Conditioned medium obtained from culturing cells overexpressing B-Raf leads to the tubule formation on Matrigel. HMVEC-Ds cultured on Matrigel-coated wells were analyzed as described in “Materials and methods” for the ability to form tubules when grown for 16 hours in (A) conditioned medium obtained from BCBL-1 cells, (B) RPMI containing 2% FBS, and (C-D) conditioned medium from culturing BCBL-1 cells obtained 48 hours after transfection of siRNA specific for B-Raf and (NS)siRNA, respectively. Representative illustrations (A-D) are at an original magnification of × 40. (E) The tubular structures were scored by counting the number of tubules formed by HMVEC-Ds in each well when grown in conditioned medium from culturing BCBL-1 cells. (F) Target HFF cells were stimulated with 1 μM β-estradiol for 24 hours at 37°C. The stimulated cells were untransfected or transfected either with siRNA specific to VEGF or (NS)siRNA. At 48 hours after transfection, conditioned medium from the above cells were used to culture HMVEC-Ds on Matrigel at 37°C. After 16 hours incubation, the number of tubules/well was counted and is represented as described above. Each reaction was done in triplicate, and each point represents the average ± SD of 3 experiments. Average values on the columns with different superscripts are statistically significant (P < .05) by least significant difference (LSD).