Orthovanadate activates the PI-3 kinase/PKB/Akt cascade and PI-3 kinase activity is required for survival in orthovanadate-treated HCD57 cells.

(A) HCD57 cells were deprived of EPO and cultured 24 hours in 0 (lanes i and ii), 10 (lanes iii and iv) or 70 μmol/L orthovanadate (lanes v and vi). The cells were either stimulated with 10 units EPO per milliliter (lanes ii, iv, and vi) or mock treated for 5 minutes (lanes i, iii, and v). Whole cell extracts were analyzed by Western blot with anti–phospho-PKB/Akt antibody. The blot was stripped and reprobed with anti-PKB/Akt antibody (lower part of panel) as a loading control. (B) HCD57 cells were deprived of EPO and treated with 50 μmol/L orthovanadate for 24 hours. The 50 μmol/L LY294002 was added to one aliquot of these cells for the 24-hour interval. For each aliquot, 10 μg of genomic DNA was analyzed by agarose gel for fragments of DNA characteristic of apoptosis (upper panel). Whole cell extracts were analyzed by Western blot with anti–phospho-PKB/Akt antibody (lower panel). The blot was stripped and reprobed with anti-PKB/Akt antibody as a loading control (bottom of lower panel).