Journal: Molecular Cancer

Article Title: A positive feed-forward loop between LncRNA-URRCC and EGFL7/P-AKT/FOXO3 signaling promotes proliferation and metastasis of clear cell renal cell carcinoma

doi: 10.1186/s12943-019-0998-y

Figure Lengend Snippet: URRCC enhances EGFL7 level by mediating histone H3 acetylation of EGFL7 promoter. a : URRCC subnetwork according to the result of Target mRNA PCR Array. Genes colored in blue are down-regulated genes after that cells were transfected with sh-URRCC. Genes colored in red are up-regulated genes after that cells were transfected with sh-URRCC. The size of the gene round represents the fold change. b : The mRNA and protein levels of EGFL7 were detected by qRT-PCR and WB assays in A498 and OSRC-2 cells after transfection with sh-control or sh-URRCC. c : The mRNA and protein levels of EGFL7 were detected by qRT-PCR and WB assays in A498 and OSRC-2 cells after transfection with mock or oe-URRCC. d : EGFL7 expression in ccRCC and normal samples from TCGA KIRC dataset. e : Representative EGFL7 IHC staining of ccRCC tissues compared to paired normal renal tissues (200×, 400×). Black scale bar represents 100 μm (200×) or 50 μm (400×). f and g : Representative EGFL7 IHC staining of xenograft tumors from sh-control, sh-URRCC, mock, and oe-URRCC groups (200×, 400×). Black scale bar represents 100 μm (200×) or 50 μm (400×). h : qRT-PCR and WB analysis of EGFL7 in A498 and OSRC-2 cells treated with DMSO or trichostatin A (TSA) (50 nM or 100 nM) for 72 h ( n = 3). i : ChIP analyses of A498 cells transfected with sh-control or sh-URRCC were conducted on the EGFL7 promoter regions using anti-acetyl-histone H3 and anti-acetyl-histone H4. Enrichment was determined relative to input controls. The data are the means ± standard deviations of three independent biological replicates. j : ChIP analyses of A498 cells transfected with sh-control or sh-URRCC were conducted on the GAPDH promoter regions using anti-acetyl-histone H3 and anti-acetyl-histone H4. Enrichment was determined relative to input controls. k : ChIP analyses of OSRC-2 cells transfected with mock or oe-URRCC were conducted on the EGFL7 promoter regions using anti-acetyl-histone H3 and anti-acetyl-histone H4. Enrichment was determined relative to input controls. The data are the means ± standard deviations of three independent biological replicates. l : EGFL7 promoter region was enriched with H3K27Ac histone mark presented with UCSC data. m : WB analysis of H3K27ac and total H3 in A498 and OSRC-2 cells treated with DMSO or TSA, β-actin was used as a loading control

Article Snippet: Immunohistochemistry for the target molecules was performed on paraffin sections using a primary antibody against EGFL7 (1:50, proteintech, 19,291–1-AP), FOXO3 (1:500, Abcam, ab12162), P-AKT (1:50, Abcam, ab131443), Ki67 (1:500, Abcam, ab92742) and the proteins in situ were visualized with 3, 3-diaminobenzidine.

Techniques: Transfection, Quantitative RT-PCR, Control, Expressing, Immunohistochemistry

Journal: Molecular Cancer

Article Title: A positive feed-forward loop between LncRNA-URRCC and EGFL7/P-AKT/FOXO3 signaling promotes proliferation and metastasis of clear cell renal cell carcinoma

doi: 10.1186/s12943-019-0998-y

Figure Lengend Snippet: URRCC is associated with EGFL7/P-AKT/FOXO3 signaling pathway. a and b : WB analysis of T-AKT, P-AKT, and FOXO3 in URRCC-overexpressing or URRCC-inhibited A498 and OSRC-2 cells compared with sh-control or mock group, β-actin was used as a loading control. c : CCK8 assays of A498 cells transfected with mock/oe-URRCC and si-NC/si-EGFL7. d : Representative images and the numbers of invasive cells per high-power field reduced after the transfection with mock/oe-URRCC and si-NC/si-EGFL7 in A498 cells. e : WB analysis for EGFL7, T-AKT, P-AKT, and FOXO3 protein levels of A498 cells transfected with mock/oe-URRCC and si-NC/si-EGFL7. β-actin was used as a loading control

Article Snippet: Immunohistochemistry for the target molecules was performed on paraffin sections using a primary antibody against EGFL7 (1:50, proteintech, 19,291–1-AP), FOXO3 (1:500, Abcam, ab12162), P-AKT (1:50, Abcam, ab131443), Ki67 (1:500, Abcam, ab92742) and the proteins in situ were visualized with 3, 3-diaminobenzidine.

Techniques: Control, Transfection

Journal: Molecular Cancer

Article Title: A positive feed-forward loop between LncRNA-URRCC and EGFL7/P-AKT/FOXO3 signaling promotes proliferation and metastasis of clear cell renal cell carcinoma

doi: 10.1186/s12943-019-0998-y

Figure Lengend Snippet: A Schematic Diagram of LncRNA-URRCC-Based Signaling Pathway in ccRCC cells proliferation and invasion. LncRNA-URRCC upregulates AKT signaling by directly targeting EGFL7 via mediating histone H3 acetylation of EGFL7 promoter, and then inducing cell growth and invasion. AKT signaling downstream FOXO3 in turn downregulates URRCC by directly binding to its promoter

Article Snippet: Immunohistochemistry for the target molecules was performed on paraffin sections using a primary antibody against EGFL7 (1:50, proteintech, 19,291–1-AP), FOXO3 (1:500, Abcam, ab12162), P-AKT (1:50, Abcam, ab131443), Ki67 (1:500, Abcam, ab92742) and the proteins in situ were visualized with 3, 3-diaminobenzidine.

Techniques: Binding Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of notch signaling pathway prevents cholestatic liver fibrosis by decreasing the differentiation of hepatic progenitor cells into cholangiocytes.

doi: 10.1038/labinvest.2015.149

Figure Lengend Snippet: Figure 3 Notch signaling pathway was activated in CLF. (a) Notch-1, 2, 3, 4 mRNA. (b) JAG1, 2, and DLL1, 3, 4 mRNA. (c) Hes1, Numb, and RBPJк mRNA. All mRNA were quantified with RT-PCR and normalized to GAPDH mRNA (n = 6 per group). (d) Notch-1, Notch-2, Notch3, Notch4, JAG1, JAG2, DLL1, RBPJк, and Numb protein expression were quantified via immunoblotting and normalized to GAPDH (n = 6 per group), and (e) densitometric analysis of protein bands. *Po0.05, **Po0.01. Sham, Sham group; 1 wM, BDL-1w group; 2 wM, BDL-2w group; 3 wM, BDL-3w group; 4 wM, BDL-4w group.

Article Snippet: Rabbit polyclonal antibodies to Notch4 (sc-5594), JAG1 (sc-8303), DLL1 (sc-9102), mouse monoclonal antibody to Sox9 (E-9, sc-166505), and goat polyclonal antibody to JAG2 (sc-34475) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of notch signaling pathway prevents cholestatic liver fibrosis by decreasing the differentiation of hepatic progenitor cells into cholangiocytes.

doi: 10.1038/labinvest.2015.149

Figure Lengend Snippet: Figure 6 Inhibition of the Notch signaling pathway reduced the progression of liver fibrosis. (a) Notch-1, Notch-2, Notch3, Notch4, JAG1, JAG2, DLL1, RBPJк, and Numb, protein bands on the left (immunoblot), and the histogram depicts the densitometric analysis of protein bands (n = 6 per group). (b) Notch-1, Notch-2, Notch3, Notch4, JAG1, JAG2, DLL1, RBPJк, and Numb mRNA were measured via RT-PCR and normalized to GAPDH mRNA (n = 6 per group). (c) Sirius Red staining (×100). (d) α-SMA immunostaining (×200). (e) Hydroxyproline content. (f) α-SMA, TNF-α, TGF-β1, MCP-1, Col(1), and Col (4) mRNA were measured by RT-PCR and normalized to GAPDH mRNA (n = 6 per group). *Po0.05, **Po0.01. BDL, single bile duct ligation group; DAPT, BDL plus DAPT group; sham, sham group.

Article Snippet: Rabbit polyclonal antibodies to Notch4 (sc-5594), JAG1 (sc-8303), DLL1 (sc-9102), mouse monoclonal antibody to Sox9 (E-9, sc-166505), and goat polyclonal antibody to JAG2 (sc-34475) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining, Immunostaining, Ligation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Androgen receptor suppresses vasculogenic mimicry in hepatocellular carcinoma via circRNA7/miRNA7‐5p/VE‐cadherin/Notch4 signalling

doi: 10.1111/jcmm.16022

Figure Lengend Snippet: AR increases the expression of miR‐7‐5p in SK cells. (A) Relative normalized expression of miRNAs by qRT‐PCR analysis in SK cells. oeAR significantly up‐regulates expression of miR‐7‐5p and shAR down‐regulates the expression of miR‐7‐5p in SK cells. (B) The effect of shmiR‐7‐5p on VM formation. The number of VM formations were counted and compared in SK and HA22T cells. (C) Quantification of VM formation in SK and HA22T cells. (D) The effect of shmiR‐7‐5p on expression of VE‐cadherin and Notch4 in SK and HA22T. Western blotting was performed as described in Materials and Methods. ## P < .01 compared with PWPI + PLV group. ** P < .01 compared with oeAR + PLV group

Article Snippet: Notch4 antibody for Western blotting analysis was purchased from Biorbyt.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: The Journal of allergy and clinical immunology

Article Title: A Jagged 1–Notch 4 molecular switch mediates airway inflammation induced by ultrafine particles

doi: 10.1016/j.jaci.2018.03.009

Figure Lengend Snippet: AM supports UFP-dependent upregulation of Th cell cytokine production by allergen-specific CD4+ T cells in a Notch4-dependent manner. A, Representative flow cytometric analysis of IL-4, IL-13 and IL-17 cytokine production by naive Il4raR576CD4+DO11.10+ T cells co-cultured with FACS-purified AM isolated from l4raR576 or Il4raR576Lyz2CreJag1Δ/Δ mice that have been pulsed with OVA323-339 peptide in the presence of UFP (10 μg/mL). Co-cultures were treated with either isotype control (Iso) Ab or an anti-Notch4 mAb, as indicated, and cytokine analysis was carried out on gated CD4+Foxp3− T cell. B, Frequencies of T cells expressing the respective cytokine upon co-culture with AM that have been either sham treated (PBS) or pulsed with OVA323-339 peptide alone or in combination with UFP (10 μg/mL). Anti-Notch4 mAb or isotype control Ab were added as indicated. Results are representative of 3 independent experiments. *P < .05, **P < .01, ***P < .001, and ****P < .0001, two-way ANOVA with post-test analysis.

Article Snippet: For anti-Notch4 antibody blocking, 150 μg Armenian hamster anti-mouse Notch4 IgG mAb (clone HMN4-14; Bio X Cell) 35 , or control Armenian hamster IgG polyclonal antibodies (Ab) (Bio X cell), were suspended in 100 μl PBS buffer and administered daily for three consecutive days during OVA aerosol challenge.

Techniques: Cell Culture, Purification, Isolation, Expressing, Co-Culture Assay

Journal: The Journal of allergy and clinical immunology

Article Title: A Jagged 1–Notch 4 molecular switch mediates airway inflammation induced by ultrafine particles

doi: 10.1016/j.jaci.2018.03.009

Figure Lengend Snippet: UFP enhances allergic airway inflammation in a Notch4-dependent manner. A, Representative PAS staining of lung tissues isolated from Il4raR576 mice sensitized and challenged with OVA alone, or together with UFP, in the presence of either isotype control (Iso) Ab or an anti-Notch4 mAb. B, Inflammation scores in lung tissues of the mouse groups described in in Fig 7, A. C–H Airway hyper-responsiveness in response to methacholine (Fig 7, C), absolute numbers of eosinophils (Fig 7, D), T cells (Fig 7, E) and neutrophils (Fig 7, F) in the BAL fluids, total (Fig 7, G) and OVA-specific (Fig 7, H) levels in the serum of the mouse groups described in Fig 7, A. I-L, Absolute numbers of lung Foxp3−CD4+T cells secreting IL-4 (Fig 7, I), IL13 (Fig 7, J), IL-17 (Fig 7, K) and IFN-γ (Fig 7, L) in the mouse groups described in Fig 7, A. M–P, Absolute numbers of lung Foxp3+CD4+Treg cells secreting IL-4 (Fig 7, M), IL13 (Fig 7, N), IL-17 (Fig 7, O) and IFN-γ (Fig 7, P) in the mouse groups described in panel Fig 7, A. Results are representative of 2 independent experiments. N=5 mice/group. *p<0.05, **<0.01, ***<0.001, ****<0.0001 by two-way ANOVA with post test analysis.

Article Snippet: For anti-Notch4 antibody blocking, 150 μg Armenian hamster anti-mouse Notch4 IgG mAb (clone HMN4-14; Bio X Cell) 35 , or control Armenian hamster IgG polyclonal antibodies (Ab) (Bio X cell), were suspended in 100 μl PBS buffer and administered daily for three consecutive days during OVA aerosol challenge.

Techniques: Staining, Isolation