Journal: Bioactive Materials

Article Title: Phosphorylation inhibition of protein-tyrosine phosphatase 1B tyrosine-152 induces bone regeneration coupled with angiogenesis for bone tissue engineering

doi: 10.1016/j.bioactmat.2020.12.025

Figure Lengend Snippet: 152RM promotes angiogenesis via the Notch signaling pathway. (A) Representative images of tube formation by ECs (with or without coculture with MSCs) after the addition of 152RM. Scale bar, 100 μm. The quantitative analysis of cumulative tube length is shown in the right panel (n = 5 per group). (B) Gene set enrichment analysis (GSEA) plots showing upregulation of the Notch signaling pathway in ECs cultured with 152RM (n = 3 per group). (C) RNA-seq analysis showed alterations in Notch signaling pathway-related gene expression in ECs cultured with 152RM (n = 3 per group). (D) Relative mRNA expression levels of Notch signaling pathway-related genes in ECs cultured with 152RM (n = 5 each). (E) Representative immunostaining images of DLL4 (red) ECs with 152RM (n = 5 per group). Scale bar, 100 μm. (F) Representative immunostaining images of Notch1 (red) ECs with 152RM (n = 5 per group). Scale bar, 100 μm. (G) Representative images of tube formation by ECs treated with 152RM (with or without Notch inhibitor). n = 5 per group. Scale bar, 100 μm. (H) Representative immunostaining images of Noggin (red) ECs with 152RM (n = 5 per group). Scale bar, 100 μm. (I) Schematic illustration of the role of 152RM in inducing the formation of type H vessels and coupling osteogenesis and angiogenesis. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; Student's t -test was employed. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with individual primary antibodies against mouse CD31 (ab28364; Abcam), endomucin (V.7C7; Santa Cruz), Ki67 (AF7617; R&D), beta-catenin (8480, CST), osterix (bs-1110R; Bioss), osteocalcin (bs-0470R; Bioss), Runx2 (bs-1134R; Bioss), DLL4 (bs-6044R; Bioss), Notch1 (bs-1335R; Bioss), Noggin (bs-2975R; Bioss), CXCR4 D1S7W; Cell Signaling Technology), integrin αvβ3 (bs-1310R; Bioss), CD90 (bs-20640R; Bioss), CD105 (bs-0579R; Bioss), CD271 (bs-0161R; Bioss), OPN (bs-23258R; Bioss), and phospho-VEGFR2(bs-2674R; Bioss) overnight at 4 °C.

Techniques: Cell Culture, RNA Sequencing Assay, Expressing, Immunostaining

Journal: Frontiers in Pharmacology

Article Title: Antiproliferative and Immunoregulatory Effects of Azelaic Acid Against Acute Myeloid Leukemia via the Activation of Notch Signaling Pathway

doi: 10.3389/fphar.2019.01396

Figure Lengend Snippet: Notch was found up-regulated after azelaic acid (AZA) treatment. (A) 120 differential expressed proteins associated in 52 functions after AZA treatment by protein mass spectrometry analysis in the heat map. (B) GO annotation with 528 differential expressed proteins. (C, D) THP-1, Molm-13, NK, and T cells were treated with 10 µM AZA for 24 h, the expression level of ICN1 and ICN2 was validated by western blot. (E) The expression of Notch1 and Notch2 in mouse spleen measured by ICH. IHC, immunohistochemistry.

Article Snippet: The following antibodies were used: CD3(Cat# 100203), CD4 (Cat# 100431), CD8 (Cat# 100761), CD107a (Cat# 328605), TRAIL (Cat# 308205), CD25 (Cat# 302605), and CD69 (Cat# 310903) were purchased from BioLegend (USA), ICN1 (Cat# CSB-PA084572), ICN2 (Cat# CSB-PA964902) were from CUSABIO (USA); GAPDH (Cat# 6004-1), β-actin (Cat# 14395-1) were from ProteinTech (USA).

Techniques: Mass Spectrometry, Expressing, Western Blot, Immunohistochemistry

Journal: Frontiers in Pharmacology

Article Title: Antiproliferative and Immunoregulatory Effects of Azelaic Acid Against Acute Myeloid Leukemia via the Activation of Notch Signaling Pathway

doi: 10.3389/fphar.2019.01396

Figure Lengend Snippet: Azelaic acid (AZA) exerts anti-leukemic effect by activating the Notch signaling pathway. (A) Notch responsive elements were transfected into 293T cells after 24 h. Cells were then treated with 10 µM AZA, 10 µM RO4929097, and combination for 24 h, the Notch signaling reporter assay was measured by dual luciferase reporter activity. (B) Validation of the RNA expression of Notch1 and Notch2, the downstream target genes HES1 and HEY1 in Molm-13 and THP-1 cells by qPCR. (C) The protein expression level of ICN1, ICN2, HEY1, and HES1 in acute myeloid leukemia (AML) cell lines after treatment of AZA and RO4929097 and their detection by western blot. ImageJ was used for the densitometric analysis. Data represent means ± SD. (D) Molm-13 cells were pretreated with 10 µM RO4939097 for 24 h, and then treated with 10 µM AZA for another 24 h. Cells were collected for apoptosis analysis. (E) NK cells and T cells were pretreated with 10 µM AZA, 10 µM RO4929097, and combination for 24h before co-culture with THP-1 cell and Molm-13 cells at an E:T ratio of 5:1. The cytotoxicity of NK and T was determined by detecting the LDH release rate. (F) NK and T cells were pre-treated with AZA and RO4929097, then co-cultured with THP-1 cell at an E:T ratio of 3:1 for 4 h. The level of TNF-α and IFN-γ in the supernatant was measured by ELISA. A Total of three independent experiments were performed. *P < 0.05, **P < 0.01,***P < 0.001.

Article Snippet: The following antibodies were used: CD3(Cat# 100203), CD4 (Cat# 100431), CD8 (Cat# 100761), CD107a (Cat# 328605), TRAIL (Cat# 308205), CD25 (Cat# 302605), and CD69 (Cat# 310903) were purchased from BioLegend (USA), ICN1 (Cat# CSB-PA084572), ICN2 (Cat# CSB-PA964902) were from CUSABIO (USA); GAPDH (Cat# 6004-1), β-actin (Cat# 14395-1) were from ProteinTech (USA).

Techniques: Transfection, Reporter Assay, Luciferase, Activity Assay, Biomarker Discovery, RNA Expression, Expressing, Western Blot, Co-Culture Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Notch system is differentially expressed and activated in pituitary adenomas of distinct histotype, tumor cell lines and normal pituitaries

doi: 10.18632/oncotarget.19046

Figure Lengend Snippet: ( A – C ) Gene expression normalized to Gapdh in AtT20 cells and mouse pituitaries determined by qRT-PCR and expressed as percentage of change of the normal pituitaries which were considered 100%. *p = 0.0003 for Notch3, Notch1,2,4 NS, n = 3–5; 3–8 for AtT20 and pituitary respectively (A), p = 0.0111 and p = 0.0105 for the Notch ligands Dll1 and Jagged1 , respectively vs pituitary and (B) p = 0.0016, 0.0184 and 0.0032 for the Notch target genes Hes1, Hes5 and Hey2 , respectively, vs. pituitary n = 2–3; 3 for AtT20 and pituitary respectively (C). ( D , E ) Active and membrane domains of NOTCH1-3 receptor levels determined by Western Blot: Bars show the mean of the receptor normalized to β actin levels, expressed as the percentage of normal mouse pituitary. *p = 0.026 for NOTCH1, p < 0.0001 for NOTCH2 and p = 0.0146 for NOTCH3 vs. pituitary, for the active domains (80KDa) n = 3–6; 5–7 for AtT20 and pituitary respectively (D). No significant differences were found for the membrane domain (110 KDa) n = 3–6; 3–7 for AtT20 and pituitary respectively (E). Representative Western blots showing the active and membrane domains of NOTCH receptors in normal pituitaries (Pit) and ATt20 cells ( F ).

Article Snippet: For mouse tissue the following antibodies were used: Rabbit polyclonal antibody against NOTCH1 and NOTCH2 (dilution 1:700, Merck Millipore), NOTCH3 (dilution 1:200, Santa Cruz Biotechnology Inc), HES1 (dilution 1:400, Merck Millipore).

Techniques: Gene Expression, Quantitative RT-PCR, Membrane, Western Blot

Journal: Oncotarget

Article Title: Notch system is differentially expressed and activated in pituitary adenomas of distinct histotype, tumor cell lines and normal pituitaries

doi: 10.18632/oncotarget.19046

Figure Lengend Snippet: ( A – C ) Gene expression determined by qRT-PCR in pituitaries of lacDrd2KO and Drd2l oxP/loxP counterparts normalized to Gapdh and expressed as percentage of change of the Drd2 loxP/loxP pituitaries considered 100%. Bars show mean values. Notch1 *p = 0.0431, Notch3 *p = 0.0089 vs. Drd2 loxp/loxp ; Notch2, 4 NS, n = 7, 5 for lacDrd2KO and Drd2 loxP/loxP mice, respectively (A). Notch ligands Dll1 and Jagged1 NS (B), and Notch target genes Hes1 (* p = 0.0106 lacDrd2KO vs. Drd2 loxP/loxP ) , Hes5 and Hey2 , NS (C); n = 7, 5 for lacDrd2KO and Drd2 loxP/loxP mice, respectively. ( D – E ) The expression of the active and membrane domains of NOTCH1-3 receptors was determined by Western blot, related to the correspondent β actin levels, and expressed as the percentage of Drd2 loxP/loxP pituitary average. *p = 0.01 for NOTCH1, NOTCH2, 3 NS active domain, p =0.0604 and p = 0.0042 for NOTCH2 and NOTCH3 membrane domains, respectively for lacDrd2KO vs Drd2 loxP/loxP m ice. n = 4, 3–4 for lacDrd2KO and Drd2 loxP/loxP . Representative Western blots showing the active and membrane domains of NOTCH receptors in lacDrd2KO and Drd2 loxP/loxP ( F ). Representative microphotographies of NOTCH1-3 and HES1 Immunohistochemistry performed in lacDrd2KO and Drd2 loxP/loxP mice ( G ).

Article Snippet: For mouse tissue the following antibodies were used: Rabbit polyclonal antibody against NOTCH1 and NOTCH2 (dilution 1:700, Merck Millipore), NOTCH3 (dilution 1:200, Santa Cruz Biotechnology Inc), HES1 (dilution 1:400, Merck Millipore).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Membrane, Western Blot, Immunohistochemistry