Fig, 4.

Roles HIF-1α in the regulation of AS expression. A, Arginine deprivation suppresses HIF-1α expression. A2058 cells were maintained in normal medium, medium containing 0.05 µg/ml ADI, or arginine-free medium with or without 100 µM DFX or 100 µM CoCl₂. Seventy-two hours after treatment, cells were harvested and subjected for Western blotting. B, A2058 cells were transfected with 100 nM scramble (control) or HIF-1α siRNA and maintained in normal medium, medium containing 0.05 µg/ml ADI or arginine-free medium with 100 µM DFX. Untransfected A2058 cells maintained in normal medium, medium containing 0.05µg/ml ADI or arginine-free medium were used for control. Levels of AS, HIF-1α, c-Myc, Sp4 and β-actin were measured by Western blottings. C, A2058, SK-MEL-2 and A375 cells were transfected with c-Myc or co-transfected with HIF-1α expression vector and pCGN (control) vector, and cell lystates were analyzed by Western blot. As an expression control, the level of total expression of each protein was examined by immunoblotting with an antibodies against whole protein as indicated or β-actin (loading control). D and E, Kinetics of HIF-1α, AS, c-Myc and Sp4 expression in A2058 cells grown in arginine-free medium (D) or 0.05 µg/ml ADI-containing medium (E) for the time courses as indicated.