American Society of Hematology

Figure 6.

$Figure 6. PSGL-1–mediated rolling on P-selectin is dependent on Syk. (A) Rolling of KG1 cells on P-selectin: effect of treatment with genistein. Results are expressed as mean percent (± SEM; n = 3) of control cells treated with vehicle alone. (B) Rolling of KG1 and 300.19-L-selectin cells on P-selectin, E-selectin, or PSGL-1. Cells were treated with vehicle (▪) or with piceatannol (▦ and □). Results are expressed as mean (%) plus or minus SEM (n = 3). (C) Inhibition of Syk expression by Syk-specific siRNA. KG1 cells were transfected with 3 μg Syk siRNA or of nontargeting siRNA. Syk/GAPDH mRNA ratio was assessed by RQ-PCR at 48 hours. Results are expressed as percent of mock-transfected cells, mean plus or minus SEM (n = 2). (D) Flow adhesion of KG1 cells on P-selectin. KG1 cells were transfected with 3 μg Syk siRNA or with nontargeting siRNA and compared with mock-transfected cells. Results are expressed as mean percent mock-treated cells plus or minus SEM (n = 3). ***P < .001. (E) Adhesion of U937 cells or neutrophils to confluent CHO–P-selectin monolayers or mock-transfected CHO monolayers. Lipid rafts were isolated by fractionation of cell lysates. Expression of Syk and PSGL-1 in lipid raft fractions (2 to 4) was detected by Western blot analysis using mAb 4D10.1 (0.2 μg/mL) or mAb PS5 (3 μg/mL). (F) Densitometric analysis of Syk in sucrose density fractions 1 to 10 prepared from U937 cells after rolling on CHO–P-selectin cell monolayers. Syk levels were normalized using PSGL-1 as a standard. Control values were determined by assessing Syk levels in fractions from U937 cells after rolling on mock-CHO cell monolayers. Results are representative of 3 experiments.$

PSGL-1–mediated rolling on P-selectin is dependent on Syk. (A) Rolling of KG1 cells on P-selectin: effect of treatment with genistein. Results are expressed as mean percent (± SEM; n = 3) of control cells treated with vehicle alone. (B) Rolling of KG1 and 300.19-L-selectin cells on P-selectin, E-selectin, or PSGL-1. Cells were treated with vehicle (▪) or with piceatannol (▦ and □). Results are expressed as mean (%) plus or minus SEM (n = 3). (C) Inhibition of Syk expression by Syk-specific siRNA. KG1 cells were transfected with 3 μg Syk siRNA or of nontargeting siRNA. Syk/GAPDH mRNA ratio was assessed by RQ-PCR at 48 hours. Results are expressed as percent of mock-transfected cells, mean plus or minus SEM (n = 2). (D) Flow adhesion of KG1 cells on P-selectin. KG1 cells were transfected with 3 μg Syk siRNA or with nontargeting siRNA and compared with mock-transfected cells. Results are expressed as mean percent mock-treated cells plus or minus SEM (n = 3). ***P < .001. (E) Adhesion of U937 cells or neutrophils to confluent CHO–P-selectin monolayers or mock-transfected CHO monolayers. Lipid rafts were isolated by fractionation of cell lysates. Expression of Syk and PSGL-1 in lipid raft fractions (2 to 4) was detected by Western blot analysis using mAb 4D10.1 (0.2 μg/mL) or mAb PS5 (3 μg/mL). (F) Densitometric analysis of Syk in sucrose density fractions 1 to 10 prepared from U937 cells after rolling on CHO–P-selectin cell monolayers. Syk levels were normalized using PSGL-1 as a standard. Control values were determined by assessing Syk levels in fractions from U937 cells after rolling on mock-CHO cell monolayers. Results are representative of 3 experiments.

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