Santa Cruz), p21 (1:1000 mouse monoclonal anti-Human Cip1; BD Biosciences), p53 (1:1000 Rabbit polyclonal anti-p53; Cell Signaling) IVL (1:1000 mouse monoclonal anti-Involucrin clone SY5; Sigma-Aldrich, St Louis, MO), β-actin (1:30,000 mouse monoclonal anti-β-actin AC-74; Sigma Aldrich), Cat# A5316, and then with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). β-actin served as a loading control.

Techniques: Expressing, Transfection, Control, Small Interfering RNA, Construct, Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry, Quantitative RT-PCR, Luciferase, Activation Assay

Journal: Oncogene

Article Title: Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor suppressor activities

doi: 10.1038/onc.2014.169

Figure Lengend Snippet: EPC2-hTERT cells were stimulated with 5 ng/ml TGF-β1 alone, or along with either GSI (1µM compound E) or DMSO (vehicle) in ( a )–( f ). Cells were transiently transfected with a 8xCSL-luc Notch reporter construct 24 h prior to TGF-β stimulation in ( c ) and siRNA directed against Notch1 (N1-A and N1-B) 24 h prior to TGF-β stimulation in ( g ). Cells were analyzed at indicated time points in ( a ) and ( d ); and 7 d after TGF-β stimulation in ( b ), ( c ), ( e )–( g ). Western blotting determined indicated molecules with β-actin serving as a loading control in ( a ) and ( b ). In ( c ), luciferase assays determined activation of the 8xCSL-luc Notch reporter construct. In ( d ), cell number was counted to determine cell proliferation. In ( e ), flow cytometry was done to determine cell-cycle. In ( f ) and ( g ), SABG assays were carried out and scored (see Supplementary Figure S12a and c for representative photomicrographs). In ( c ), *, P <0.05 vs. TGF-β1 (−) and GSI (−); #, P <0.05 vs. TGF-β1 (+) and GSI (−); (n=3). In ( d ), *, P <0.05 vs. TGF-β1 (−) and GSI (−); #, P <0.05 vs. TGF-β1 (+) and GSI (−); (n=3). In ( e ), representative histogram plots are shown. Histograms show proportions of cells in G0/G1, S and G2/M cell-cycle phases. *, P < 0.05 vs. TGF-β (−) and GSI (−); ns not significant vs. TGF-β (−) and GSI (−); #, P < 0.05 vs. TGF-β (+) and GSI (−); (n=3). In ( f ), *, P <0.05 vs. TGF-β1 (−) and GSI (−); #, P <0.05 vs. TGF-β1 (+) and GSI (−); (n=6). In ( g ), *, P <0.05 vs. TGF-β1 (−) and Scr. (−); #, P <0.05 vs. TGF-β1 (+) and Scr.; (n=6).

Article Snippet: Following electrotransfer, Immobilon-P membranes (Millipore) were incubated with primary antibodies for Notch1 (1:1000 rat monoclonal 5B5; Cell Signaling, Danvers, MA), ICN1 Val1744 (1:1000 rabbit monoclonal anticleaved NOTCH1 Val1744 D3B8; Cell Signaling), ICN3 (1:1000 rat monoclonal anti-NOTCH3 8G5; Cell Signaling), JAG1 (1:1000 rabbit monoclonal anti-Jagged1 28H8; Cell Signaling), pRb (1:1000 rabbit polyclonal anti-Phospho-Rb Ser 780 ; Cell Signaling), p16 (1:1000 mouse monoclonal anti-Human p16 G175–1239; BD Biosciences), p15 (1:200 mouse monoclonal anti-p15 15P06; Santa Cruz), p21 (1:1000 mouse monoclonal anti-Human Cip1; BD Biosciences), p53 (1:1000 Rabbit polyclonal anti-p53; Cell Signaling) IVL (1:1000 mouse monoclonal anti-Involucrin clone SY5; Sigma-Aldrich, St Louis, MO), β-actin (1:30,000 mouse monoclonal anti-β-actin AC-74; Sigma Aldrich), Cat# A5316, and then with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). β-actin served as a loading control.

Techniques: Transfection, Construct, Western Blot, Control, Luciferase, Activation Assay, Flow Cytometry

Journal: Oncogene

Article Title: Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor suppressor activities

doi: 10.1038/onc.2014.169

Figure Lengend Snippet: TE11 and EN60 cells carrying ICN1 Tet-On were subjected to soft agar colony formation assays in ( a ) and xenograft transplantation experiments in ( b ) and ( c ). In ( a ), cells were grown for 2 weeks in soft agar in the presence [DOX (+)] or absence [DOX (−)] of 1 µg/ml DOX and photomicrographed. Colony number and size were determined per low-power field under light microscopy. *, P < 0.01 vs. DOX (−); n=6. In ( b ) and ( c ), immunodeficient mice underwent xenograft transplantation and fed with DOX-containing pellets (20 mg/kg) to induce ICN1. Tumor growth was monitored for indicated time periods in ( b ). Representative images for H&E and immunohistochemistry for Notch1 in resulting xenograft tumors are shown in ( c ). Note tumors in mice treated with DOX display less-differentiated SCC featuring smaller ESCC cells. Tumors grown in DOX-untreated control mice display well-differentiated SCC with keratin pearl formation. Scale bar, 100 µm.

Article Snippet: Following electrotransfer, Immobilon-P membranes (Millipore) were incubated with primary antibodies for Notch1 (1:1000 rat monoclonal 5B5; Cell Signaling, Danvers, MA), ICN1 Val1744 (1:1000 rabbit monoclonal anticleaved NOTCH1 Val1744 D3B8; Cell Signaling), ICN3 (1:1000 rat monoclonal anti-NOTCH3 8G5; Cell Signaling), JAG1 (1:1000 rabbit monoclonal anti-Jagged1 28H8; Cell Signaling), pRb (1:1000 rabbit polyclonal anti-Phospho-Rb Ser 780 ; Cell Signaling), p16 (1:1000 mouse monoclonal anti-Human p16 G175–1239; BD Biosciences), p15 (1:200 mouse monoclonal anti-p15 15P06; Santa Cruz), p21 (1:1000 mouse monoclonal anti-Human Cip1; BD Biosciences), p53 (1:1000 Rabbit polyclonal anti-p53; Cell Signaling) IVL (1:1000 mouse monoclonal anti-Involucrin clone SY5; Sigma-Aldrich, St Louis, MO), β-actin (1:30,000 mouse monoclonal anti-β-actin AC-74; Sigma Aldrich), Cat# A5316, and then with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). β-actin served as a loading control.

Techniques: Transplantation Assay, Light Microscopy, Immunohistochemistry, Control

Journal: Oncogene

Article Title: Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor suppressor activities

doi: 10.1038/onc.2014.169

Figure Lengend Snippet: a . Like classic oncogenes (e.g. Ras and Raf), ICN1 induces senescence via intact cell-cycle checkpoint functions. In normal human esophageal keratinocytes, the p16 INK4A -Rb pathway may have a predominant role in ICN1-induced senescence; however, the p14 ARF , p53 and others may constitute alternative pathways to mediate senescence when the p16 INK4A -Rb pathway is impaired. When cell-cycle checkpoint functions are fully impaired (e.g. concurrent Rb and p53 inactivation), cells fail to undergo senescence in response to ICN1, resulting in malignant transformation. b . Endogenous Notch1 may serve as a tumor suppressor by mediating TGF-β-induced senescence. TGF-β has been implicated in replicative senescence as well as oncogene-induced senescence , . TGF-β requires p53 to transactivate p21 . Notch1 may be targeted for inactivation by HPV oncogenes E6 and E7 during malignant transformation. E6 may suppress Notch1 by degrading p53 . E7 also targets Rb for degradation or sequestration. We find that HPV E6 and E7 inhibit TGF-β signaling to prevent the activation of endogenous Notch1 and induction of p16 INK4A and p21. While TGF-β may induce CDKIs independent of Notch1, Notch1 may regulate the SABG activity independent of cell-cycle regulation. Since oncogeneinduced senescence may involve autophagy and lysosomal functions , Notch signaling may regulate the activity of SABG, a lysosomal enzyme , .

Article Snippet: Following electrotransfer, Immobilon-P membranes (Millipore) were incubated with primary antibodies for Notch1 (1:1000 rat monoclonal 5B5; Cell Signaling, Danvers, MA), ICN1 Val1744 (1:1000 rabbit monoclonal anticleaved NOTCH1 Val1744 D3B8; Cell Signaling), ICN3 (1:1000 rat monoclonal anti-NOTCH3 8G5; Cell Signaling), JAG1 (1:1000 rabbit monoclonal anti-Jagged1 28H8; Cell Signaling), pRb (1:1000 rabbit polyclonal anti-Phospho-Rb Ser 780 ; Cell Signaling), p16 (1:1000 mouse monoclonal anti-Human p16 G175–1239; BD Biosciences), p15 (1:200 mouse monoclonal anti-p15 15P06; Santa Cruz), p21 (1:1000 mouse monoclonal anti-Human Cip1; BD Biosciences), p53 (1:1000 Rabbit polyclonal anti-p53; Cell Signaling) IVL (1:1000 mouse monoclonal anti-Involucrin clone SY5; Sigma-Aldrich, St Louis, MO), β-actin (1:30,000 mouse monoclonal anti-β-actin AC-74; Sigma Aldrich), Cat# A5316, and then with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). β-actin served as a loading control.

Techniques: Transformation Assay, Activation Assay, Activity Assay

Journal: Frontiers in Oncology

Article Title: HIF-2α regulates proliferation, invasion, and metastasis of hepatocellular carcinoma cells via VEGF/Notch1 signaling axis after insufficient radiofrequency ablation

doi: 10.3389/fonc.2022.998295

Figure Lengend Snippet: Expression of HIF-2α, Notch1, and VEGF in HC and their paracancerous tissues of patients after insufficient RFA as revealed by immunohistochemistry. Brown color indicates positive staining. Magnification 400×.

Article Snippet: Our study incorporated the following reagents/materials at different stages: DMEM medium, fetal bovine serum (Gibco, USA), tetrazolium blue (MTT) powder (Dongguan Science and Technology Biology Company), Transwell chambers, Matrigel matrix glue (Corning, USA), mouse anti-VEGF monoclonal antibody (Proteintech), rabbit anti-human HIF-2α, rabbit anti-human Notch1, rabbit anti-human β-actin (monoclonal antibodies; CST), and PT2385 (MCE).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Frontiers in Oncology

Article Title: HIF-2α regulates proliferation, invasion, and metastasis of hepatocellular carcinoma cells via VEGF/Notch1 signaling axis after insufficient radiofrequency ablation

doi: 10.3389/fonc.2022.998295

Figure Lengend Snippet: HIF-2α, Notch1, and VEGF were involved in the increased invasion and proliferation of HCC induced by insufficient RFA. (A) Representative photos of HCC after insufficient RFA were detected by a phase contrast microscope and crystal violet staining. Magnification 400×. (B) Detection of cell proliferation by MTT. (C) Quantitation analysis of Transwell assay. (D–F) Detection of the mRNA expression of HIF-2α, Notch1, and VEGF. **P < 0.01, ***P < 0.001.

Article Snippet: Our study incorporated the following reagents/materials at different stages: DMEM medium, fetal bovine serum (Gibco, USA), tetrazolium blue (MTT) powder (Dongguan Science and Technology Biology Company), Transwell chambers, Matrigel matrix glue (Corning, USA), mouse anti-VEGF monoclonal antibody (Proteintech), rabbit anti-human HIF-2α, rabbit anti-human Notch1, rabbit anti-human β-actin (monoclonal antibodies; CST), and PT2385 (MCE).

Techniques: Microscopy, Staining, Quantitation Assay, Transwell Assay, Expressing

Journal: Frontiers in Oncology

Article Title: HIF-2α regulates proliferation, invasion, and metastasis of hepatocellular carcinoma cells via VEGF/Notch1 signaling axis after insufficient radiofrequency ablation

doi: 10.3389/fonc.2022.998295

Figure Lengend Snippet: Inhibition of HIF-2α by PT2385 suppressed the VEGF and Notch1 signaling pathway in HCC after insufficient RFA. (A–C) Detection of mRNA expression by RT-qPCR. (D, E) Detection of protein levels by western blotting. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Our study incorporated the following reagents/materials at different stages: DMEM medium, fetal bovine serum (Gibco, USA), tetrazolium blue (MTT) powder (Dongguan Science and Technology Biology Company), Transwell chambers, Matrigel matrix glue (Corning, USA), mouse anti-VEGF monoclonal antibody (Proteintech), rabbit anti-human HIF-2α, rabbit anti-human Notch1, rabbit anti-human β-actin (monoclonal antibodies; CST), and PT2385 (MCE).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Western Blot

Journal: Oncogene

Article Title: The miR-106b-25 cluster mediates breast tumor initiation through activation of NOTCH1 via direct repression of NEDD4L

doi: 10.1038/s41388-018-0239-7

Figure Lengend Snippet: (A) Western blot analysis for NOTCH1-ICD and Full length (FL) NOTCH1 in a panel of breast cancer cell lines with stable overexpression of the miR-106b-25 cluster (CLR) including miR-106b, miR-93, and miR-25 or a non-silencing control (NS). (B) Notch Luciferase reporter assay using the RBP-Jk reporter transfected into MCF7, T47D, HCC1937, Sum159, and Sum149 NS and Cluster expressing lines. Statistical significance assessed by a student T-test on triplicate samples of a representative experiment (n=3). (C) qRT-PCR analysis in MCF7 and Sum159 NS and CLR cells showing an increase in NOTCH1 direct target genes with CLR overexpression. Statistical significance assessed by a student T-test on six replicate samples of a representative experiment (n=2).

Article Snippet: Sample aliquots (1 mg of total protein in 1 ml) were subjected to immunoprecipitation with either 5 μl of anti-NOTCH1 antibody (Cell Signaling Technologies, #4380) or non-specific rabbit IgG (Santa Cruz Biotechnology, sc-3888).

Techniques: Western Blot, Over Expression, Control, Luciferase, Reporter Assay, Transfection, Expressing, Quantitative RT-PCR

Journal: Oncogene

Article Title: The miR-106b-25 cluster mediates breast tumor initiation through activation of NOTCH1 via direct repression of NEDD4L

doi: 10.1038/s41388-018-0239-7

Figure Lengend Snippet: (A) Flow cytometry analysis in MCF7 cells treated with 30nM of either non-targeting siRNAs or pooled siRNAs that target NOTCH1 and transiently transfected with either a negative mimic or a combination of miR-106b, miR-93, and miR-25 mimics (20 nM total) to assess the percentage of CD44 + /CD24 − cells. (B) Secondary mammosphere assay in MCF7- and Sum159-NS and Cluster (CLR) stable cell lines in the presence of DAPT (5μM) or a DMSO vehicle control. (C) Left: Secondary mammosphere assay in MCF7 NS and CLR stable cell lines with stable shRNA knock down of NOTCH1 . Right: Primary mammosphere assay in Sum-159 NS and Cluster stable cell lines with stable shRNA knock down of NOTCH1 . Statistical significance on all mammosphere assays was assessed by one-way ANOVA followed by a Tukey post-test on triplicate samples of a representative experiment (n=3). (D) MCF7-NS and CLR cells with stable shRNA KD of Notch1 or a scramble control (SCR) were transplanted into the 4th mammary fat pad of NOG/SCID mice at limiting dilutions, data represents presence of tumors at week 5 post tumor cell implantation.

Article Snippet: Sample aliquots (1 mg of total protein in 1 ml) were subjected to immunoprecipitation with either 5 μl of anti-NOTCH1 antibody (Cell Signaling Technologies, #4380) or non-specific rabbit IgG (Santa Cruz Biotechnology, sc-3888).

Techniques: Flow Cytometry, Transfection, Stable Transfection, Control, shRNA, Knockdown

Journal: Oncogene

Article Title: The miR-106b-25 cluster mediates breast tumor initiation through activation of NOTCH1 via direct repression of NEDD4L

doi: 10.1038/s41388-018-0239-7

Figure Lengend Snippet: (A) Western blot analysis, using a NEDD4L antibody, in MCF7 and Sum159 NS and CLR cell lines with stable HA-NEDD4L expression (ND4L). Due to the rapid turnover of the exogenous protein in MCF7 cells, MG132 (25μM) was added to MCF7 NS and CLR cells for 4hrs to stabilize HA-NEDD4L and allow its detection (B) Western blot analysis showing that Notch-ICD levels are decreased in MCF7 and Sum159 Cluster cell lines with stable rescue of NEDD4L (C) Western blot analysis to identify polyubiquitinated NOTCH1 species in MCF7-CLR-EV (low NEDD4L) vs MCF7-CLR-ND4L (high NEDD4L) cells after NOTCH1 immunoprecipitation. (D) Quantification of the number of secondary mammospheres after 7 days in culture in MCF7 and Sum159 NS, CLR, and CLR-ND4L rescue lines. Statistics were assessed by ANOVA followed by a Tukey post-test of triplicate samples of a representative experiment (n=2) and by a Fisher’s LSD test of triplicate samples from a representative experiment (n=2), respectively.

Article Snippet: Sample aliquots (1 mg of total protein in 1 ml) were subjected to immunoprecipitation with either 5 μl of anti-NOTCH1 antibody (Cell Signaling Technologies, #4380) or non-specific rabbit IgG (Santa Cruz Biotechnology, sc-3888).

Techniques: Western Blot, Expressing, Immunoprecipitation

Journal: Oncogene

Article Title: The miR-106b-25 cluster mediates breast tumor initiation through activation of NOTCH1 via direct repression of NEDD4L

doi: 10.1038/s41388-018-0239-7

Figure Lengend Snippet: (A–C) In the TCGA breast invasive carcinoma dataset we plotted (A) the normalized log2 miRNA expression for each microRNA in normal adjacent breast tissue (n=87) vs. breast tumor tissue (n=762), statistics derived from KS test. (B) Top: The normalized log2 expression of each miRNA in matched normal and tumor samples are plotted, with black lines corresponding to samples where expression is decreased in matched tumor tissue and aqua lines corresponding to samples where expression is increased in matched tumor tissue. Bottom: Box and whisker plots of the above data with statistics derived from KS test C) Pearson correlations between individual miRNAs within the cluster (log2 normalized) and NEDD4L mRNA expression extracted from TCGA RNAseqV2 (Log2 normalized) (top), and Pearson correlations between miRNA expression (miRNA-seq) and NOTCH1 protein expression (from TCGA RPPA) (bottom). (D) Pearson correlation demonstrates a negative association between NEDD4L mRNA expression and NOTCH1 RPPA protein expression in the TCGA breast cancer dataset (E) Differential expression of NEDD4L mRNA (RSEM value) in normal vs. breast tumor vs. metastatic tissue in same TCGA dataset as A–C (F) Kaplan-Meier of Distant Relapse Free Survival (DRFS) in 216 individuals with early primary breast cancer, compiled from GEO dataset (GSE22219), stratified into NEDD4L high (above the median) vs. low (below the median).

Article Snippet: Sample aliquots (1 mg of total protein in 1 ml) were subjected to immunoprecipitation with either 5 μl of anti-NOTCH1 antibody (Cell Signaling Technologies, #4380) or non-specific rabbit IgG (Santa Cruz Biotechnology, sc-3888).

Techniques: Expressing, Derivative Assay, Whisker Assay, Quantitative Proteomics