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: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Fully human monoclonal antibody targeting activated ADAM10 on colorectal cancer cells

doi: 10.1016/j.biopha.2023.114494

Figure Lengend Snippet: 1H5 inhibits Notch cleavage. Sandwich ELISA was used to measure the levels of total (A) and cleaved (B) Notch1 in COLO205 cells upon treatment with 1H5. The data represent mean of triplicate experiments, and the bar plots show the effect of treatment with 1H5 relative to untreated control, mean ± SEM. Comparison of notch levels between treated and untreated groups was performed using independent t test. Total Notch1 levels did not significantly differ between the two groups (A), p = 0.162. On the other hand, the mAb-treated group showed significant decrease of the cleaved Notch1 levels when compared with the untreated control (B), p < 0.001.

Article Snippet: To detect NICD1, we used PathScan ® cleaved Notch1 (Val1744) sandwich ELISA Kit (Cell Signaling Technologies).

Techniques: Sandwich ELISA, Control, Comparison

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: Inhibition of the Notch pathway aggravates DSS-induced colitis. CT: control, LY411,575 (Notch inhibitor): 10 mg/kg LY411,575 gavage, DSS: 2.5% DSS water, DSS-LY411,575: LY411,575 was intragastric administrated at the last 4 days of DSS feeding (once per day). (A) DAI scores of each group. DAI scores were compared by the repeated measures ANOVA with Bonferroni post hoc multiple comparisons. (B) Serum FD-4 concentration in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (C) Histological scores of distal colons in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (D) Representative colonic H&E staining image of each group (scale bar = 50 μm). (E) Representative colonic IF staining image of claudin-1 ( green ) and claudin-3 ( green ) (scale bar = 25 μm). (F) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) (scale bar = 25 μm). Quantification of N1ICD and Hes1 were determined by the ratio of positive staining cells per crypt. Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Inhibition, Control, Concentration Assay, Staining

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: Notch pathway is inhibited in the colon under VDd or VDR KO conditions. (A, B) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDR KO (A) and VDd (B) mice (scale bar = 25 μm). (C, D) Real-time PCR analysis of the gene expression of Notch ligands, receptors, and effectors in the colon in VDR KO (C) and VDd (D) mice. Expression levels (VDR KO vs. WT or VDd vs. CT) were compared using paired t -test. Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Staining, Real-time Polymerase Chain Reaction, Expressing

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR modulates the Notch pathway in colitis. (A, B) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDd-DSS and respective control colitis mice (scale bar = 25 μm). (C, D) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDR KO-DSS and respective control colitis mice. To activate VDR, mice were administered paricalcitol (PAR) 7 days before drinking DSS water and were sustained until sacrifice. (E) DAI scores of each group. DAI scores were compared by the repeated measures ANOVA with Bonferroni post hoc multiple comparisons. (F) Histological scores of distal colons in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (G) Representative colonic H&E staining image of each group (scale bar = 50 μm). (H) Representative colonic IF staining image of claudin-1 ( green ), claudin-3 ( green ), and Hes1 ( red ) (scale bar = 25 μm). Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR signaling regulates the Notch pathway and TJs in Caco-2 cells. TNF-α (100 ng/mL) was added to mimic inflammatory stimulation, and 100 nM PAR was used to activate VDR; the solvate was added as control (CT). Specific siRNA of VDR (SiVDR) was used to downregulate VDR, negative control (NC) sequence was used as control (scale bar = 10 μm). (A) Representative IF staining image of claudin-1 ( green ) and claudin-3 ( green ) in Caco-2 cells treated with or without PAR before TNF-α intervention. (B) Representative IF staining image of N1ICD ( red ) and Hes1 ( red ) in Caco-2 cells with or without VDR downregulation before TNF-α intervention. (C) Representative IF staining image of claudin-1 ( green ), claudin-3 ( green ), N1ICD ( red ), and Hes1 ( red ) in Caco-2 cells after VDR downregulation.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Control, Negative Control, Sequencing, Staining

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR-mediated TJ protection is partly dependent on the Notch pathway. LY411,575 (1 μM) was used to inhibit the Notch pathway in Caco-2 cells, and 100 nM PAR was used to activate VDR. The solvate was added as control (CT). Representative IF staining image of N1ICD ( red ), Hes1 ( red ), claudin-1 ( green ), and claudin-3 ( green ) in Caco-2 cells treated with or without LY411,575 before PAR intervention (scale bar = 10 μm).

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Control, Staining

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR signaling positively regulates the Notch-1/Hes1 pathway. (A) Representative IF staining image of claudin-1 ( green ), claudin-3 ( green ), N1ICD ( red ), and Hes1 ( red ) in cultured intestinal organoids (scale bar = 10 μm). (B) Western blot analysis of N1ICD and Hes1 proteins in cultured intestinal organoids from WT and VDR KO mice. (C) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in cultured intestinal organoids from WT and VDR KO mice. (D) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in Caco-2 cells after TNF-α intervention. (E, F) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in Caco-2 cells after downregulation of VDR with siVDR (E) or activation of VDR with PAR (F) . Data are presented as the mean ± SEM from three separate experiments, the statistical analysis performed with paired t -test. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Staining, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR positively regulates Notch-1 transcription. (A) Western blot analysis of VDR, Notch-1, N1ICD, and Hes1 in Caco-2 cells after siVDR and PAR intervention. (B) Graphs of the predicted VDR binding region in the Notch-1 promoter. (C) Dual-luciferase reporter assay of VDR to Notch-1 promoter. Notch-1 promoter (Notch-1) and negative control (CON) were constructed in a GV238 vector. The VDR cDNA and vector (pcDNA3.1) were linked to upregulate VDR (pVDR). Data are presented as the mean ± SEM from three separate experiments. Statistical analysis was performed using ANOVA with Tukey’s post-test. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Western Blot, Binding Assay, Luciferase, Reporter Assay, Negative Control, Construct, Plasmid Preparation