Figure 2
Figure 2. VEGF up-regulates HLX and UNC5B in endothelial cells. (A) Real-time RT-PCR analysis of UNC5B, PLXNA1, SEMA3G, HES1, and HLX mRNA: HUVECs were cultured to density, starved overnight in EBM-2 medium without supplements and then induced with 100 ng of VEGF/mL for 1, 2, 4, 8, 16, 32, and 56 hours. Total RNA was isolated and mRNA levels were determined by real-time RT-PCR. All values were normalized to β2-microglobulin mRNA as internal standard. Results displayed represent the mean of fold induction ± SEM of the respective mRNA levels calculated from triplicate wells. One representative experiment of 3 performed is shown. (B) Western blot analysis of HLX: Total cell lysates were prepared from HUVECs induced with 100 ng/mL VEGF for 4, 6, and 24 hours and HUVECs transduced with adenoviruses encoding HLX (Ad.HLX) for 72 hours. Cells were harvested and proteins were separated by SDS-PAGE. Membranes were probed with anti-HLX and anti-GAPDH antibodies. The left panel displays one exemplary blot, the right panel the quantification obtained from 3 independent experiments. (C) Flow cytometric analysis of UNC5B surface expression after VEGF induction: HUVECs starved as described in panel A were induced with VEGF-A for 56 hours or kept without stimulation. Cells were detached from the plates, stained with UNC5B and Alexa Fluor 647–labeled goat–anti-human IgG antibodies, and subjected to flow cytometry. The left panel displays representative dot blots of untreated, 56-hour VEGF-induced and control samples transduced for 56 hours with an UNC5B adenovirus. The right panel shows the quantification of 3 independent experiments performed in triplicates. Mean percentage of positive cells ± SEM calculated from at least 3 independent experiments is displayed. (D) Immunocytochemistry analysis of HLX and UNC5B after VEGF induction: HUVECs were induced for 24 and 48 hours; cells were fixed and stained with anti-HLX and anti-UNC5B antibodies. The top panels display the VEGF induction, the lower panels a control experiment using transduction with HLX and UNC5B-expressing adenoviruses. (E) Up-regulation of UNC5B by VEGF-A is HLX-dependent: HUVECs transduced with lentiviruses containing shRNA.con and shRNA.HLX for 48 hours were starved overnight and then induced with VEGF for 24, 48, or 56 hours as indicated. Cells were harvested and UNC5B mRNA and protein were analyzed by real-time RT-PCR and flow cytometry, respectively. The left and middle panels display fold induction of UNC5B mRNA ± SD or HLX mRNA ± SD (as a control) in comparison to noninduced control shRNA cells as calculated from 1 experiment representative of 4 performed in triplicates. The right panel displays the percentage of UNC5B-positive cells ± SD as measured by flow cytometry in a representative experiment performed in triplicates.

VEGF up-regulates HLX and UNC5B in endothelial cells. (A) Real-time RT-PCR analysis of UNC5B, PLXNA1, SEMA3G, HES1, and HLX mRNA: HUVECs were cultured to density, starved overnight in EBM-2 medium without supplements and then induced with 100 ng of VEGF/mL for 1, 2, 4, 8, 16, 32, and 56 hours. Total RNA was isolated and mRNA levels were determined by real-time RT-PCR. All values were normalized to β2-microglobulin mRNA as internal standard. Results displayed represent the mean of fold induction ± SEM of the respective mRNA levels calculated from triplicate wells. One representative experiment of 3 performed is shown. (B) Western blot analysis of HLX: Total cell lysates were prepared from HUVECs induced with 100 ng/mL VEGF for 4, 6, and 24 hours and HUVECs transduced with adenoviruses encoding HLX (Ad.HLX) for 72 hours. Cells were harvested and proteins were separated by SDS-PAGE. Membranes were probed with anti-HLX and anti-GAPDH antibodies. The left panel displays one exemplary blot, the right panel the quantification obtained from 3 independent experiments. (C) Flow cytometric analysis of UNC5B surface expression after VEGF induction: HUVECs starved as described in panel A were induced with VEGF-A for 56 hours or kept without stimulation. Cells were detached from the plates, stained with UNC5B and Alexa Fluor 647–labeled goat–anti-human IgG antibodies, and subjected to flow cytometry. The left panel displays representative dot blots of untreated, 56-hour VEGF-induced and control samples transduced for 56 hours with an UNC5B adenovirus. The right panel shows the quantification of 3 independent experiments performed in triplicates. Mean percentage of positive cells ± SEM calculated from at least 3 independent experiments is displayed. (D) Immunocytochemistry analysis of HLX and UNC5B after VEGF induction: HUVECs were induced for 24 and 48 hours; cells were fixed and stained with anti-HLX and anti-UNC5B antibodies. The top panels display the VEGF induction, the lower panels a control experiment using transduction with HLX and UNC5B-expressing adenoviruses. (E) Up-regulation of UNC5B by VEGF-A is HLX-dependent: HUVECs transduced with lentiviruses containing shRNA.con and shRNA.HLX for 48 hours were starved overnight and then induced with VEGF for 24, 48, or 56 hours as indicated. Cells were harvested and UNC5B mRNA and protein were analyzed by real-time RT-PCR and flow cytometry, respectively. The left and middle panels display fold induction of UNC5B mRNA ± SD or HLX mRNA ± SD (as a control) in comparison to noninduced control shRNA cells as calculated from 1 experiment representative of 4 performed in triplicates. The right panel displays the percentage of UNC5B-positive cells ± SD as measured by flow cytometry in a representative experiment performed in triplicates.

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