Figure 3
Figure 3. Shed syndecan-1 in conditioned medium of HPSE-high myeloma cells facilitates endothelial invasion and ERK phosphorylation. (A) Endothelial invasion is enhanced by shed syndecan-1. An equal number of endothelial cells were seeded on invasion chambers coated with Matrigel and cells allowed to migrate in the presence of medium conditioned by HPSE-low or HPSE-high CAG cells, or medium from HPSE-high cells pretreated with Hep III or IP SDC1 or with medium from cells expressing high levels of an enzymatically inactive form of heparanase (M225). After overnight incubation, cells that invaded through the Matrigel were fixed, stained, and counted. Data are mean ± SD of 3 independent experiments. *P < .01 vs HPSE-low. **P < .05 vs HPSE-high. ***P < .01 vs HPSE-high. (B left panel) Pretreatment of medium with Hep III diminishes ERK activation. Serum-deprived endothelial cells were treated with conditioned medium from HPSE-low or HPSE-high myeloma cells with or without pretreatment with Hep III. At the designated time points, cells were washed and total cell lysates were subjected to immunoblotting with antibodies against p-ERK or t-ERK. (Right panel) ERK signaling is lost after depletion of syndecan-1 from conditioned medium. Medium conditioned by HPSE-high cells was subjected to immunodepletion with control antibody or antibody to syndecan-1. Serum-deprived endothelial cells were treated with this medium for 15 minutes, lysed, and subjected to immunoblotting with antibodies against p-ERK or t-ERK. (C) ERK activation stimulates invasion of endothelial cells. Equal number of endothelial cells along with dimethyl sulfoxide as control, ERK inhibitor PD98059 (50μM), or ERK inhibitor II negative control (50μM) were loaded into the upper compartment of the Matrigel invasion chamber and medium from HPSE-low or HPSE-high cells added to the lower compartment. After overnight incubation, cells that invaded through the Matrigel were fixed, stained, and counted. Data represent mean ± SD of 3 independent experiments. *P < .01 vs HPSE-low without addition of PD98059. **P < .01 vs HPSE-high without addition of PD98059.

Shed syndecan-1 in conditioned medium of HPSE-high myeloma cells facilitates endothelial invasion and ERK phosphorylation. (A) Endothelial invasion is enhanced by shed syndecan-1. An equal number of endothelial cells were seeded on invasion chambers coated with Matrigel and cells allowed to migrate in the presence of medium conditioned by HPSE-low or HPSE-high CAG cells, or medium from HPSE-high cells pretreated with Hep III or IP SDC1 or with medium from cells expressing high levels of an enzymatically inactive form of heparanase (M225). After overnight incubation, cells that invaded through the Matrigel were fixed, stained, and counted. Data are mean ± SD of 3 independent experiments. *P < .01 vs HPSE-low. **P < .05 vs HPSE-high. ***P < .01 vs HPSE-high. (B left panel) Pretreatment of medium with Hep III diminishes ERK activation. Serum-deprived endothelial cells were treated with conditioned medium from HPSE-low or HPSE-high myeloma cells with or without pretreatment with Hep III. At the designated time points, cells were washed and total cell lysates were subjected to immunoblotting with antibodies against p-ERK or t-ERK. (Right panel) ERK signaling is lost after depletion of syndecan-1 from conditioned medium. Medium conditioned by HPSE-high cells was subjected to immunodepletion with control antibody or antibody to syndecan-1. Serum-deprived endothelial cells were treated with this medium for 15 minutes, lysed, and subjected to immunoblotting with antibodies against p-ERK or t-ERK. (C) ERK activation stimulates invasion of endothelial cells. Equal number of endothelial cells along with dimethyl sulfoxide as control, ERK inhibitor PD98059 (50μM), or ERK inhibitor II negative control (50μM) were loaded into the upper compartment of the Matrigel invasion chamber and medium from HPSE-low or HPSE-high cells added to the lower compartment. After overnight incubation, cells that invaded through the Matrigel were fixed, stained, and counted. Data represent mean ± SD of 3 independent experiments. *P < .01 vs HPSE-low without addition of PD98059. **P < .01 vs HPSE-high without addition of PD98059.

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