ING4 involvement in MM-induced in vitro angiogenesis. Using an angiogenic in vitro assay (ANGIOkit TCS Biologicals, Buckingham, United Kingdom), endothelial-like cells were stimulated with the conditioned media (dilution 1:2) of RPMI-8226 previously transfected with a nonspecific control siRNA (siRNA Cy) or anti-ING4 siRNA and incubated in normoxic (A) or hypoxic (B) conditions. Blocking anti–IL-8 or anti-OPN or nonspecific anti-IgG Abs were added in the conditioned media of RPMI-8226 transfected with siRNA anti-ING4 incubated in normoxic condition. VEGF and suramin treatments were used as positive and negative controls, respectively. Every 3 days the medium was replaced, and after 13 days cells were fixed and stained with anti-CD31 Ab according to the manufacturer's protocol. (Images were obtained on a Nikon Eclipse TE300 microscope at 10×/0.13 NA using a DS-US digital slight at 4×/0.12 NA objective lens. Original magnification, 10×). Vessel formation was quantified as described in “Cells and cell culture conditions; In vitro angiogenesis assay.” Graphs on the left, middle, and right represent the mean plus or minus SD of the number of capillary junctions, tubules, and the tubule length, respectively, of 2 independent experiments tested twice (control represents D-MEM medium nonconditioned by HMCLs; *P < .05).