Journal: Science Advances

Article Title: Epigenetically heterogeneous tumor cells direct collective invasion through filopodia-driven fibronectin micropatterning

doi: 10.1126/sciadv.aaz6197

Figure Lengend Snippet: ( A ) RNA-seq normalized gene counts for the canonical Notch receptors and ligands. Ordinary two-way ANOVA with Tukey’s correction. n = 3. ( B ) Western blots, H1299 parental, follower, and leader cells and H1299 cells FACS sorted for JAG1-low/IL13RA2 + (J LOW IL + ) or JAG1-high/IL13RA2 − (J HIGH IL − ). Probed for MYO10, JAG1 full-length (FL), JAG1 intracellular domain (ICD), and IL13RA2. n = 3. ( C ) JAG1 immunofluorescence of 3D invasion of H1299 parental, follower, or leader cell spheroids. Scale bars, 50 μm. n = 3. ( D ) qPCR, leader cell mRNA expressing shCtrl or shJAG1, normalized to actin. n = 3. ( E ) Western blots of JAG1 and MYO10 expressions in H1299 leaders expressing shCtrl or shJAG1. n = 3. ( F ) Western blots of JAG1 and MYO10 in H1299 leaders (L), followers expressing an empty vector (F Ctrl) or JAG1 (F JAG1). n = 4. ( G ) Quantification of MYO10 methylation status using qPCR. n = 3. ( H and I ) Violin plots quantifying 2D filopodia length with representative images of leader cell filopodia in leaders expressing shRNA-JAG1 and/or mCherry-MYO10 (H) or followers expressing JAG1 and/or mCherry-MYO10 (I), n = 2, Kruskal-Wallis test with Dunn’s multiple comparison test. Scale bars, 10 mm. ( J ) 3D spheroid invasion of leaders expressing shRNA-JAG1 and/or mCherry-MYO10. n = 4. ( K ) Representative images and quantification of 3D collective spheroid invasion follower cells expressing JAG1 and/or mCherry-MYO10. n = 3. (B, E, and F) Actin as loading control. (D, G, J, and K) Ordinary one-way ANOVA with Tukey’s correction. (A to K) n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: JAG1-PE (Sino Biological, 11648-MM03-P) was used at 5 μl per 100 μl, and IL13RA2-APC (Miltenyi Biotec, 130-104-505) was used at 2 μl per 100 μl.

Techniques: RNA Sequencing Assay, Western Blot, Immunofluorescence, Expressing, Plasmid Preparation, Methylation, shRNA

Journal: Journal of Diabetes Research

Article Title: The Involvement of Notch1-RBP-Jk/Msx2 Signaling Pathway in Aortic Calcification of Diabetic Nephropathy Rats

doi: 10.1155/2017/8968523

Figure Lengend Snippet: The mRNA expressions of Notch1 (a), RBP-Jk (b), Msx2 (c), and Jagged1 (d) in aortic tissues. Data presented as mean ± standard deviation (SD). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus Nor group; Δ p > 0.05, # p < 0.05, ## p < 0.01, and ### p < 0.001. Nor group: normal controls; DN + VDN group: diabetic nephropathy rats with vitamin D3/nicotine-induced vascular calcification.

Article Snippet: The following primary antibodies were used for immunohistochemical analysis: monoclonal rabbit anti-rat RBP-Jk, Msx2, and N1-ICD (Shanghai Bioworld Tech Inc., China); monoclonal rabbit anti-rat Jagged1 and Runx2 (Beijing BioTech Inc., China); monoclonal rabbit anti-rat Notch1 (Wuhan Proteintech Group Inc., China); and monoclonal mouse anti-rat α -SMA (Wuhan Boster Bio-Tech., Ltd, China).

Techniques: Standard Deviation

Journal: Journal of Diabetes Research

Article Title: The Involvement of Notch1-RBP-Jk/Msx2 Signaling Pathway in Aortic Calcification of Diabetic Nephropathy Rats

doi: 10.1155/2017/8968523

Figure Lengend Snippet: Detection of Notch1, RBP-Jk, Msx2, Jagged1, and N1-ICD levels by immunohistochemical analysis. The representative images and quantitative analysis of Notch1 (a-b), RBP-Jk (c-d), Msx2 (e-f), Jagged1 (g-h), and N1-ICD (i-j) in aortic tissues. The left panel of histological images: Nor group; right panel: DN + VDN group at 8, 12 , and 16 weeks, respectively. Data presented as mean ± standard deviation (SD). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus Nor group; Δ p > 0.05, # p < 0.05, ## p < 0.01, and ### p < 0.001. Nor group: normal controls; DN + VDN group: diabetic nephropathy rats with vitamin D3/nicotine-induced vascular calcification.

Article Snippet: The following primary antibodies were used for immunohistochemical analysis: monoclonal rabbit anti-rat RBP-Jk, Msx2, and N1-ICD (Shanghai Bioworld Tech Inc., China); monoclonal rabbit anti-rat Jagged1 and Runx2 (Beijing BioTech Inc., China); monoclonal rabbit anti-rat Notch1 (Wuhan Proteintech Group Inc., China); and monoclonal mouse anti-rat α -SMA (Wuhan Boster Bio-Tech., Ltd, China).

Techniques: Immunohistochemical staining, Standard Deviation

Journal: Nature Communications

Article Title: Inhibiting WNT and NOTCH in renal cancer stem cells and the implications for human patients

doi: 10.1038/s41467-020-14700-7

Figure Lengend Snippet: a , b Treatment schemes and quantifications of tumor volumes of PDX4 and PDX1 tumors ( n = 3 tumors in each group) treated with 100 mg/kg ICG-001 (green diamond), 10 mg/kg DAPT (blue squares), combination (black diamond, PDX1 only) or vehicle (red circles) every three days. Data are shown as mean, error bars represent s.d., p -values: *<0.05, *<0.01, ***<0.001 by two-way ANOVA with Tukey post-test (two-sided). c , d HE and Ki-67 staining of representative sections of tumors of PDX1 and PDX4 (scale bars, 50 µm). e , f Immunofluorescence of β-CATENIN (scale bars, 50 µm) and in situ hybridization for AXIN2 (scale bars 100 µm) in PDX4 and PDX1 tumors in mice treated with 100 mg/kg ICG-001. g , h Immunofluorescence of NOTCH1 and JAG1 in PDX4 and PDX1 tumors in mice treated with 10 mg/kg DAPT (scale bars, 50 µm).

Article Snippet: Section were incubated overnight at 4 °C with antibodies against CA9 (1:200, Abcam), CD10 (1:30, Dako), CD44 (1:400, BD biosciences), CXCR4 (1:200, Abcam), MET (1:50, Cell Signaling Technology), VCAM1 (1:250, Abcam), E-Cadherin (1:1000, BD biosciences), Epcam (1:500, Abcam), Ki-67 (1:200, Thermo Fisher), or JAG1 (1:200, Atlas Antibodies) primary antibodies and Alexa-conjugated secondary antibodies (1:200, Dianova).

Techniques: Staining, Immunofluorescence, In Situ Hybridization

Journal: Clinical chemistry

Article Title: Potential Involvement of Jagged1 in Metastatic Progression of Human Breast Carcinomas.

doi: 10.1373/clinchem.2015.246686

Figure Lengend Snippet: Fig. 1. Impact of Jagged1 expression on survival analysis in breast cancer patients with negative lymph nodes. N0 indicates negative lymph nodes status.

Article Snippet: Deparaffinized tissue microarray sections were treated for 5 min in citrate buffer (pH 6.0, Biogenex) at 120 °C in a steamer and incubated for 16 h at 4 °C with rabbit polyclonal antihuman Jagged1 antibody (CD339/Jagged1, 110 –125, Acris Antibodies) diluted 1:500 in Dako REALTM antibody diluent (Dako).

Techniques: Expressing

Journal: Clinical chemistry

Article Title: Potential Involvement of Jagged1 in Metastatic Progression of Human Breast Carcinomas.

doi: 10.1373/clinchem.2015.246686

Figure Lengend Snippet: Fig. 3. Jagged1 expression in CTCs of breast cancer patients. Representative images of Jagged1-positive CTC in the peripheral blood of metastatic breast cancer patient stained with Jagged1 (orange)/ keratins (green)/CD45 (red) and counterstained with DAPI (blue) to visualize cells’ nuclei. Tumor cells are defined as keratin(+), DAPI(+), and CD45(−) cells. Magnification 1000×.

Article Snippet: Deparaffinized tissue microarray sections were treated for 5 min in citrate buffer (pH 6.0, Biogenex) at 120 °C in a steamer and incubated for 16 h at 4 °C with rabbit polyclonal antihuman Jagged1 antibody (CD339/Jagged1, 110 –125, Acris Antibodies) diluted 1:500 in Dako REALTM antibody diluent (Dako).

Techniques: Expressing, Staining

Journal: Clinical chemistry

Article Title: Potential Involvement of Jagged1 in Metastatic Progression of Human Breast Carcinomas.

doi: 10.1373/clinchem.2015.246686

Figure Lengend Snippet: Fig. 4. Impact of CTCs and Jagged1(+)CTCs on survival analysis. Correlation of CTCs and CTCs expressing Jagged1 to progression-free survival (PFS) in the total cohort of patients, patients treated with bisphosphonates and patients with bone metastasis.

Article Snippet: Deparaffinized tissue microarray sections were treated for 5 min in citrate buffer (pH 6.0, Biogenex) at 120 °C in a steamer and incubated for 16 h at 4 °C with rabbit polyclonal antihuman Jagged1 antibody (CD339/Jagged1, 110 –125, Acris Antibodies) diluted 1:500 in Dako REALTM antibody diluent (Dako).

Techniques: Expressing

Journal: Experimental and therapeutic medicine

Article Title: Study of dietary‑induced progression of psoriasis‑like mice based on gut macrophage polarization.

doi: 10.3892/etm.2023.11976

Figure Lengend Snippet: Figure 5. Protein expressions of Jagged1/Notch1/HES5 and TLR‑2/IκB‑α/NF‑κB p65 signaling pathway on skin lesions of imiquimod‑induced psoriasis model. (A and a) Western blot analysis of Notch signaling and NRP1 in skin lesions. The mammalian Notch signaling pathway consists of four Notch receptors (Notch 1, 2, 3, and 4) and five Notch ligands (Jagged 1, and 2, and Delta‑like 1, 3 and 4). HES5 is an important downstream target gene of Notch signaling pathway. NRP1 is involved in keratinocyte hyperproliferation of psoriasis accompanied by inflammation activated angiogenesis. (B and b) Western blot analysis of TLR‑2/IκB‑α/NF‑κB p65 signaling in skin lesions. Values are expressed as mean ± SD (*P<0.05, **P<0.01 vs. the normal control; #P<0.05, ##P<0.01, ###P<0.001 vs. the psoriasis group) and were statistically analyzed with one‑way ANOVA. NC, normal control; P, psoriasis; P + SF, psoriasis + SF; SF, stimulating food; p‑, phosphorylated.

Article Snippet: Proteins were transferred onto a PVDF membrane (MilliporeSigma), and then incubated with primary antibodies specific for NF‐κB p65 (cat. no. 6956), phospho‐NF‐κB p65 (cat. no. 3033), Toll‐like receptor‐2 (cat. no. 2229S), IKBα (cat. no. 9242), Jagged‐1 (cat. no. 70109s), Notch‐1 (cat. no. 3608s; all CST; 1:1,000), Hes‐5 (cat. no. ab25374; Abcam; 1:1,000) and anti‐β‐actin (cat. no. BM0627; Wuhan Boster Biological Technology, Ltd.; 1:2,000) overnight at 4 ̊C and then followed by incubation with horseradish peroxidase‐conjugated secondary anti‐mouse or anti‐rabbit IgG (cat. nos.

Techniques: Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Regulation of C-C chemokine receptor 5 (CCR5) stability by Lys 197 and by transmembrane protein aptamers that target it for lysosomal degradation

doi: 10.1074/jbc.RA117.001067

Figure Lengend Snippet: Lysine 197 reduces the stability of CCR5 in the absence of traptamers. a, BaF3 cells expressing empty (MSCVn) vector or BaF3 cells expressing WT or K197M CCR5 were established in parallel, and cell-surface expression of each receptor was analyzed by flow cytometry of nonpermeabilized cells by using 2D7 anti-CCR5 antibody. The geometric mean fluorescence intensity for each sample was determined and normalized to cells expressing WT CCR5. Average and S.D. (error bars) for three experiments are shown. b, BaF3 cells expressing the empty vector, WT CCR5, or the indicated CCR5 point mutant were established in parallel. Detergent lysates were subjected to SDS-PAGE and Western blotting for CCR5 or Hsp90 as a loading control. c, the cells studied in a were subjected to cell-surface biotinylation, and biotinylated cell-surface proteins were recovered from cell extracts with streptavidin beads. The pellet (cell-surface) and supernatant (intracellular) fractions were subjected to SDS-PAGE and immunoblotted using an anti-CCR5 antibody. Samples were also probed for Jag1 or Hsp90 as loading controls. d, BaF3 cells expressing WT CCR5 or CCR5/K197M were treated with cyclohexamide (CHX), and at the indicated times, a portion of the culture was pelleted and lysed. Cell lysates were electrophoresed and immunoblotted for CCR5. The filters were stripped and reprobed for actin as a loading control. Numbers to the right of b, c, and d indicate the size in kilodaltons of molecular mass standards.

Article Snippet: Antibodies Unconjugated and FITC-conjugated mouse anti-human CCR5 monoclonal antibody 2D7, which recognizes a conformation-specific N-terminal epitope, mouse anti-CXCR4 mAb 12G5, and monoclonal antibodies recognizing profilin, Hsp90 (catalog no. 610418), β-catenin (catalog no. 610153), or Jag1 (catalog no. 612346) were purchased from BD Biosciences.

Techniques: Expressing, Plasmid Preparation, Flow Cytometry, Fluorescence, Mutagenesis, SDS Page, Western Blot

Journal: Oncology Letters

Article Title: MicroRNA-26b acts as an antioncogene and prognostic factor in cervical cancer

doi: 10.3892/ol.2019.9965

Figure Lengend Snippet: Clinicopathological features and miR-26b expression in 54 paired cervical cancer tissues.

Article Snippet: Then, the blots were transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and blocked with 5% skim milk powder at room temperature for 1 h. The membrane was incubated with rabbit JAG1 polyclonal antibody (cat. no. PAB807Mu01; 1:500; Wuhan USCN Business Co., Ltd., Wuhan, China) and mouse GAPDH momoclonal antibody (cat. no. sc-32233; 1:2,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C overnight.

Techniques: Expressing

Journal: Oncology Letters

Article Title: MicroRNA-26b acts as an antioncogene and prognostic factor in cervical cancer

doi: 10.3892/ol.2019.9965

Figure Lengend Snippet: RT-qPCR detection of miR-26b and JAG1 expression levels in cervical cancer tissues and cells. (A) The relative expression level of miR-26b was determined in CC tissues and PT by RT-qPCR (normalized by U6). (B) miR-26b expression levels in HeLa and JAR are expressed as relative to that of Ect1/E6E7 cells. (C) JAG1 mRNA expression levels in HeLa and JAR are compared to that of normal cervical epithelial cells Ect1/E6E7. (D) Correlation between miR-26b and JAG1 mRNA expression levels is presented. **P<0.01, ***P<0.001. miR-26b, microRNA-26b; JAG1, Jagged1; CC, cervical cancer; PT, paracancerous tissues.

Article Snippet: Then, the blots were transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and blocked with 5% skim milk powder at room temperature for 1 h. The membrane was incubated with rabbit JAG1 polyclonal antibody (cat. no. PAB807Mu01; 1:500; Wuhan USCN Business Co., Ltd., Wuhan, China) and mouse GAPDH momoclonal antibody (cat. no. sc-32233; 1:2,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C overnight.

Techniques: Quantitative RT-PCR, Expressing

Journal: Oncology Letters

Article Title: MicroRNA-26b acts as an antioncogene and prognostic factor in cervical cancer

doi: 10.3892/ol.2019.9965

Figure Lengend Snippet: JAG1 was a target of miR-26b in cervical cancer cells. (A) Two binding sites of miR-26b on 3′UTR of JAG1 were predicted by TargetScan. (B) Changes in the luciferase activity of cells co-transfected with miR-26b mimic (or negative control) and WT or MUT S1/S2 in HeLa and JAR cells are presented. (C) The expression levels of JAG1 mRNA and protein were calculated by RT-qPCR and western blotting when transfected with miR-26b mimic or inhibitor. *P<0.05, **P<0.01, ***P<0.001. JAG1, Jagged1; miR-26b, microRNA-26b; 3′UTR, 3′-untranslated regions; WT, wild-type of JAG1 3′UTR; MUT S1/S2, mutant site 1/2 of JAG1 3′UTR.

Article Snippet: Then, the blots were transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and blocked with 5% skim milk powder at room temperature for 1 h. The membrane was incubated with rabbit JAG1 polyclonal antibody (cat. no. PAB807Mu01; 1:500; Wuhan USCN Business Co., Ltd., Wuhan, China) and mouse GAPDH momoclonal antibody (cat. no. sc-32233; 1:2,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C overnight.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Negative Control, Expressing, Quantitative RT-PCR, Western Blot, Mutagenesis

Journal: Oncology Letters

Article Title: MicroRNA-26b acts as an antioncogene and prognostic factor in cervical cancer

doi: 10.3892/ol.2019.9965

Figure Lengend Snippet: JAG1 restored partial impact of miR-26b in cervical cancer cells. (A) The expression levels of JAG1 were measured by RT-qPCR and western blotting when cervical cancer cells were transfected with miR-26b mimic or miR-26b mimic and JAG1 overexpression plasmid. (B) Migration and invasion were calculated when cervical cancer cells were transfected with miR-26b mimic or miR-26b mimic and JAG1. (C) Kaplan-Meier survival curves of cervical cancer patients with high or low miR-26b expression level. *P<0.05, **P<0.01, ***P<0.001. JAG1, Jagged1; miR-26b, microRNA-26b.

Article Snippet: Then, the blots were transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and blocked with 5% skim milk powder at room temperature for 1 h. The membrane was incubated with rabbit JAG1 polyclonal antibody (cat. no. PAB807Mu01; 1:500; Wuhan USCN Business Co., Ltd., Wuhan, China) and mouse GAPDH momoclonal antibody (cat. no. sc-32233; 1:2,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Over Expression, Plasmid Preparation, Migration

Journal: Oncology Letters

Article Title: Linc-OIP5 loss regulates migration and invasion in MDA-MB-231 breast cancer cells by inhibiting YAP1/JAG1 signaling

doi: 10.3892/ol.2019.11071

Figure Lengend Snippet: Differential expression of Linc-OIP5 , YAP1, and JAG1 in breast cancer cells with different degrees of malignancy. (A) Relative expression of Linc-OIP5 in MDA-MB-231 and MCF-7 cells. *P<0.05. (B) YAP1 and JAG1 expression levels were significantly higher in MDA-MB-231 cells compared with the MCF-7 cells. Expression was normalized to GAPDH . **P<0.01. Immunofluorescence analysis confirming that expression of (C) YAP1 and (D) JAG1 were significantly higher in MDA-MB-231 cells compared with MCF-7 cells. Immunofluorescence staining for YAP1 and JAG1 proteins (green) and nuclei (blue). *P<0.05; Magnification ×100. (E) Western blots showing that YAP1 and JAG1 expression levels cells were significantly upregulated in MDA-MB-231 cells compared with MCF-7 cells. Protein expression was normalized against β-actin. ***P<0.001.

Article Snippet: The following primary antibodies were used for immunofluorescence staining: YAP1 (1:200 dilution; cat. no. GTX129151; GeneTex, Inc.) and JAG1 (1:100 dilution; cat. no. GTX48691; GeneTex, Inc.).

Techniques: Quantitative Proteomics, Expressing, Immunofluorescence, Staining, Western Blot

Journal: Oncology Letters

Article Title: Linc-OIP5 loss regulates migration and invasion in MDA-MB-231 breast cancer cells by inhibiting YAP1/JAG1 signaling

doi: 10.3892/ol.2019.11071

Figure Lengend Snippet: YAP1 and JAG1 expression are downregulated in MDA-MB-231 cells transfected with Linc-OIP5 siRNA. (A) Western blotting confirmed that Linc-OIP5 knockdown significantly decreased the expression of YAP1 and JAG1 in MDA-MB-231 cells. Expression levels were normalized to β-actin. **P<0.01, ***P<0.001. (B) Reverse transcription-quantitative PCR demonstrated that the mRNA expression levels of YAP1 and JAG1 were prominently decreased in MDA-MB-231 cells treated with Linc-OIP5 siRNA. mRNA expression levels were normalized to GAPDH . **P<0.01, ***P<0.001. NC, negative control; si, small interfering.

Article Snippet: The following primary antibodies were used for immunofluorescence staining: YAP1 (1:200 dilution; cat. no. GTX129151; GeneTex, Inc.) and JAG1 (1:100 dilution; cat. no. GTX48691; GeneTex, Inc.).

Techniques: Expressing, Transfection, Western Blot, Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control