![Figure 6. CDS2 regulates angiogenesis in vitro. (A) Schematic diagram that shows the HUVEC invasion assay used to model angiogenesis in vitro. HUVECs are plated on the surface of a collagen gel that contains VEGFA and other factors (see “PIP2 quantitation and delivery into cultured HUVECs or zebrafish embryos”), which they invade and grow into. Invasion is imaged and quantified at specific depths. (B-D) CDS2 or CDS1 knockdown inhibits ERK activation and in vitro angiogenesis of HUVECs. (B) Sample DIC microscopy images of HUVECs transfected with control (ctrl), CDS1, or CDS2 small interfering RNA (siRNA) for 48 hours and then transferred into a 3-dimensional (3D) invasion assay for an additional 24 hours. Images show HUVECs in the monolayer at the surface of the collagen gel (monolayer) and at 2 separate depths (depth 1, depth 2) to which some of the cells have migrated within the collagen gel. Images such as these are collected and used for quantitation of HUVEC angiogenic invasion. (C) Quantitation of the percentage of invading control, CDS1, or CDS2 siRNA-treated HUVECs with the use of the assay shown in panel A. The percentage of invading cells was normalized to control siRNA-treated HUVECs. (D) Protein extracts from panels B and C were assayed by immunoblotting with the use of anti-ERK1/2 and anti–phospho-ERK1/2 antibodies. (E) CDS overexpression promotes increased HUVEC invasion of 3D collagen matrices in vitro. Quantitation of the percentage of invading HUVECs transduced with either green fluorescent protein (GFP; control), zCDS1, or zCDS2 producing lentiviral vectors, using the assay shown in panel A. The percentage of invading cells was normalized to control HUVECs. (F) Exogenous phosphatidylinositol-4,5-bisphosphate (PIP2) promotes increased HUVEC invasion of 3D collagen matrices in vitro. Quantitation of the percentage of invading HUVECs that were either untreated (control) or treated with carrier alone (carrier only) or carrier-loaded phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2; carrier + PIP2] before plating on the collagen gel with the use of the assay shown in panel A. Scale bars = 25 μm (B). CDS indicates CDP-diacylglycerol synthetase.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/120/2/10.1182_blood-2012-02-408328/4/zh89991293850006.jpeg?Expires=1724148693&Signature=cMESxh~xNJ8KWyHDzQhY1h2FDeotXMSaJ~2z-vsJaJy~x2gjQBwZpSQKc9osAfSLcNYgXRT3ne4rtDbrysvPlkdb9X2V7QLZP2uqQ99M7Lv3QaPJdD0CpOfAJCbmUqLzISJKFCui-fyVkrA1QRzKw2LPOKqC1Ho5EptszHx1XW5iMr2NQaTvDehmmRgqOqJXxc0Jj0cb~7bF2I~3tDv0tWPsRLxzYjoJlKQ6o5B2OC9Q3xnVLS5phhMKnD9XXR4Mi~uUYmTvsEfGN9fOKL7w6D8Rvyd4pm6cxTneykDraPBYET5RS92X1WqdrjY4Cq1M4Cm3agYMzMgyWjY5Li-HVg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
CDS2 regulates angiogenesis in vitro. (A) Schematic diagram that shows the HUVEC invasion assay used to model angiogenesis in vitro. HUVECs are plated on the surface of a collagen gel that contains VEGFA and other factors (see “PIP2 quantitation and delivery into cultured HUVECs or zebrafish embryos”), which they invade and grow into. Invasion is imaged and quantified at specific depths. (B-D) CDS2 or CDS1 knockdown inhibits ERK activation and in vitro angiogenesis of HUVECs. (B) Sample DIC microscopy images of HUVECs transfected with control (ctrl), CDS1, or CDS2 small interfering RNA (siRNA) for 48 hours and then transferred into a 3-dimensional (3D) invasion assay for an additional 24 hours. Images show HUVECs in the monolayer at the surface of the collagen gel (monolayer) and at 2 separate depths (depth 1, depth 2) to which some of the cells have migrated within the collagen gel. Images such as these are collected and used for quantitation of HUVEC angiogenic invasion. (C) Quantitation of the percentage of invading control, CDS1, or CDS2 siRNA-treated HUVECs with the use of the assay shown in panel A. The percentage of invading cells was normalized to control siRNA-treated HUVECs. (D) Protein extracts from panels B and C were assayed by immunoblotting with the use of anti-ERK1/2 and anti–phospho-ERK1/2 antibodies. (E) CDS overexpression promotes increased HUVEC invasion of 3D collagen matrices in vitro. Quantitation of the percentage of invading HUVECs transduced with either green fluorescent protein (GFP; control), zCDS1, or zCDS2 producing lentiviral vectors, using the assay shown in panel A. The percentage of invading cells was normalized to control HUVECs. (F) Exogenous phosphatidylinositol-4,5-bisphosphate (PIP2) promotes increased HUVEC invasion of 3D collagen matrices in vitro. Quantitation of the percentage of invading HUVECs that were either untreated (control) or treated with carrier alone (carrier only) or carrier-loaded phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2; carrier + PIP2] before plating on the collagen gel with the use of the assay shown in panel A. Scale bars = 25 μm (B). CDS indicates CDP-diacylglycerol synthetase.
