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

Journal: Stem Cell Research & Therapy

Article Title: Ang II-AT2R increases mesenchymal stem cell migration by signaling through the FAK and RhoA/Cdc42 pathways in vitro

doi: 10.1186/s13287-017-0617-z

Figure Lengend Snippet: Effect of different concentrations of Ang II on migration of human bone marrow MSCs. a Nondirectional migration ability of human bone marrow MSCs after stimulations with different concentrations of Ang II (10 –8 , 10 –7 , 10 –6 , 10 –5 , and 3 × 10 –5 M) examined using the scratch assay. Wound sites (areas cleared of cells in the center of the scratched area) were observed and photographed at 0 and 24 h (200×). b Quantitative results of wound healing. c Directional migration ability of human bone marrow MSCs after stimulations with the different concentrations of Ang II indicated examined using the Transwell migration assay. Migrated cells on the bottom surfaces of the Transwell inserts were stained with crystal violet and observed under a microscope (200×). d Quantitative results of cell migration. n = 3; * p < 0.05 vs normal. Ang II angiotensin II

Article Snippet: Modified Boyden chamber assays were conducted using Transwell polyester membrane filter inserts with 8-μm pores (Corning Inc., Corning, NY, USA) at a density of 500,000 cells/ml per Transwell (upper chamber) as described previously [ ].

Techniques: Migration, Wound Healing Assay, Transwell Migration Assay, Staining, Microscopy

Journal: Stem Cell Research & Therapy

Article Title: Ang II-AT2R increases mesenchymal stem cell migration by signaling through the FAK and RhoA/Cdc42 pathways in vitro

doi: 10.1186/s13287-017-0617-z

Figure Lengend Snippet: Effect of AT1R and AT2R antagonists on Ang II-mediated migration of MSCs. a Nondirectional migration ability of MSCs after stimulation with 100 nM Ang II following pretreatment with Losartan (5 μM) and/or PD-123319 (5 μM) examined with the scratch assay. Wound sites (areas cleared of cells in the center of the scratched area) were observed and photographed at 0 and 24 h (200×). b Quantitative results of wound healing. c Directional migration ability of MSCs after stimulation with 100 nM Ang II following pretreatment with Losartan (5 μM) and/or PD-123319 (5 μM) examined using the Transwell migration assay. Migrated cells on the bottom surfaces of the Transwell inserts were stained with crystal violet and observed under a microscope (200×). d Quantitative results of cell migration. Significant differences were found compared with the normal group ( n = 3; * p < 0.05) and compared with the control group ( n = 3; # p < 0.05). Ang II angiotensin II

Article Snippet: Modified Boyden chamber assays were conducted using Transwell polyester membrane filter inserts with 8-μm pores (Corning Inc., Corning, NY, USA) at a density of 500,000 cells/ml per Transwell (upper chamber) as described previously [ ].

Techniques: Migration, Wound Healing Assay, Transwell Migration Assay, Staining, Microscopy, Control

Journal: Stem Cell Research & Therapy

Article Title: Ang II-AT2R increases mesenchymal stem cell migration by signaling through the FAK and RhoA/Cdc42 pathways in vitro

doi: 10.1186/s13287-017-0617-z

Figure Lengend Snippet: Roles of FAK and Rho GTPases in the migration of MSCs. a Nondirectional migration ability of MSCs after stimulation with Ang II and/or inhibitors of FAK and the Rho GTPases examined using the scratch assay. Wound sites (areas cleared of cells in the center of the scratched area) were observed and photographed at 0 and 24 h (200×). b Quantitative results of wound healing. c Directional migration ability of MSCs after stimulation with Ang II and/or inhibitors of FAK and the Rho GTPases examined using the Transwell migration assay. Migrated cells on the bottom surfaces of the Transwell inserts were stained with crystal violet and observed under a microscope (200×). d Quantitative results of cell migration. n = 3; * p < 0.05 vs normal, # p < 0.05 vs the control group. Ang II angiotensin II

Article Snippet: Modified Boyden chamber assays were conducted using Transwell polyester membrane filter inserts with 8-μm pores (Corning Inc., Corning, NY, USA) at a density of 500,000 cells/ml per Transwell (upper chamber) as described previously [ ].

Techniques: Migration, Wound Healing Assay, Transwell Migration Assay, Staining, Microscopy, Control

Journal: Viruses

Article Title: Host–Pathogen Responses to Pandemic Influenza H1N1pdm09 in a Human Respiratory Airway Model

doi: 10.3390/v12060679

Figure Lengend Snippet: Characterization of wdNHBE cells by histology and immunocytochemistry. ( a ) Hematoxylin and eosin staining of a transverse section of NHBE cells differentiated at the air liquid interface on the apical surface of a transwell membrane. Nuclei ( n ) are stained purple and membranes, cytoplasm and cilia are stained pink. Basal cells ( b ), ciliated cells ( c ) with surface cilia, goblet cells ( g ) and the porous polyester transwell membrane ( m ). ( b ) En face images of wdNHBE cells stained with MUC5AC (green, goblet cells) and MUC5B (red, secretory cells). The Merge of MUC5AC, MUC5B and phalloidin (F-actin)-stained cytoskeleton (pink) depicts the partial colocalization of MUC5AC and MUC5B secretion (yellow, indicated by arrows). En face Z-stacks of 19 slices (9 µm) were captured using a 40x oil immersion objective and maximum intensity projections generated, 20 µm scale bars indicate cell size. ( c ) Confocal microscopy of wdNHBE cells stained with acetylated tubulin (TubAc, green, cilia on the surface of ciliated cells) and CLDN4 (red, tight junctions). Merge of TubAc, CLDN4 and F-actin. Maximum intensity projections of en face Z-stacks, 19 slices (9 µm) imaged using a 40x oil immersion objective with 20 µm scale bars are shown.

Article Snippet: Cells were passaged at 65–70% confluence as previously described and undifferentiated NHBE cells seeded at 2.1 × 10 5 cells/cm 2 on the apical surface of a 6.5 mm diameter transwell polyester membrane (0.33 cm 2 ; 0.4 μm pores; Corning, Corning, NY, USA) that had been precoated with Gibco Collagen I Rat Protein, Tail (5 μg/cm 2 ; (ThermoFisher Scientific, Waltham, MA, USA).

Techniques: Immunocytochemistry, Staining, Membrane, Generated, Confocal Microscopy