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: International Journal of Molecular Sciences

Article Title: Indoxyl Sulfate Inhibits Osteogenesis in Bone Marrow Mesenchymal Stem Cells through the AhR/Hes1 Pathway

doi: 10.3390/ijms25168770

Figure Lengend Snippet: IS upregulated the expression of hairy/enhancer of split-1 ( Hes1 ) through AhR. ( A ) qRT-PCR revealed the mRNA levels of Notch receptors and Hes1 in D1 cells treated with IS for at least 4 and 8 h or left untreated. ( B ) Western blotting revealed the protein levels of cleaved-Notch1 and Hes1 in D1 cells treated with IS or a vehicle control for 1 and 2 days. ( C ) qRT-PCR indicated the level of Hes1 expression in control or IS-treated D1 cells subjected to AhR antagonist (CH-223191) treatment (upper panel) or Ahr knockdown (KD) (lower panel) for 4 and 8 h. Data are presented as mean ± standard deviation values ( n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001; * compared with the control. # p < 0.05, ## p < 0.01, ### p < 0.001; # compared with the IS-treated group; Student t -test ( A , B ) and analysis of variance ( C )).

Article Snippet: The membrane was blocked with 5% skim milk–Tris-buffered saline with 0.1% Tween 20 for 1 h. After washing, the membrane was incubated overnight at 4 °C with primary antibodies against Runx2 (Abcam, Cambridge, UK, Cat# ab23981), Bmp2 (Abcam, Cat# ab14933), Alp (Abcam, Cat# ab203106), Oc (Abcam, Cat# ab93876), AhR (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, Cat# sc-133088), Hes1 (NOVUS Biologicals, Centennial, CO, USA, Cat# NBP1-47791), and cleaved-Notch1 (Elabscience Biotechnology Co., Ltd., Wuhan, China, Cat# E-AB-30054).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Knockdown, Standard Deviation

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: Journal of Feline Medicine and Surgery

Article Title: Immunohistochemical evaluation of the activation of hepatic progenitor cells and their niche in feline lymphocytic cholangitis

doi: 10.1177/1098612X17699723

Figure Lengend Snippet: Primary antibodies and antigen retrieval and washing buffer methods

Article Snippet: Notch1/NICD , Mouse , Monoclonal , mN1a , Merck/Millipore , 1:200 , TE, pH 9.0 , TBS and TBS/T.

Techniques: