Figure 2

DUSP3 downregulation affects HUVEC cell angiogenic sprouting but does not affect proliferation. (A) 72 hours after transfection with siCTL, siDUSP3-1 or siDUSP3-2, HUVECs were trypsinized and 7.5 × 10³ from each transfection condition were plated (in triplicate) and cultured for an additional 24 hours. ³H-Thymidine was added for the last 4 h before cell harvesting. Radioactivity was counted using a scintillation analyzer. (Ai) Statistical analysis from three independent experiments. Data are reported as mean ± SEM. (Aii) Western blot analysis using anti-DUSP3 antibody and anti-GAPDH of one representative experiment showing DUSP3 depletion after siDUSP3-1 and siDUSP3-2 transfection. Quantification of DUSP3 protein expression in siCTL, siDUSP3-1 and siDUSP3-2 transfection conditions are shown as a ratio of densitometry values of DUSP3 on GAPDH bands. (Bi) Snapshots from phase contrast time-lapse movies of tube formation assay of HUVECs transfected with siCTL or with siDUSP3 and seeded on Matrigel solidified matrix (Additional files 1 and 2). (Bii) Quantitative analysis of the snapshots shown in (Bi) obtained by measuring the tube lengths (left panel) and number of intersections (right panel) from 10 fields. (Biii) Western blot and quantification of DUSP3 protein expression in siCTL and siDUSP3-1 transfection conditions represented as a ratio of DUSP3 on GAPDH.