Journal: Nature Communications

Article Title: Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma

doi: 10.1038/s41467-017-01500-9

Figure Lengend Snippet: Lineage tracing reveals mesenchymal traits and Notch1 activation in ESCC lesions. a Schematic of cell-lineage tracing experiments. b Macroscopic and fluorescent images of representative tdTomato-labeled esophagus bearing 4NQO-induced tumors (arrows). Scale bar, 1 mm. c qRT-PCR analysis for indicated genes comparing EpCAM neg to EpCAM pos cells from a representative tdTomato-labeled tumor. * P < 0.0001 and # P < 0.05 vs. EpCAM pos , n = 3. d Representative IF images for Zeb1 and E-cadherin (E-cad) in normal mucosa (top) and ESCC (bottom). Dashed line denotes interface between stroma and basal keratinocytes (top) or invasive ESCC cells (bottom). Note E-cadherin downregulation in ESCC cells with nuclear Zeb1 (arrowheads) at the tumor invasive front. Scale bar, 50 µm. e H&E, multicolor IF for E-cadherin and Zeb1, and IHC for ICN1 (ICN1 Val1744 ) in representative single-cell-derived organoids from normal mucosa or 4NQO-induced tumors. Note that spherical organoids from 4NQO-untreated control mice exhibit a differentiation gradient with predominant luminal keratinization, whereas tumor-derived organoids (neoplastic) display irregular morphology with increased cellularity and diminished keratinization. Zeb1 expression was robust in tumor organoids, particularly at invasive protrusions with decreased E-cadherin expression and detectable ICN1 expression. Box denotes area magnified in panel below. Scale bars, 20 µm. f Representative IHC image for ICN1 4NQO-induced IEN (dysplasia) containing spindle-shaped cells (arrowheads). Scale bar, 50 µm. g Quantification of ICN1 IHC scoring in normal mucosa and 4NQO-induced lesions in mice of indicated genotype. * P < 0.05 vs. p53 +/+ ( K5Cre ERT2 ;R26tdTomato lsl/lsl ) normal, # P < 0.0005 vs. p53 −/− ( K5Cre ERT2 ;R26tdTomato lsl/lsl ; p53 loxP/loxP ) normal; † P < 0.05 vs. p53 −/− IEN; ns not significant vs. p53 +/+ normal. Data in b – f represent at least three independent 4NQO-induced lesions and >20 organoids from at least two independent experimental replicates. In g , n = 5 normal, n = 8 IEN, and n = 5 invasive ESCC in p53 +/+ esophagi. N.D. not detectable. n = 3 normal, n = 7 IEN, n = 5 invasive ESCC, and n = 4 metastatic tumors in p53 −/− esophagi. Two independent experimental replicates were carried out. Bar diagrams represent mean ± s.d. in c and mean ± s.e.m. in g . Student’s t -test was used for paired data comparisons in c , g . ANOVA with Tukey’s post hoc test was used for multiple comparisons in g

Article Snippet: RNA isolation, cDNA synthesis, and qRT-PCR were done using StepOnePlusTM Real-Time PCR System (Applied Biosystems) by TaqMan ® Gene Expression Assays (Applied Biosystems) for NOTCH1 (Hs01062014_m1), Notch1 (Mm00435249_m1), NOTCH3 (Hs00166432_m1), Notch3 (Mm00435270_m1), IVL (Hs00846307_s1), CK13 (s00999762_m1), CDH1 (Hs00170423_m1), CDH2 (Hs00983062 _m1), ZEB1 (Hs00232783_m1), SNAI1 (Hs00195591_m1), and PAI1 (Hs01126606_m1), and SYBR ® Green PCR for human ACTB (β-Actin) as well as murine Cdh1 (5′-TCAAGCTCGCGGATAACCAGAACA-3′ and 5′-ATTCCCGCCTTCATGCAGTTGTTG-3′), Cdh2 (5′-ATGGCCTTTCAAACACAGCCACAG-3′ and 5′-ACAATGACGTCCACCCTGTTCTCA-3′), Zeb1 (5′-TGAGCACACAGGTAAGAGGCC-3′ and 5′-GGCTTTTCCCCAGAGTGCA-3′), Zeb2 (5′-TGATAGCCTTGCAAACCCTCTGGA-3′ and 5′-TCCTTCATTTCTTCTGGACCGGCT-3′), Twist (5′-AGCTGAGCAAGATTCAGACCCTCA-3′ and 5′-TGCAGCTTGCCATCTTGGAGT-3′), and Gapdh (5′-GGTGGTCTCCTCTGACTTCAACA-3′ and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′) and as described , , .

Techniques: Activation Assay, Labeling, Quantitative RT-PCR, Derivative Assay, Control, Expressing

Journal: Nature Communications

Article Title: Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma

doi: 10.1038/s41467-017-01500-9

Figure Lengend Snippet: Notch1 promotes ESCC tumorigenesis. a Schematic of 8×CSL-GFP reporter. Notch activation permits GFP reporter expression via concatemeric CSL-binding sites. Constitutively active UBC promoter drives tRFP expression concurrently. Cells without tRFP represent host-derived stromal cells. b Representative flow cytometry scatter plots determining 8×CSL -mediated GFP reporter activation, indicating Notch active population of tRFP-labeled ESCC cells in TE11 and EN60 xenograft tumors. On average, 43.5 ± 0.5% s.d. of EN60 and 11.2 ± 6.5% s.d. of TE11 cells comprised the live GFP Pos /tRFP Pos fraction across three independent tumors. c , d Tumor growth curves in immunodeficient mice carrying TE11 tumors of indicated genotypes. Upon xenograft transplantation, mice were treated with DOX to induce ICN1 or NOTCH 1-directed shRNA. Tumor growth was monitored for indicated time periods. In c , * P < 0.01 vs. zeo and DOX (−), # P < 0.05 vs. zeo and DOX (+), n = 6–8 per group. In d , * P < 0.05 vs. DOX (−); n = 6 per group. e TE11 cells with CRISPR/Cas9-mediated NOTCH1 deletion. Immunoblotting confirms ICN1 loss in NOTCH1 -deleted TE11 (N1 Δ ), but not non-targeted control TE11 (N1 WT ) cells. Bar graph shows tumor formation rate in immunodeficient mice 7 weeks after xenograft transplantation. * P < 0.05 vs. N1 WT , n = 8 per group. f Esophageal keratinocytes isolated from 4NQO-induced IEN (dysplasia) lesions or ESCC tumors of Notch1 loxP/loxP mice were utilized to generate 3D esophageal organoids in the presence or absence of ex vivo Cre-mediated recombination. qRT-PCR analysis confirms inhibition of Notch1 gene expression upon ex vivo Cre-mediated recombination in ESCC organoids. * P < 0.000 vs. Cre (−), n = 3. Organoid formation rate was evaluated at day 14 post-plating and is represented as relative to Cre (−) for IEN and ESCC. * P < 0.000 vs. Cre (−) IEN; # P < 0.001 vs. Cre (−) ESCC. Line graphs represent mean ± s.e.m. in c , d . Bar diagrams represent mean ± s.d. in f . At least two independent replicates were performed for all experiments. Repeated measures ANOVA with Tukey’s post hoc test were used for multiple comparisons in c , d . Chi square with Fisher’s exact test was used for percentage comparisons in e . Student’s t -test was used for paired data comparisons in f

Article Snippet: RNA isolation, cDNA synthesis, and qRT-PCR were done using StepOnePlusTM Real-Time PCR System (Applied Biosystems) by TaqMan ® Gene Expression Assays (Applied Biosystems) for NOTCH1 (Hs01062014_m1), Notch1 (Mm00435249_m1), NOTCH3 (Hs00166432_m1), Notch3 (Mm00435270_m1), IVL (Hs00846307_s1), CK13 (s00999762_m1), CDH1 (Hs00170423_m1), CDH2 (Hs00983062 _m1), ZEB1 (Hs00232783_m1), SNAI1 (Hs00195591_m1), and PAI1 (Hs01126606_m1), and SYBR ® Green PCR for human ACTB (β-Actin) as well as murine Cdh1 (5′-TCAAGCTCGCGGATAACCAGAACA-3′ and 5′-ATTCCCGCCTTCATGCAGTTGTTG-3′), Cdh2 (5′-ATGGCCTTTCAAACACAGCCACAG-3′ and 5′-ACAATGACGTCCACCCTGTTCTCA-3′), Zeb1 (5′-TGAGCACACAGGTAAGAGGCC-3′ and 5′-GGCTTTTCCCCAGAGTGCA-3′), Zeb2 (5′-TGATAGCCTTGCAAACCCTCTGGA-3′ and 5′-TCCTTCATTTCTTCTGGACCGGCT-3′), Twist (5′-AGCTGAGCAAGATTCAGACCCTCA-3′ and 5′-TGCAGCTTGCCATCTTGGAGT-3′), and Gapdh (5′-GGTGGTCTCCTCTGACTTCAACA-3′ and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′) and as described , , .

Techniques: Activation Assay, Expressing, Binding Assay, Derivative Assay, Flow Cytometry, Labeling, Transplantation Assay, shRNA, CRISPR, Western Blot, Control, Isolation, Ex Vivo, Quantitative RT-PCR, Inhibition, Gene Expression

Journal: Nature Communications

Article Title: Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma

doi: 10.1038/s41467-017-01500-9

Figure Lengend Snippet: Notch1 facilitates expansion of CD44H cells with mesenchymal properties. a qRT-PCR analysis for indicated genes comparing EN60 intratumoral CD44L and CD44H cells. * P < 0.05 vs. CD44L; # P < 0.0001 vs. CD44L; n = 3 per group. b Representative flow cytometry scatter plot determining CD44H cells in EN60 tumors grown for 4 weeks with or without DOX-induced ICN1 (EN60- ICN1 TetOn ). c Experimental design of serial transplantation experiments and tumor formation rates by CD44L and CD44H cells purified from parental xenograft tumors carrying DOX-inducible ICN1 (EN60- ICN1 TetOn and TE11- ICN1 TetOn ). Parental tumors were grown in mice without DOX treatment and dissociated for FACS-purification of CD44L and CD44H cells. Purified cells were serially transplanted (10 3 cells per injection site) into recipient mice to monitor tumor formation. Recipient mice were treated with or without DOX. TE11 carried DNMAML1 or zeo (empty vector control). * P < 0.05 for CD44L and DOX (+) vs. CD44L and DOX (−), n = 10–12 per group; ns not significant for CD44L and DOX (+) vs. CD44H (with or without DOX treatment), n = 10 per group; # P < 0.05 vs. TE11- ICN1 TetOn -zeo CD44L and DOX (+), n = 10 per group. d Flow cytometry analysis for EpCAM-negative cells in TE11 with NOTCH1 deletion (N1 Δ ) or wild-type control (N1 WT ). Cells were treated with or without 5 ng/ml TGFβ for 72 h. * P < 0.05 vs. TGFβ (−); # P < 0.0001 vs. N1 WT and TGFβ (+), n = 3. Data are presented as mean ± s.e.m. e Multicolor IF for E-cadherin and Zeb1 in representative single-cell-derived organoids from 4NQO-induced ESCC tumors of Notch1 loxP/loxP mice with or without ex vivo Cre-mediated recombination. Zeb1 expression diminished in organoids upon Cre-mediated Notch1 deletion (arrowheads). Box denotes area that is magnified in panel below. KC keratinized core of organoids. Scale bars, 20 µm. Bar diagrams represent mean ± s.d. in a , d . At least two independent replicates were performed for all experiments. Student’s t -test was used for paired data comparisons in a , d . Chi square with Fisher’s exact test was used for percentage comparisons in c

Article Snippet: RNA isolation, cDNA synthesis, and qRT-PCR were done using StepOnePlusTM Real-Time PCR System (Applied Biosystems) by TaqMan ® Gene Expression Assays (Applied Biosystems) for NOTCH1 (Hs01062014_m1), Notch1 (Mm00435249_m1), NOTCH3 (Hs00166432_m1), Notch3 (Mm00435270_m1), IVL (Hs00846307_s1), CK13 (s00999762_m1), CDH1 (Hs00170423_m1), CDH2 (Hs00983062 _m1), ZEB1 (Hs00232783_m1), SNAI1 (Hs00195591_m1), and PAI1 (Hs01126606_m1), and SYBR ® Green PCR for human ACTB (β-Actin) as well as murine Cdh1 (5′-TCAAGCTCGCGGATAACCAGAACA-3′ and 5′-ATTCCCGCCTTCATGCAGTTGTTG-3′), Cdh2 (5′-ATGGCCTTTCAAACACAGCCACAG-3′ and 5′-ACAATGACGTCCACCCTGTTCTCA-3′), Zeb1 (5′-TGAGCACACAGGTAAGAGGCC-3′ and 5′-GGCTTTTCCCCAGAGTGCA-3′), Zeb2 (5′-TGATAGCCTTGCAAACCCTCTGGA-3′ and 5′-TCCTTCATTTCTTCTGGACCGGCT-3′), Twist (5′-AGCTGAGCAAGATTCAGACCCTCA-3′ and 5′-TGCAGCTTGCCATCTTGGAGT-3′), and Gapdh (5′-GGTGGTCTCCTCTGACTTCAACA-3′ and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′) and as described , , .

Techniques: Quantitative RT-PCR, Flow Cytometry, Transplantation Assay, Purification, Injection, Plasmid Preparation, Control, Derivative Assay, Ex Vivo, Expressing

Journal: Nature Communications

Article Title: Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma

doi: 10.1038/s41467-017-01500-9

Figure Lengend Snippet: TGFβ signaling facilitates ESCC tumor growth, Notch1 signaling, and EMT. a Growth curve for mice bearing TE11 or HNSCC PDX tumors receiving either ID11 anti-TGFβ blocking antibody or control IgG via intraperitoneal injection at indicated time points (arrows). ID11 neutralizes all TGFβ isoforms. * P < 0.05 vs. IgG, n = 5–8 per group. b , c p53 +/+ mice were treated with 4NQO for 16 weeks. Six weeks following 4NQO withdrawal, mice were treated with ID11 anti-TGFβ blocking antibody or control IgG via intraperitoneal injection three times weekly for a period of 2 weeks. In b , quantification of ICN1 IHC scoring in esophageal epithelium with representative IHC images for IgG-treated and anti-TGFβ blocking antibody-treated animals. * P < 0.05; n = 4–5 per group. Scale bar, 50 µm. In c , multicolor IF for E-cadherin and Zeb1 in representative 4NQO-induced IEN lesions from IgG-treated and anti-TGFβ blocking antibody-treated animals. Data indicate mean ± s.e.m. in a , b . At least two independent replicates were performed for all experiments. Repeated measures ANOVA with Tukey’s post hoc test were used for multiple comparisons in a . Student’s t -test was used for paired data comparisons in b

Article Snippet: RNA isolation, cDNA synthesis, and qRT-PCR were done using StepOnePlusTM Real-Time PCR System (Applied Biosystems) by TaqMan ® Gene Expression Assays (Applied Biosystems) for NOTCH1 (Hs01062014_m1), Notch1 (Mm00435249_m1), NOTCH3 (Hs00166432_m1), Notch3 (Mm00435270_m1), IVL (Hs00846307_s1), CK13 (s00999762_m1), CDH1 (Hs00170423_m1), CDH2 (Hs00983062 _m1), ZEB1 (Hs00232783_m1), SNAI1 (Hs00195591_m1), and PAI1 (Hs01126606_m1), and SYBR ® Green PCR for human ACTB (β-Actin) as well as murine Cdh1 (5′-TCAAGCTCGCGGATAACCAGAACA-3′ and 5′-ATTCCCGCCTTCATGCAGTTGTTG-3′), Cdh2 (5′-ATGGCCTTTCAAACACAGCCACAG-3′ and 5′-ACAATGACGTCCACCCTGTTCTCA-3′), Zeb1 (5′-TGAGCACACAGGTAAGAGGCC-3′ and 5′-GGCTTTTCCCCAGAGTGCA-3′), Zeb2 (5′-TGATAGCCTTGCAAACCCTCTGGA-3′ and 5′-TCCTTCATTTCTTCTGGACCGGCT-3′), Twist (5′-AGCTGAGCAAGATTCAGACCCTCA-3′ and 5′-TGCAGCTTGCCATCTTGGAGT-3′), and Gapdh (5′-GGTGGTCTCCTCTGACTTCAACA-3′ and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′) and as described , , .

Techniques: Blocking Assay, Control, Injection

Journal: Nature Communications

Article Title: Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma

doi: 10.1038/s41467-017-01500-9

Figure Lengend Snippet: TGFβ-mediated Notch1 activation permits EMT and suppresses differentiation. a Representative flow cytometry scatter and histogram plots for 8×CSL-GFP reporter activity in indicated subpopulations of EPC2T cells treated with TGFβ or CaCl 2 for 72 h. b , c Flow cytometry determined CD44H cells induced in FACS-purified CD44L cells with indicated conditions or genotypes. Cells were treated with TGFβ or DOX for 7 days. * P < 0.0001 vs. TGFβ (−); # P < 0.005 vs. TGFβ (+) and DMSO, GFP , or non-silencing scrambled (NS) shRNA in b ; n = 3. * P < 0.05 vs. TGFβ (−) and DOX (−); ns not significant vs. TGFβ (−) and DOX (−); # P < 0.05 vs. TGFβ (+) and DOX (−) in c ; n = 3. d Representative H&E images of OTC reconstituted with CD44L or CD44H cells. Epi epithelia, Str stroma. Scale bar, 50 μm. e Representative flow cytometry scatter plots of purified CD44H cells treated with SB431542 or vehicle (control) for 14 days. SB431542 increased CD44L cell content (upper left quadrant) to 0.35 ± 0.08% as compared to 1.2 ± 0.1% s.d. in vehicle ( P < 0.0005, n = 3). f qRT-PCR analysis for indicated genes in FACS-purified CD44H cells treated with or without SB431542. mRNA level for each gene in vehicle-treated cells was set as 1. * P < 0.0005 vs. vehicle, n = 3. g Heat map of gene array results from EPC2T derivative (EPC2T- ICN1 TetOn ) treated with DOX and/or TGFβ ( n = 3 per condition). “Differentiation” genes were significantly increased (≥1.5-fold) by DOX-induced ectopic ICN1 alone, but suppressed by TGFβ ( P < 0.05 by two-way ANOVA). “EMT” genes activated by TGFβ (≥1.5-fold) were significantly augmented by ectopic ICN1 ( P < 0.05 by two-way ANOVA). h qRT-PCR analysis validates gene array results in g . mRNA level for DOX (−) and TGFβ (−) was set to 1. * P < 0.001 vs. TGFβ (−) and DOX (−); # P < 0.0001 vs. TGFβ (−) and DOX (+); ns not significant vs. TGFβ (−) and DOX (−); n = 3. All bar diagrams indicate mean ± s.d. Student’s t -test was used for paired data comparisons in e , f . At least two independent replicates were performed for all experiments. ANOVA with Tukey’s post hoc test were used for multiple comparisons in b , c , h

Article Snippet: RNA isolation, cDNA synthesis, and qRT-PCR were done using StepOnePlusTM Real-Time PCR System (Applied Biosystems) by TaqMan ® Gene Expression Assays (Applied Biosystems) for NOTCH1 (Hs01062014_m1), Notch1 (Mm00435249_m1), NOTCH3 (Hs00166432_m1), Notch3 (Mm00435270_m1), IVL (Hs00846307_s1), CK13 (s00999762_m1), CDH1 (Hs00170423_m1), CDH2 (Hs00983062 _m1), ZEB1 (Hs00232783_m1), SNAI1 (Hs00195591_m1), and PAI1 (Hs01126606_m1), and SYBR ® Green PCR for human ACTB (β-Actin) as well as murine Cdh1 (5′-TCAAGCTCGCGGATAACCAGAACA-3′ and 5′-ATTCCCGCCTTCATGCAGTTGTTG-3′), Cdh2 (5′-ATGGCCTTTCAAACACAGCCACAG-3′ and 5′-ACAATGACGTCCACCCTGTTCTCA-3′), Zeb1 (5′-TGAGCACACAGGTAAGAGGCC-3′ and 5′-GGCTTTTCCCCAGAGTGCA-3′), Zeb2 (5′-TGATAGCCTTGCAAACCCTCTGGA-3′ and 5′-TCCTTCATTTCTTCTGGACCGGCT-3′), Twist (5′-AGCTGAGCAAGATTCAGACCCTCA-3′ and 5′-TGCAGCTTGCCATCTTGGAGT-3′), and Gapdh (5′-GGTGGTCTCCTCTGACTTCAACA-3′ and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′) and as described , , .

Techniques: Activation Assay, Flow Cytometry, Activity Assay, Purification, shRNA, Control, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma

doi: 10.1038/s41467-017-01500-9

Figure Lengend Snippet: NOTCH1 activation and ZEB1 expression in invasive ESCC predicts poor prognosis. a , b Representative IHC images for ICN1 and ZEB1 in ESCC cells in a deep invasive tumor nest and ESCC cells invading into lymphatic vessels. a ESCC #55; b ESCC #62 in Supplementary Data . c Representative IF image for ICN1 and CD44 in invasive ESCC cells. Scale bars, 50 μm in a – c . d Survival curve for 185 post-surgical ESCC patients with or without ICN1 expression at the tumor invasive front. e Model of the role of Notch1 in EMT in ESCC. Notch1 activation promotes tumorigenicity and heterogeneity in SCC via EMT. Notch1 drives squamous-cell differentiation by inducing Notch3 in normal squamous epithelia as well as SCC cells. Notch3 limits EMT. In response to TGFβ from the tumor microenvironment (e.g., cancer-associated fibroblasts and inflammatory cells), however, transcriptional repression of Notch3 by ZEB1 permits Notch1-mediated induction of CD44H cells via EMT. Notch activation may result in the generation and maintenance of CD44H cells possessing mesenchymal properties and enhanced malignant potential. EMT allows neoplastic cells to cope with stress during carcinogenesis and disease progression (e.g., genotoxic stress induced by 4NQO). CD44H cells produce pro-tumorigenic cytokines (e.g., IL-6) and tissue remodeling factors (e.g., MMP13, LOX, and POSTN) (Supplementary Fig. )

Article Snippet: RNA isolation, cDNA synthesis, and qRT-PCR were done using StepOnePlusTM Real-Time PCR System (Applied Biosystems) by TaqMan ® Gene Expression Assays (Applied Biosystems) for NOTCH1 (Hs01062014_m1), Notch1 (Mm00435249_m1), NOTCH3 (Hs00166432_m1), Notch3 (Mm00435270_m1), IVL (Hs00846307_s1), CK13 (s00999762_m1), CDH1 (Hs00170423_m1), CDH2 (Hs00983062 _m1), ZEB1 (Hs00232783_m1), SNAI1 (Hs00195591_m1), and PAI1 (Hs01126606_m1), and SYBR ® Green PCR for human ACTB (β-Actin) as well as murine Cdh1 (5′-TCAAGCTCGCGGATAACCAGAACA-3′ and 5′-ATTCCCGCCTTCATGCAGTTGTTG-3′), Cdh2 (5′-ATGGCCTTTCAAACACAGCCACAG-3′ and 5′-ACAATGACGTCCACCCTGTTCTCA-3′), Zeb1 (5′-TGAGCACACAGGTAAGAGGCC-3′ and 5′-GGCTTTTCCCCAGAGTGCA-3′), Zeb2 (5′-TGATAGCCTTGCAAACCCTCTGGA-3′ and 5′-TCCTTCATTTCTTCTGGACCGGCT-3′), Twist (5′-AGCTGAGCAAGATTCAGACCCTCA-3′ and 5′-TGCAGCTTGCCATCTTGGAGT-3′), and Gapdh (5′-GGTGGTCTCCTCTGACTTCAACA-3′ and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′) and as described , , .

Techniques: Activation Assay, Expressing, Cell Differentiation, Biomarker Discovery

Journal: Scientific reports

Article Title: TM2D3, a mammalian homologue of Drosophila neurogenic gene product Almondex, regulates surface presentation of Notch receptors.

doi: 10.1038/s41598-023-46866-7

Figure Lengend Snippet: Figure 3. Requirement of the ligand-binding domain of Notch1 for the activation and increased cell surface expression of Notch1 by transient co-transfection of TM2D3. (a, b) Activation of Notch1 with a full-length ectodomain but not those that lack EGF repeats (Notch1 LNR, LNR CC > SS, and ΔE) (Supplementary Fig. S5 online) by co-transfection of TM2D3. Cells were transfected with vectors for the indicated proteins or empty vector (Vector) as a control. Immunoblotting was performed with the indicated antibodies. Notch1 LNR CC > SS and ΔE are constitutively active. (c) Inability of TM2D3 to activate Notch1 that lacks EGF repeats 11 and 12. Experiments were conducted as in a and b. (d, e) Increased Notch1 expression at cell surface by co-transfection of TM2D3 and its independence on EGF repeats 11 and 12. Cells transfected with vectors for the indicated proteins or empty vector (Vector) as a control were incubated with or without a non-membrane permeable biotinylation reagent as indicated. After fractionation with an avidin agarose, immunoblotting was performed with the indicated antibodies. (f) Cell surface expression of TM2D3. Experiments were conducted as in (d) and (e).

Article Snippet: An antibody against the C-terminus of human NOTCH1 (sc-6014), an antibody against the ECD of murine Notch1 (8G10; sc-32756), and antibodies against the ECD of Notch2 (sc-518169 and sc-5545) were obtained from Santa Cruz Biotechnology.

Techniques: Ligand Binding Assay, Activation Assay, Expressing, Cotransfection, Transfection, Plasmid Preparation, Control, Western Blot, Incubation, Membrane, Fractionation, Avidin-Biotin Assay

Journal: Scientific reports

Article Title: TM2D3, a mammalian homologue of Drosophila neurogenic gene product Almondex, regulates surface presentation of Notch receptors.

doi: 10.1038/s41598-023-46866-7

Figure Lengend Snippet: Figure 4. Physical association of Notch1 and TM2D3. (a) Physical association of TM2D3 and Notch1. 293 T cells were transfected with vectors for the indicated proteins or empty vector (Vector) as a control. Immunoprecipitation (IP) and immunoblotting were performed with the indicated antibodies. (b, c) Requirement of NRR of Notch1 for physical association with TM2D3. Experiments were conducted as in (a). (d) Requirement of LNR-A of NOTCH1 for physical association with TM2D3. A schematic diagram of NOTCH1 constructs used is shown in Supplementary Fig. S5 online. Experiments were conducted as in a. (e) Requirement of C-terminal portion including transmembrane 2 domain of TM2D3 for the activation of Notch1. Experiments were conducted as in (a). (f) No dependency on any one region of TM2D3 for the physical association with Notch1. Experiments were conducted as in (b).

Article Snippet: An antibody against the C-terminus of human NOTCH1 (sc-6014), an antibody against the ECD of murine Notch1 (8G10; sc-32756), and antibodies against the ECD of Notch2 (sc-518169 and sc-5545) were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, Construct, Activation Assay

Journal: Cancer Science

Article Title: Notch1 activation promotes renal cell carcinoma growth via PI 3 K / A kt signaling

doi: 10.1111/j.1349-7006.2012.02291.x

Figure Lengend Snippet: Clinicopathological features of the 125 clear cell renal cell carcinoma ( CCRCC ) patients and their correlations with N otch1 expression

Article Snippet: Control and Notch1 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) transfections were conducted with X‐tremeGENE (Roche Applied Science, Mannheim, Germany).

Techniques: Expressing

Journal: Cancer Science

Article Title: Notch1 activation promotes renal cell carcinoma growth via PI 3 K / A kt signaling

doi: 10.1111/j.1349-7006.2012.02291.x

Figure Lengend Snippet: Notch1 immunostaining correlates with p A kt and K i‐67 in clear cell renal cell carcinoma ( CCRCC ) samples

Article Snippet: Control and Notch1 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) transfections were conducted with X‐tremeGENE (Roche Applied Science, Mannheim, Germany).

Techniques: Immunostaining

Journal: Cancer Science

Article Title: Notch1 activation promotes renal cell carcinoma growth via PI 3 K / A kt signaling

doi: 10.1111/j.1349-7006.2012.02291.x

Figure Lengend Snippet: Notch1‐induced clear cell renal cell carcinomas (CCRCCs) cell growth is mediated by regulating PI3K/Akt pathway. (A) The confirmation of transfection efficiency. Western blot analysis of ICN1, pAkt in ACHN cells transiently transfected with increasing amounts of pcDNA3‐ICN1 plasmid (0, 0.5, 1 and 2 μg). glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as loading control. (B) Western blot analysis of ICN1, HA‐tag, pAkt, and GAPDH in three CCRCC cell lines transfected with pcDNA3 empty vector control (Con) or pcDNA3‐ICN1 (ICN1), tumor cells treated with LY294002 (LY) without or with pcDNA3‐ICN1 transfection, and tumor cells transfected with HA‐tagged DN‐Akt in the absence or presence of pcDNA3‐ICN1 cotransfection. GAPDH was used as loading control. (C) Cell proliferation assay using CCK‐8 was performed to evaluate the growth of the six groups of cells as described in (B). Data are mean ± standard deviation (SD) of three independent experiments. (D) Left, cell‐cycle distribution examined by propidium iodide staining and flow cytometry of the six groups of cells as described in (B). Right, the percentage of cells present in each phase of the cell cycle is shown in bar graph. Data are representative of three independent experiments. (E) Colony formation assay was performed to analyze the anchorage‐independent growth of the six groups of cells as described in (B). Left, macroscopically visible colonies were counted in three randomly chosen fields per group. Data are mean ± SD. Right, representative fields from each group are shown. Original magnification, ×100. In C–E, *P < 0.05, compared with Con group.

Article Snippet: Control and Notch1 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) transfections were conducted with X‐tremeGENE (Roche Applied Science, Mannheim, Germany).

Techniques: Transfection, Western Blot, Plasmid Preparation, Control, Cotransfection, Proliferation Assay, CCK-8 Assay, Standard Deviation, Staining, Flow Cytometry, Colony Assay

Journal: Cancer Science

Article Title: Notch1 activation promotes renal cell carcinoma growth via PI 3 K / A kt signaling

doi: 10.1111/j.1349-7006.2012.02291.x

Figure Lengend Snippet: Representative immunostaining for Notch1, pAkt and Ki‐67 in clear cell renal cell carcinoma (CCRCC) tissue microarrays. Scale bar, 25 μm.

Article Snippet: Control and Notch1 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) transfections were conducted with X‐tremeGENE (Roche Applied Science, Mannheim, Germany).

Techniques: Immunostaining

Journal: Cancer Science

Article Title: Notch1 activation promotes renal cell carcinoma growth via PI 3 K / A kt signaling

doi: 10.1111/j.1349-7006.2012.02291.x

Figure Lengend Snippet: Notch1 pathway components are upregulated in primary clear cell renal cell carcinomas (CCRCCs). (A) Western blot analysis of Jagged1, ICN1, Hes1 and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) in paired normal (N) and tumor (T) tissues from 16 CCRCC patients. GAPDH was used as loading control. (B) Densitometric quantification of the Western blot bands. The ratios of the intensity of Jagged1, ICN1 and Hes1 to that of GAPDH are shown as mean ± standard deviation (SD) of three independent experiments. *P < 0.05, significant increases (tumor versus normal tissue).

Article Snippet: Control and Notch1 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) transfections were conducted with X‐tremeGENE (Roche Applied Science, Mannheim, Germany).

Techniques: Western Blot, Control, Standard Deviation

Journal: Cancer Science

Article Title: Notch1 activation promotes renal cell carcinoma growth via PI 3 K / A kt signaling

doi: 10.1111/j.1349-7006.2012.02291.x

Figure Lengend Snippet: PI3K/Akt pathway is regulated by Notch1 activation. (A) Western blot analysis of Jagged1, ICN1, pAkt, tAkt and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) in HKC, 786‐0 and ACHN cells. (B) ACHN cells with Notch1 activation were assessed for Akt activity. Western blot analysis of ICN1, pAkt, tAkt and GAPDH in cells transfected with pcDNA3 empty vector control (Con) or pcDNA3‐ICN1 (ICN1), and cells stimulated with human IgG or Jagged1 Fc. (C) ACHN cells with Notch1 inhibition were assessed for Akt activity. Western blot analyses of ICN1, pAkt, tAkt and GAPDH in cells transfected with control (Con) siRNA or Notch1 siRNA, and cells treated with dimethylsulfoxide (DMSO) or N‐(N‐[3,5‐difluorophenacetyl]‐L‐alanyl)‐S‐phenylglycine t‐butyl ester (DAPT). In A–C, GAPDH was used as loading control.

Article Snippet: Control and Notch1 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) transfections were conducted with X‐tremeGENE (Roche Applied Science, Mannheim, Germany).

Techniques: Activation Assay, Western Blot, Activity Assay, Transfection, Plasmid Preparation, Control, Inhibition

Journal: Molecular cancer therapeutics

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

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

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

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

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

Journal: Molecular cancer therapeutics

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

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

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

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

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

Journal: Molecular cancer therapeutics

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

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

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

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

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

Journal: Molecular cancer therapeutics

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

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

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

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

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

Journal: Molecular cancer therapeutics

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

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

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

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

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

Journal: Circulation Research

Article Title: Notch Pathway Targets Proangiogenic Regulator Sox17 to Restrict Angiogenesis

doi: 10.1161/circresaha.115.303142

Figure Lengend Snippet: Figure 4. Sox17 expression is inversely regulated by Notch signaling. A, Immunofluorescence images for Sox17 (red) and green fluorescent protein (GFP; green) of human umbilical vein endothelial cells transfected with mock (control) or Notch1 intracellular domain (N1ICD) vector, which can express GPF simultaneously by a dual promoter system. Transfected cells are distinguished as GFP expression. Arrows highlight reduced Sox17 protein expression only in individual cells transfected with N1ICD vector but not in nontransfected cells. (B) Quantification of Sox17 protein expression in A. Images for Sox17 (red) and isolectin-B4 (IB4; green) of P5 retina. (C) Reduced Sox17 expression in N1ICDiGOF mutants. D, Increased Sox17- expressing endothelial cells on α-delta–like ligand 4 (Dll4) antibody treatment compared with IgG treatment. Scale bars, 100 μm. Ctrl indicates control.

Article Snippet: In Vitro Modulation of Notch Signaling For in vitro activation of Notch signaling, human umbilical vein endothelial cells were transfected with EF.hICN1.CMV.GFP vector (17623, Addgene) containing human Notch1 intracellular domain or EF.CMV.RFP vector (17619, Addgene) as a negative control.

Techniques: Expressing, Immunofluorescence, Transfection, Control, Plasmid Preparation

Journal: Circulation Research

Article Title: Notch Pathway Targets Proangiogenic Regulator Sox17 to Restrict Angiogenesis

doi: 10.1161/circresaha.115.303142

Figure Lengend Snippet: Figure 6. Notch pathway regulates Sox17 expression mainly at the post-transcriptional level. Immunoblotting and quantification of Sox17 signal (A, C, E, G, I) and transcript level of Sox17 (B, D, F, H, J). A to D, Reduced Sox17 protein level on Notch activation. A and B, Human umbilical vein endothelial cells (HUVECs) transfected with control or Notch1 intracellular domain (N1ICD) vector (n=4). C and D, P5 lungs from control and N1ICDiGOF pups (n=3). E to J, Increased Sox17 protein level after Notch inactivation. E and F, HUVECs treated with vehicle (control) or N-[N-(3,5-difluorophenacetyl)-L-alanyl]–S-phenylglycine t-butylester (DAPT; n=4). G and H, P5 lungs treated with IgG or α-delta-like ligand 4 (Dll4) antibody (Ab; n≥3). I, Images showing Sox17 (red) and platelet endothelial cell adhesion molecule (PECAM; blue) of Lewis lung carcinoma tumors treated with IgG or α-Dll4 Ab. Quantification of Sox17 intensity in tumor vessels (n=20; right). J, Transcript level of Sox17 in tumor endothelial cells (tECs) of tumors treated with IgG or α-Dll4 Ab (n=3). Data are presented as mean±SD; #P<0.01; *P<0.05. Scale bars, 100 μm.

Article Snippet: In Vitro Modulation of Notch Signaling For in vitro activation of Notch signaling, human umbilical vein endothelial cells were transfected with EF.hICN1.CMV.GFP vector (17623, Addgene) containing human Notch1 intracellular domain or EF.CMV.RFP vector (17619, Addgene) as a negative control.

Techniques: Expressing, Western Blot, Activation Assay, Transfection, Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: Anti-Tumor Effects of Second Generation β-Hydroxylase Inhibitors on Cholangiocarcinoma Development and Progression

doi: 10.1371/journal.pone.0150336

Figure Lengend Snippet: (A) Protein expression levels of ASPH, activated Notch1, JAG1, HEY1, and HES1 in 5 CCA cell lines. (B) ASPH, cyclin D1, EpCAM, HES1, HEY1, and cleaved caspase-3 in HEK293 cells transfected with WT-ASPH plasmid at concentrations of 0, 0.5, 1, 1.5, 2, and 2.5 μg. (C) Semi-quantitation of immunoblotting results depicted in ( B ). (D) Expression levels of ASPH, cyclin D1, EpCAM, CD44, HES1, HEY1, and cleaved caspase-3 in H1, RBE, and ETK1 cells infected with lentivirus containing either shLuc or shASPH. (E) pCS2-Notch1-full-length-6MT (pCS2-Notch1-F.L.-6MT), EV, ASPH, ASPH H675Q , and 12XCSL-DsRedExpressDL (Notch reporter) were co-transfected into HEK293 cells and image of red fluorescence was quantified under a fluorescence microscope. (F) Quantitation of red fluorescence signals are presented. Constitutive active Notch1 (pCS2-Notch1-ΔEMV-6MT) was used as a positive control. Transfection of Notch reporter construct alone was used as a negative control.

Article Snippet: Plasmids of murine Notch1 reporter construct (12XCSL-DsRedExpressDL, #47683), constitutive active Notch1 (pCS2-Notch1ΔEMV-6MT, #41737), full length Notch1 (pCS2-Notch1 F.L.-6MT, #41728), and human intracellular domain of Notch1 (EF.hICN1.CMV.GFP, #17623) used in rescue experiments were purchased from Addgene (Cambridge, Massachusetts).

Techniques: Expressing, Transfection, Plasmid Preparation, Quantitation Assay, Western Blot, Infection, Fluorescence, Microscopy, Positive Control, Construct, Negative Control

Journal: PLoS ONE

Article Title: Anti-Tumor Effects of Second Generation β-Hydroxylase Inhibitors on Cholangiocarcinoma Development and Progression

doi: 10.1371/journal.pone.0150336

Figure Lengend Snippet: (A) Relative CCND1 mRNA expression in H1, RBE, SSP25, and ETK1 cells infected with lentivirus containing shLuc or shASPH. (B) Immunoblotting of ASPH, cyclin D1, and intracellular domain of Notch1 (ICN) in RBE-shLuc, RBE-shASPH, ETK1-shLuc, and ETK1-shASPH transfected cells with or without overexpression of ICN. (C) Relative cell proliferation in RBE-shLuc, RBE-shASPH, ETK1-shLuc, and ETK1-shASPH in the presence or absence of 10 μM of the γ-secretase inhibitor DAPT using the MTT assay. (D) Relative cell proliferation rate in RBE-shLuc, RBE-shASPH, RBE-shASPH transfected cells overexpressing ICN. ***, p <0.001; **, p <0.01; *, p <0.05.

Article Snippet: Plasmids of murine Notch1 reporter construct (12XCSL-DsRedExpressDL, #47683), constitutive active Notch1 (pCS2-Notch1ΔEMV-6MT, #41737), full length Notch1 (pCS2-Notch1 F.L.-6MT, #41728), and human intracellular domain of Notch1 (EF.hICN1.CMV.GFP, #17623) used in rescue experiments were purchased from Addgene (Cambridge, Massachusetts).

Techniques: Expressing, Infection, Western Blot, Transfection, Over Expression, MTT Assay

Journal: PLoS ONE

Article Title: Anti-Tumor Effects of Second Generation β-Hydroxylase Inhibitors on Cholangiocarcinoma Development and Progression

doi: 10.1371/journal.pone.0150336

Figure Lengend Snippet: (A) Gross morphology and histology (H&E staining) of rat livers inoculated with BDE-Neu-CL#24-shRNA-luciferase (shLuc) or BDE-Neu-CL#24-shRNA-ASPH (shASPH). (B) Tumor volume in rat livers of BDE-Neu cell clone (#24) generated intrahepatic CCA tumors. (C) (Upper) Expression of ASPH, activated Notch1, HES1, and HEY1 in shLuc and shASPH treated in BDE-Neu-CL#24 generated tumors. (Lower) Relative expression abundance was shown by the density measurements. ( D ) (Upper) Representative IHC images of activated Notch1 in shLuc and shASPH treated rat intrahepatic CCA. (Lower) Number of positive nuclei containing an activated Notch1 signal was calculated. (E) H1 xenograft tumor growth rate and progression was determined in nude mice receiving DMSO or MO-I-1151 treatment every other day at 25 mg/kg. **, p <0.01; *, p <0.05.

Article Snippet: Plasmids of murine Notch1 reporter construct (12XCSL-DsRedExpressDL, #47683), constitutive active Notch1 (pCS2-Notch1ΔEMV-6MT, #41737), full length Notch1 (pCS2-Notch1 F.L.-6MT, #41728), and human intracellular domain of Notch1 (EF.hICN1.CMV.GFP, #17623) used in rescue experiments were purchased from Addgene (Cambridge, Massachusetts).

Techniques: Staining, shRNA, Luciferase, Generated, Expressing

Journal: Frontiers in Immunology

Article Title: NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome

doi: 10.3389/fimmu.2014.00249

Figure Lengend Snippet: NOTCH1 and PKCθ act in the same functional pathway to mediate allograft rejection . We monitored the influence on rejection kinetics of murine skin allografts by recipient mice deficient for various proteins. Grafts were visually scored for signs of rejection using a scale of 1–10, with 1 being completely rejected. An allograft was considered fully rejected when it was >80% necrotic. Graphs represent allograft score ( y axis), with a score of “1” being fully rejected and a score of “10” being fully accepted versus time in days after skin grafting ( x axis). Wild-type BALB/c skin was grafted onto (A) wild-type C57BL/6 mice (BALB/c → BL6), which served as a baseline for graft rejection for subsequent allograft combinations; (B) p50 null mice (BALB/c → p50 null ); (C) PKCθ null mice (BALB/c → PKCθ null ); (D) NOTCH1 conditional knock-out (N1KO) mice (BALB/c → N1KO); (E) wild-type C57BL/6 mice whose NOTCH signaling was abrogated by administering γ-secretase inhibitor (GSI LY411,575; BALB/c → WT + GSI); and (F) PKCθ null mice whose NOTCH signaling was abrogated by administering γ-secretase inhibitor (GSI LY411,575) in chow (BALB/c → PKCθ null + GSI). (G) Day to complete rejection was compared between different groups of recipient mice. For each animal grafted with BALB/c skin, an internal control of C57BL/6 skin was also grafted (BL6 → BL6) to monitor integrity of the grafting technique. Data represent the mean + SEM ( n = 3–16 mice/group). *** P < 0.001; one-way ANOVA with Tukey’s post-test applied.

Article Snippet: For shRNA experiments, freshly isolated CD4 + T cells were transfected with scrambled control shRNA or NOTCH1 shRNA (Santa Cruz Biotechnology) using the Amaxa Nucleoporator system according to manufacturer’s instructions.

Techniques: Functional Assay, Knock-Out, Control

Journal: Frontiers in Immunology

Article Title: NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome

doi: 10.3389/fimmu.2014.00249

Figure Lengend Snippet: NOTCH1 associates with PKCθ in stimulated T cells . NOTCH1 and PKCθ co-localize and interact following T cell stimulation. (A) Purified CD4 + T cells from C57BL/6 mice were incubated for 30 min with Dynal beads pre-coated with anti-mouse CD3ε and anti-mouse CD28 on glass slides. Cells were fixed, quenched, permeabilized, blocked, and stained with antibodies to NOTCH1 (green) and PKCθ (red). Proteins were visualized using species-specific, fluorescently conjugated secondary antibodies. *Indicates bead. (B) Jurkat T cells were stimulated with plate-bound anti-human CD3ε and anti-human CD28 for the indicated time periods, and subjected to co-immunoprecipitation with anti-NOTCH1. Immunoprecipitates (upper panel) and 1/100 of input (lower panels) were immunoblotted with anti-PKCθ and anti-NOTCH1. Data are representative of at least three independent experiments.

Article Snippet: For shRNA experiments, freshly isolated CD4 + T cells were transfected with scrambled control shRNA or NOTCH1 shRNA (Santa Cruz Biotechnology) using the Amaxa Nucleoporator system according to manufacturer’s instructions.

Techniques: Cell Stimulation, Purification, Incubation, Staining, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome

doi: 10.3389/fimmu.2014.00249

Figure Lengend Snippet: NOTCH1 interacts with CARMA1 and BCL10 following stimulation with anti-CD3ε and anti-CD28 . (A) Jurkat T cells were stimulated with plate-bound anti-human CD3ε and anti-human CD28 and subjected to sucrose gradient centrifugation. All fractions were analyzed by dot blot with anti-cholera toxin-conjugated HRP (upper panel). Pooled fractions were immunoblotted with indicated antibodies (lower panel). (B) Jurkat T cells were stimulated as in (A) for the indicated time periods, followed by co-immunoprecipitation with antibodies specific for NOTCH1, CARMA1, or BCL10. Eluates were subjected to immunoblotting with indicated antibodies. 1/100 of input (shown) was immunoblotted with anti-CARMA1, anti-BCL10, and anti-NOTCH1. Arrow heads beside IB: NOTCH1 represent 120 kDa, transmembrane form (upper arrow head) and 110 kDa intracellular form (lower arrow head) of NOTCH1. Data are representative of at least three independent experiments.

Article Snippet: For shRNA experiments, freshly isolated CD4 + T cells were transfected with scrambled control shRNA or NOTCH1 shRNA (Santa Cruz Biotechnology) using the Amaxa Nucleoporator system according to manufacturer’s instructions.

Techniques: Gradient Centrifugation, Dot Blot, Immunoprecipitation, Western Blot

Journal: Frontiers in Immunology

Article Title: NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome

doi: 10.3389/fimmu.2014.00249

Figure Lengend Snippet: Cytosolic N1IC can interact with CARMA1 in a bifluorescence complementation assay . Jurkat T cells were transfected with various constructs of nuclear and/or cytosolic proteins and their physical association was assessed microscopically by their ability to reconstitute two halves of a yellow fluorescent protein reporter. Twenty-four hours after transfection, cells were plated onto glass bottom culture dishes, stimulated with anti-human CD3ε and anti-human CD28, and fluorescent images of live cells were captured using a Zeiss LSM 510 confocal microscope. (A) Nuclear proteins, cFos and cJun, known to interact in the nucleus were co-expressed as a control; (B) NOTCH1 with an additional nuclear localization signal, N1IC–NLS was co-expressed with nuclear cJun; (C) NOTCH1 with an additional nuclear export signal, N1IC–NES, was co-expressed with nuclear cJun; (D) NOTCH1 with an additional nuclear export signal, N1IC–NES was co-expressed with CARMA1, a cytosolic protein; (E) NOTCH1 with an additional nuclear localization signal, N1IC–NLS was CARMA1; (F) NOTCH1 with an additional nuclear export signal, N1IC–NES was expressed alone. Upper panel of all images represent fluorescent channel only; lower panel of all images represent merged fluorescent and DIC images. Data are representative of at least three separate experiments.

Article Snippet: For shRNA experiments, freshly isolated CD4 + T cells were transfected with scrambled control shRNA or NOTCH1 shRNA (Santa Cruz Biotechnology) using the Amaxa Nucleoporator system according to manufacturer’s instructions.

Techniques: Transfection, Construct, Microscopy, Control

Journal: Frontiers in Immunology

Article Title: NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome

doi: 10.3389/fimmu.2014.00249

Figure Lengend Snippet: NOTCH1 deficiency prevents the formation of the CBM complex . (A) NOTCH1 expression was efficiently reduced when Jurkat T cells were incubated with viral supernatants. NOTCH1-knock-down Jurkat T cells, or mock-infected control cells, were stimulated with plate-bound anti-human CD3ε and anti-human CD28 for 1 h, harvested for co-immunoprecipitation with indicated antibodies, then subjected to immunoblotting with (B,C) anti-NOTCH1, anti-CARMA1, anti-BCL10, or anti-PKCθ; 1/100 of input was immunoblotted with indicated antibodies, or with (D) antibodies specific for NOTCH1, phosphorylated and total IκBα, phosphorylated and total ERK, and phosphorylated and total p38 MAPK. (E) NOTCH1-knock-down Jurkat T cells, or mock-infected control cells, were stimulated with plate-bound anti-human CD3ε and anti-human CD28 for the times indicated. EMSAs were performed on nuclear extracts using radiolabeled oligonucleotides containing the NF-κB binding sequence. Red filled diamond represents competition with cold probe. Data are representative of at least three independent experiments.

Article Snippet: For shRNA experiments, freshly isolated CD4 + T cells were transfected with scrambled control shRNA or NOTCH1 shRNA (Santa Cruz Biotechnology) using the Amaxa Nucleoporator system according to manufacturer’s instructions.

Techniques: Expressing, Incubation, Knockdown, Infection, Control, Immunoprecipitation, Western Blot, Binding Assay, Sequencing

Journal: Frontiers in Immunology

Article Title: NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome

doi: 10.3389/fimmu.2014.00249

Figure Lengend Snippet: The RAM domain of NOTCH1 mediates its interaction with CARMA1 . (A) GFP–N1IC mutant expression plasmids were transfected into 293T cells and sub-cellular localization of NOTCH1 was examined using fluorescence microscopy. (B) VSV–CARMA1 expression plasmids were transiently co-transfected with GFP empty vector as a control, or with GFP–N1IC mutant plasmids in 293T cells, as indicated. Transfected cells were harvested and cell lysates were co-immunoprecipitated with antibodies specific for GFP. Co-immunoprecipitated proteins were then immunoblotted with anti-VSV or anti-GFP. Ig heavy chain indicates immunoglobulin heavy chain.

Article Snippet: For shRNA experiments, freshly isolated CD4 + T cells were transfected with scrambled control shRNA or NOTCH1 shRNA (Santa Cruz Biotechnology) using the Amaxa Nucleoporator system according to manufacturer’s instructions.

Techniques: Mutagenesis, Expressing, Transfection, Fluorescence, Microscopy, Plasmid Preparation, Control, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome

doi: 10.3389/fimmu.2014.00249

Figure Lengend Snippet: NOTCH1 increases NF-κB activity through its direct interaction with CARMA1 in the cytoplasm . (A) 293T cells were transiently transfected with GFP-N1IC–NLS or GFP-N1IC–NES and sub-cellular localization was determined using fluorescence microscopy. (B) CARMA1 expression plasmids were transiently co-transfected with GFP empty vector as a control, or with GFP-N1IC–NLS or GFP-N1IC–NES in 293T cells. Cytoplasmic extracts from transfected cells were co-immunoprecipitated with anti-GFP. 1/100 of input (shown) was immunoblotted with anti-VSV and anti-GFP. (C) Vectors expressing cytosolic N1IC–NES, membrane-tethered N1ΔE–PM, and/or cytosolic CARMA1 were co-transfected with an NF-κB luciferase reporter construct into Jurkat T cells, stimulated with PMA and CaI for 3 h, then subjected to a luciferase reporter gene assay. Ig heavy chain indicates immunoglobulin heavy chain. Data are mean + SEM of at least three independent experiments.

Article Snippet: For shRNA experiments, freshly isolated CD4 + T cells were transfected with scrambled control shRNA or NOTCH1 shRNA (Santa Cruz Biotechnology) using the Amaxa Nucleoporator system according to manufacturer’s instructions.

Techniques: Activity Assay, Transfection, Fluorescence, Microscopy, Expressing, Plasmid Preparation, Control, Immunoprecipitation, Membrane, Luciferase, Construct, Reporter Gene Assay

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: Lineage Plasticity in SCLC Generates Non-Neuroendocrine Cells Primed for Vasculogenic Mimicry

doi: 10.1016/j.jtho.2023.07.012

Figure Lengend Snippet: Non-NE CDX cells ex vivo are VM-competent and require NOTCH signaling. ( A ) Representative immunoblots of CDX NE and non-NE cell lysates. There are two to three independent replicate tumors per CDX. Tubulin loading control was run subsequently for each marker illustrated on the same blot. ( B ) Representative brightfield images of tubule-forming assay with CDX NE and non-NE cells. There were two to three independent replicate tumors per CDX. Scale bars, 500 μ m. ( C ) Representative immunofluorescence of CDX non-NE cells in tubule-forming assay stained for human mitochondria (yellow) and nuclear DAPI (blue) in ( B ). There were two to three independent replicate tumors per CDX. Scale bars, 100 μ m. ( D-F ) Representative images of HUVECs ( D ), CDX17 ( E ), and CDX30P ( F ) non-NE cells labeled with Cell Tracker Green forming hollow tubules when grown on Matrigel for 72 hours. Confocal microscopy images are illustrated after Z-stack software reconstruction (Imaris). Tubule length and diameter dimensions are illustrated, scale bars 50 μ m. ( G ) Representative images of empty-vector control and NICD expressing CDX31P cells three weeks after dox induction. Scale bars, 250 μ m. ( H ) Percentage of adherent cells in control versus NICD-expressing CDX31P cells three weeks after dox induction in the suspension cells (four replicates from one tumor sample). Data are mean (± SEM) (***p < 0.001 two-tailed unpaired Student’s t test. ( I ) Representative immunoblots in control and NICD CDX31P cells with or without dox. Tubulin loading control was run subsequently for each marker illustrated on the same blot. ( J ) Reverse transcription quantitative PCR analysis of NE ( ASCL1, SYP, NCAm, cHgA, MYCL ) and non-NE ( HEY1, REST, YAP1, FOXC2, MYC ) markers in control versus NICD-expressing CDX31P cells. Mean values are illustrated (black lines) in which each circle represents one independent analysis, error bars are (± SEM). ( K ) Representative images of tubule-forming assay with control and NICD CDX31P cells 3 weeks after dox induction. Scale bars, 200 μ m. There are three independent replicate tumors. CDX, circulating tumor cell-derived explant; DAPI, 4’,6-diamidino-2-phenylindole; dox, doxycycline; HUVECs, human umbilical vein endothelial cells; NE, neuroendocrine; NICD, NOTCH 1 intracellular domain; PCR, polymerase chain reaction; SEM, standard error of the mean; VM, vasculogenic mimicry.

Article Snippet: The human NOTCH1 intracellular domain (hN1ICD) doxycycline-inducible expression plasmid (pLIX-hN1ICD) was a gift from Julien Sage (Addgene #91897 [Watertown, MA]).

Techniques: Ex Vivo, Western Blot, Control, Marker, Immunofluorescence, Staining, Labeling, Confocal Microscopy, Software, Plasmid Preparation, Expressing, Suspension, Two Tailed Test, Reverse Transcription, Real-time Polymerase Chain Reaction, Derivative Assay, Polymerase Chain Reaction