American Society of Hematology

Figure 5.

$Figure 5. Ephrin B2 knockdown with specific siRNA inhibits viability in KS cells and HUVECs grown in the presence of VEGF but not IGF, EGF, or bFGF. (A) Ephrin B2 siRNAs block ephrin B2 expression in KS-SLK cells. Cells were transfected with various siRNA to ephrin (EFN) B2 and controls. After 48 hours the cells were harvested and crude cell lysates fractionated on 4% to 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot was performed with monoclonal antibody to ephrin B2 generated in-house. The membrane was stripped and reprobed with β-actin monoclonal antibody (Sigma) to illustrate equivalent loading and transfer. (B) Three-day cell viability assay of KS-SLK cultures in the presence of ephrin B2 and EphB4 siRNAs. Cells (1 × 105/well) in 24-well plates were treated with 0, 10, and 100 ng/mL siRNAs as indicated on the graph. Viability of cultures was determined by MTT assay as described in “Patients, materials, and methods.” Shown are the mean ± standard deviation of duplicate samples. (C) ELISA of cytosolic nucleosomes 48 hours after transfection of cells with siRNA as indicated. KS 6-3 and KS 38 are early passage isolates generated in our laboratory from AIDS-KS patient biopsies. (D) Colorimetric assay of caspase 8 (extrinsic cell death) and caspase 9 (intrinsic cell death) activity in KS-SLK cells treated as in panel C. Means ± standard deviation of triplicate determinations are shown for both panels C and D. (E) Ephrin B2 siRNA blocks VEGF-mediated induction of ephrin B2 in HUVECs. HUVECs cultured in endothelial basal medium (EBM-2) supplemented with 5% FCS and 10 ng/mL VEGF were transfected with the indicated siRNAs. Cell lysates were prepared as in panel A and subjected to sequential Western blot with ephrin B2 and β-actin. (F) HUVECs were seeded on 8-well chamber slides coated with fibronectin. The HUVECs were grown overnight in endothelial growth medium (EGM-2), which contains all growth supplements (see “Patients, materials, and methods”). On the following day, the medium was replaced with EBM-2 supplemented with 5% FCS and either VEGF (10 ng/mL) alone or EGF, FGF, and IGF in combination as indicated. After 2 hours of incubation at 37°C, the cells were transfected using Lipofectamine 2000 (Invitrogen) in Opti-MEM medium containing 10 nM of siRNA to ephrin B2, EphB4, or green fluorescence protein (GFP) as control. The cells were returned to EBM-2 supplemented as described in “Patients, materials, and methods” with VEGF alone or EGF, FGF, and IGF in combination, following the 2-hour transfection in Opti-MEM-1. After a further 48 hours, the cells were stained with crystal violet. Digital images were captured immediately after staining using a Nikon Coolpix 5000 with microscope adaptor. Original magnification × 10. TR indicates transfection reagent.$

Ephrin B2 knockdown with specific siRNA inhibits viability in KS cells and HUVECs grown in the presence of VEGF but not IGF, EGF, or bFGF. (A) Ephrin B2 siRNAs block ephrin B2 expression in KS-SLK cells. Cells were transfected with various siRNA to ephrin (EFN) B2 and controls. After 48 hours the cells were harvested and crude cell lysates fractionated on 4% to 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot was performed with monoclonal antibody to ephrin B2 generated in-house. The membrane was stripped and reprobed with β-actin monoclonal antibody (Sigma) to illustrate equivalent loading and transfer. (B) Three-day cell viability assay of KS-SLK cultures in the presence of ephrin B2 and EphB4 siRNAs. Cells (1 × 10⁵/well) in 24-well plates were treated with 0, 10, and 100 ng/mL siRNAs as indicated on the graph. Viability of cultures was determined by MTT assay as described in “Patients, materials, and methods.” Shown are the mean ± standard deviation of duplicate samples. (C) ELISA of cytosolic nucleosomes 48 hours after transfection of cells with siRNA as indicated. KS 6-3 and KS 38 are early passage isolates generated in our laboratory from AIDS-KS patient biopsies. (D) Colorimetric assay of caspase 8 (extrinsic cell death) and caspase 9 (intrinsic cell death) activity in KS-SLK cells treated as in panel C. Means ± standard deviation of triplicate determinations are shown for both panels C and D. (E) Ephrin B2 siRNA blocks VEGF-mediated induction of ephrin B2 in HUVECs. HUVECs cultured in endothelial basal medium (EBM-2) supplemented with 5% FCS and 10 ng/mL VEGF were transfected with the indicated siRNAs. Cell lysates were prepared as in panel A and subjected to sequential Western blot with ephrin B2 and β-actin. (F) HUVECs were seeded on 8-well chamber slides coated with fibronectin. The HUVECs were grown overnight in endothelial growth medium (EGM-2), which contains all growth supplements (see “Patients, materials, and methods”). On the following day, the medium was replaced with EBM-2 supplemented with 5% FCS and either VEGF (10 ng/mL) alone or EGF, FGF, and IGF in combination as indicated. After 2 hours of incubation at 37°C, the cells were transfected using Lipofectamine 2000 (Invitrogen) in Opti-MEM medium containing 10 nM of siRNA to ephrin B2, EphB4, or green fluorescence protein (GFP) as control. The cells were returned to EBM-2 supplemented as described in “Patients, materials, and methods” with VEGF alone or EGF, FGF, and IGF in combination, following the 2-hour transfection in Opti-MEM-1. After a further 48 hours, the cells were stained with crystal violet. Digital images were captured immediately after staining using a Nikon Coolpix 5000 with microscope adaptor. Original magnification × 10. TR indicates transfection reagent.

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