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: Oncotarget

Article Title: Inhibition of SDF-1-induced migration of oncogene-driven myeloid leukemia by the L-RNA aptamer (Spiegelmer), NOX-A12, and potentiation of tyrosine kinase inhibition

doi: 10.18632/oncotarget.22409

Figure Lengend Snippet: ( A ) Transwell migration assay ( n = 1); error bars (standard deviation) represent intra-experimental variability, with multiple quantifications taken for one Transwell migration assay. Ba/F3.p210 cells were stimulated with mSDF-1 (100 ng/mL) in the absence or presence of NOX-A12 (100 nM). Data are presented as fold migration/control, where control is normalized to a value of 1. Transwell migration assay incubation time was overnight for Ba/F3.p210 cells. Data shown are mean +/− S.D. ( B ) Effect of mSDF-1 (100 ng/mL) on proliferation of Ba/F3.p210 cells following approximately 2 days of treatment. ( C ) Transwell migration assay: Ba/F3.p210 cells stimulated with mSDF-1 (100 ng/mL) in the presence of 25 nM NOX-A12, 1000 nM imatinib, or a combination of both. Transwell migration assay incubation time was overnight to 24 hours. Results shown are a composite of 3–4 independent experiments and error bars (standard deviation) represent inter-experimental variability. Data shown are mean +/− S.D. 2-sided t -test, p values: Control versus SDF-1 is statistically significant ( p = 0.00076). * SDF-1 versus SDF-1+imatinib+NoxA12 is statistically significant ( p = 0.00032). * SDF-1+imatinib versus SDF-1+imatinib+NOX-A12 is statistically significant ( p = 0.00003). * SDF-1+NOX-A12 versus SDF-1+imatinib+NOX-A12 is statistically significant ( p = 0.01758). Control versus NOX-A12 in the absence of SDF-1 is not statistically significant ( p = 0.79296). ( D ) Transwell migration assay ( n = 1); error bars (standard deviation) represent intra-experimental variability, with multiple quantifications taken for one Transwell migration assay. Ba/F3.p210 cells were stimulated with mSDF-1 (100 ng/mL) in the absence or presence of NOX-A12 (50 nM). Data are presented as number of migrated cells. Transwell migration assay incubation time was 24 hr. Data shown are mean +/− S.D. ( E ) Effect of NOX-A12 (25–50 nM) on proliferation of Ba/F3.p210 cells following approximately 24 hr of treatment. Data are presented as cell concentration (cell number/mL). Data shown are mean +/− S.D.

Article Snippet: For murine Ba/F3.p210 and 32D.p210 cells, the lower chamber of a Transwell plate (6.5mm Transwell® with 5.0μm Pore Polycarbonate Membrane Insert, Sterile (Product #3421), Corning Costar Corporation, Cambridge, MA) was filled with 600 μL of migration (starvation) media (RPMI 1640/PBS+0.01% bovine serum albumin (BSA)), followed by placement of inserts into the wells.

Techniques: Transwell Migration Assay, Standard Deviation, Migration, Control, Incubation, Concentration Assay

Journal: Oncotarget

Article Title: Inhibition of SDF-1-induced migration of oncogene-driven myeloid leukemia by the L-RNA aptamer (Spiegelmer), NOX-A12, and potentiation of tyrosine kinase inhibition

doi: 10.18632/oncotarget.22409

Figure Lengend Snippet: ( A ) Transwell migration assay ( n = 1); error bars (standard deviation) represent intra-experimental variability, with multiple quantifications taken for one Transwell migration assay. SUP-B15 cells were stimulated with hSDF-1 (100 ng/mL) in the presence of NOX-A12 (1–100 nM). Data are presented as number of migrated cells. Transwell migration assay incubation time was 24 hours for SUP-B15. Data shown are mean +/− S.D. ( B ) Transwell migration assay ( n = 1); error bars (standard deviation) represent intra-experimental variability, with multiple quantifications taken for one Transwell migration assay. MOLM14 cells were stimulated with hSDF-1 (50 ng/mL) in the presence of NOX-A12 (100 nM). Data are presented as fold migration/control, where control is normalized to a value of 1. Transwell migration assay incubation time was overnight. Data shown are mean +/− S.D.

Article Snippet: For murine Ba/F3.p210 and 32D.p210 cells, the lower chamber of a Transwell plate (6.5mm Transwell® with 5.0μm Pore Polycarbonate Membrane Insert, Sterile (Product #3421), Corning Costar Corporation, Cambridge, MA) was filled with 600 μL of migration (starvation) media (RPMI 1640/PBS+0.01% bovine serum albumin (BSA)), followed by placement of inserts into the wells.

Techniques: Transwell Migration Assay, Standard Deviation, Incubation, Migration, Control