Figure 4: Flunarizine does not require Tie2 or downstream signalling to reduce Angpt-2.

(A) Cropped immunoblot for Angiopoietin-2 (Angpt-2), tTie2 and GAPDH as a loading control from HUVECs transfected with control siRNA or Tie2 siRNA and stimulated with 10 μM Flunarizine (FLU) or vehicle for 24 hrs (n = 4). (B) HUVECs were treated with 10 μM FLU (+) or vehicle (−) for 24 hrs after transfection with Tie2 siRNA (+) or control siRNA (−) and the concentration of Angpt-2 in the supernatant was determined by ELISA (n = 5–6). Angpt-2 concentration in the supernatant of cells pretreated with (C) 1 μM Wortmannin or control for 1 h or with (D) 50 μM LY294002 for 1 h and stimulated with 10 μM FLU or vehicle for 12 hrs was measured by enzyme-linked immunosorbent assay (ELISA) (n = 6). (E) Real-time transendothelial electrical impedance from HUVECs 24 hrs after transfection with Tie2 siRNA or control siRNA, who were pretreated with 10 μM Flunarizine (FLU) or vehicle for 1 h and stimulated with 10 ng/mL TNFα, was recorded with an electric cell-substrate impedance sensing (ECIS) device (ibidi). Bar graphs represent n = 8 measurements per conditions after 5 hrs of TNF challenge. Columns are presented as mean ± SEM.