Figure 2
Dasatinib inhibits other adhesion-dependent responses but does not block TNF signaling. (A-G) Human neutrophils pretreated with the indicated concentrations of dasatinib were stimulated with 20 ng/mL of human TNF while adherent to a fibrinogen (Fbg)–coated surface (A-E and top part of G) or plated on a poly-RGD–coated surface (F and bottom part of G), followed by microscopic observation (A) and quantification (B) of cell spreading, measurement of cell adhesion (C), assessment of lactoferrin release (D), direct analysis of total cellular tyrosine phosphorylation and phosphorylation of Syk Tyr352 by immunoblotting (E-F), or analysis of Syk phosphorylation by immunoprecipitation (IP), followed by immunoblotting for phosphotyrosine (PY) residues (G). (H-K) Human neutrophils pretreated with the indicated concentrations of dasatinib were stimulated with TNF in suspension, followed by analysis of gelatinase release by in-gel zymography (H), flow cytometric analysis of expression (I) and activation (J) of CD11b, or phosphorylation of ERK and the p38 MAPK by immunoblotting (K). Bar graphs in panels B through D show mean and SD of representative experiments, and dose-response curves in panels B through D and bar graphs in panels I and J show mean and SEM of percent response. Mean fluorescence intensity values of isotype control–stained samples were subtracted in panels I and J, and the resulting fluorescence was expressed as a percentage of that in the indicated samples. Each set of data was obtained from 3-9 independent experiments. The mean and SEM of phosphorylation of the various MAPKs at 10nM, 100nM, and 1μM dasatinib after subtraction of unstimulated control values corresponded to 101% ± 18%, 94% ± 17%, and 56% ± 8% (p38 MAPK) and 89% ± 31%, 85% ± 26%, and 89% ± 20% (ERK) of that in the absence of dasatinib, respectively.

Dasatinib inhibits other adhesion-dependent responses but does not block TNF signaling. (A-G) Human neutrophils pretreated with the indicated concentrations of dasatinib were stimulated with 20 ng/mL of human TNF while adherent to a fibrinogen (Fbg)–coated surface (A-E and top part of G) or plated on a poly-RGD–coated surface (F and bottom part of G), followed by microscopic observation (A) and quantification (B) of cell spreading, measurement of cell adhesion (C), assessment of lactoferrin release (D), direct analysis of total cellular tyrosine phosphorylation and phosphorylation of Syk Tyr352 by immunoblotting (E-F), or analysis of Syk phosphorylation by immunoprecipitation (IP), followed by immunoblotting for phosphotyrosine (PY) residues (G). (H-K) Human neutrophils pretreated with the indicated concentrations of dasatinib were stimulated with TNF in suspension, followed by analysis of gelatinase release by in-gel zymography (H), flow cytometric analysis of expression (I) and activation (J) of CD11b, or phosphorylation of ERK and the p38 MAPK by immunoblotting (K). Bar graphs in panels B through D show mean and SD of representative experiments, and dose-response curves in panels B through D and bar graphs in panels I and J show mean and SEM of percent response. Mean fluorescence intensity values of isotype control–stained samples were subtracted in panels I and J, and the resulting fluorescence was expressed as a percentage of that in the indicated samples. Each set of data was obtained from 3-9 independent experiments. The mean and SEM of phosphorylation of the various MAPKs at 10nM, 100nM, and 1μM dasatinib after subtraction of unstimulated control values corresponded to 101% ± 18%, 94% ± 17%, and 56% ± 8% (p38 MAPK) and 89% ± 31%, 85% ± 26%, and 89% ± 20% (ERK) of that in the absence of dasatinib, respectively.

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