Journal: Microvascular research

Article Title: Semaphorin 5A promotes angiogenesis by increasing endothelial cell proliferation, migration, and decreasing apoptosis

doi: 10.1016/j.mvr.2009.10.005

Figure Lengend Snippet: Sema5A induces migration of endothelial cells through Met kinase activity. (A) Increased migration of HMEC-1 cells treated with Sema5A. Migration of HMEC-1 cells in response to Sema5A was examined using a transwell chamber assay (see Materials and methods for description). The values represent the average number of cells migrated±SEM. *Significantly different from HMEC-1 cells cultured in media alone (p<0.05). (B) HMEC-1 cells undergoing migration in response to Sema5A were captured using a light microscope at 200× magnification. HMEC-1 treated with a. media alone, b. 10 ng/ml of Sema5A and c. 10 ng/ml of VEGF-A. (C) Sema5A–induced migration of endothelial cells is mediated through Met receptor. Migration of HMEC-1 cells in response to Sema5A with or without neutralizing antibody for Met receptor was examined using a transwell chamber assay. The values are percent cells migrated in the presence of Sema5A with or without Met antibody ± SEM. *Significantly different from endothelial cells treated with Sema5A alone.

Article Snippet: HMEC-1 cells (1×10 5 ) were plated in duplicate onto the upper transwell chamber (6.5 mm, Corning Costar, Cambridge, MA).

Techniques: Migration, Activity Assay, Transwell Chamber Assay, Cell Culture, Light Microscopy

Journal: Journal of Cellular and Molecular Medicine

Article Title: Cell surface‐anchored syndecan‐1 ameliorates intestinal inflammation and neutrophil transmigration in ulcerative colitis

doi: 10.1111/jcmm.12934

Figure Lengend Snippet: Shed SDC 1 and inflammatory responses induced by PMA in HT 29 cells. PMA was used to induce SDC 1 shedding in HT 29 cells. ( A ) Levels of Sdc1 in six intestinal epithelial cells were detected by western blot. GAPDH was used as the loading control. ( B ) Level of cell surface SDC 1 was detected by immunofluorescence. SDC 1 (red), cell nucleus (blue), original magnifications: 40×. ( C ) Levels of shed SDC 1 in the cell culture supernatant were detected by ELISA (upper) and dot blot (lower). ( D ) Levels of secreted TNF ‐α, IL ‐1β, IL ‐6 and IL ‐8 in the cell culture supernatant were detected by PCR . ( E ) Levels of P65 and phosphorylated P65 were detected by Western blot. ( F ) Secretion of CXCL ‐1 was detected by ELISA . ( G ) Neutrophils were isolated from human venous blood, and their transmigration was measured with use of transwell inserts. Crystal violet staining was used to observe the cell quantity change. Values represent mean ± S.E.M ( n = 3), * P < 0.05, ** P < 0.01, based on t ‐test.

Article Snippet: For the migration assay, 0.5 × 10 6 neutrophils in 10% FBS medium were added to the upper insert of each transwell polyester membrane filter (6.5‐mm‐diameter inserts, 3.0‐μm pore size; BD, NY, USA) and 500 μl cell culture supernatant was collected from the epithelial cells at the indicated time‐point (when secretion of CXCL‐1 was highest), and added to the matched lower chamber.

Techniques: Western Blot, Immunofluorescence, Cell Culture, Enzyme-linked Immunosorbent Assay, Dot Blot, Isolation, Transmigration Assay, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Cell surface‐anchored syndecan‐1 ameliorates intestinal inflammation and neutrophil transmigration in ulcerative colitis

doi: 10.1111/jcmm.12934

Figure Lengend Snippet: Secreted CXCL ‐1 induced by LPS and sequent neutrophil transmigration was inhibited by cell surface‐anchored SDC 1 in Caco‐2 cells. LPS was use to induce secretions of CXCL ‐1, and expression of CXCL ‐1 was assessed by ELISA . ( A ) The level of secreted CXCL ‐1 was down‐regulated by Sdc1. ( B and C ) Cell culture supernatants containing high concentrations of CXCL ‐1 were used to induce migration of human neutrophils. CXCL ‐1 secretion promoted migration of neutrophils; this promoting effect was diminished when cells were transfected with mut‐ SDC 1. The transmigration was measured with use of transwell inserts, crystal violet staining was used to observe the cell quantity change. Values represent mean ± S.E.M ( n = 3) and were analysed by Duncan's multiple range test for multiple comparison in anova (* P < 0.05, ** P < 0.01. #significance between wt‐ SDC 1 and mut‐ SDC 1, # P < 0.05).

Article Snippet: For the migration assay, 0.5 × 10 6 neutrophils in 10% FBS medium were added to the upper insert of each transwell polyester membrane filter (6.5‐mm‐diameter inserts, 3.0‐μm pore size; BD, NY, USA) and 500 μl cell culture supernatant was collected from the epithelial cells at the indicated time‐point (when secretion of CXCL‐1 was highest), and added to the matched lower chamber.

Techniques: Transmigration Assay, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Migration, Transfection, Staining