Journal: Cell Death & Disease
Article Title: Identification of a novel tumor angiogenesis inhibitor targeting Shh/Gli1 signaling pathway in Non-small cell lung cancer
Figure Lengend Snippet: Pristimerin treatment decreases Shh-induced pericyte motilities of angiogenesis. a Pristimerin suppresses Shh-mediated adhesion ability of endothelial cells. The cells that pre-treated with Shh (100 ng/mL) and pristimerin (125, 250, and 500 nM) were seeded at 24-well plate. After 1-h culture, the un-attached cells were removed and the attached cells were stained with 0.1% crystal violet. b Pristimerin restrains Shh-induced pericyte migration to newly formed networks in a three-dimensional co-culture system of HUVEC and HBVP. HUVECs (red) were seeded on Matrigel-coated 96-well and cultured for 2 h to allow the formation of tube network. And HBVPs (green) pre-treated with or without pristimerin were added to the endothelial tubes. The recruitment of HBVPs to HUVEC tubes were observed and photographed at 0, 2, and 12 h. Scale bar: 20 μm. c Pristimerin suppresses the nucleus translocation of Gli1in HBVPs. HBVPs were stimulated with Shh (100 ng/mL) and pristimerin for 24 h. After staining with 4% paraformaldehyde, the HBVPs were labeled with Gli1 antibody and Alexa Fluor 488 Donkey anti-Rabbit IgG. DAPI was used to recognize the nucleus, and the images were taken with a laser scanning confocal microscope. d Pristimerin inhibits the distribution of Gli1 in the nucleus. HBVPs were collected and lysed with a NE-PERTM Nucleus and Cytoplasmic Extraction Kit following the manufacturer’s protocol, and then applied for western blotting assay. β-actin and Lamin B were set as loading control. e Pristimerin blocks the downstream of Shh/Gli1 signaling pathway. After being treated with Shh and pristimerin for 24 h, HBVP cells were lysed using RIPA buffer and subjected to western blotting assay. β-actin was set as loading control. The data are presented as mean ± SEM, n = 3. *** P
Article Snippet: Antibodies against α-SMA (19245S), Ki67 (9449T), Akt (4685S), p-AktThr308 (13038S), ERK (4695S), p-ERKThr202/Tyr204 (4370T), PDGFR-β (3174T), p-PDGFR-βTyr857 (3170T), β-actin (4970T), Alexa Fluor 594 Donkey anti-Goat IgG (889S), Alexa Fluor 488 Donkey anti-Rabbit IgG (4416S) and HRP-conjugated anti-rabbit IgG antibody (4412S) were purchased from Cell Signaling Technology (Danvers, MA, USA).
Techniques: Staining, Migration, Co-Culture Assay, Cell Culture, Translocation Assay, Labeling, Microscopy, Western Blot