STAT1 plays a critical role in VEGF-induced down-regulation of MMP-9. (A) A total of 10⁷ B-CLL cells were untreated or treated with 200 ng/mL VEGF for the indicated times and mRNA expression analyzed by reverse-transcribed polymerase chain reaction. Quantitative values represent the average of 3 samples after normalizing untreated cells values to 1. (B) A total of 3 × 10⁶ B-CLL cells were lysed before (constitutive [Const.]) or after 30 minutes of treatment with medium (untreated [Unt.]) or the indicated stimuli. STAT1 tyrosine 701 phosphorylation (p-Y-STAT1) and total STAT1 (t-STAT1) were analyzed by Western blotting, and normalized average values (n = 3) are shown. V+R2 inhib. indicates VEGF + VEGFR2 inhibitor I. (C) A total of 20 × 10⁶ B-CLL cells treated with or without VEGF for 30 minutes were lysed, and the nuclear and cytosolic fractions separated and analyzed by Western blotting. The H4 histone was used as an internal nuclear marker. p-Y-STAT1 indicates tyrosine 701 phosphorylated STAT1. (D) B-CLL cells were untransfected (None) or transfected with STAT1 siRNA or control siRNA, lysed, and analyzed by Western blotting to determine STAT1 gene silencing efficiency. Actin was used as an internal loading control. Numbers represent the average t-STAT1/actin ratio after normalizing untransfected values to 1. *P ≤ .05 compared with untransfected, untreated control. (E) B-CLL cells untransfected or transfected with STAT1 or control siRNA were treated with medium or 200 ng/mL VEGF for 24 hours. The conditioned media was concentrated and MMP-9 levels analyzed by gelatin zymography. Normalized average values (n = 3) are shown. (F) B-CLL cells untransfected or transfected with STAT1 or control siRNA were treated with medium or VEGF and added to Transwell filters (5 μm pore size) coated with Matrigel. Cell migration was determined after 24 hours by flow cytometry. Values are the percentage of total cells added. *P ≤ .05. **P ≤ .01.