ROCK1/PTEN/PI3K signaling pathway regulates AITC-mediated dephosphorylation and mitochondrial translocation of cofilin and apoptosis. (A) U937 cells were treated without or with various concentrations of AITC for 24 h, or treated with 20 μM AITC for different time intervals as indicated. Total cellular extracts were prepared and subjected to Western blot analysis using antibodies against ROCK1 and PTEN. CF represents cleavage fragment. Cells were pretreated with 20 μM Y27632, a ROCK1 inhibitor, for 2 h, followed by treating with 20 μM AITC for 24 h. (B) Total cellular extracts, cytosolic and mitochondrial fractions were prepared and subjected to Western blot analysis. (C) Cell lysates were prepared and subjected to immunoprecipitation using anti-ROCK1 antibody. The associated PTEN was determined using immunoblotting. (D) Apoptosis was determined by Annexin V/PI staining and flow cytometry. Error bars represent means ± SD (n=3). **P < 0.01. (E) Total cellular extracts and cytosolic fractions were prepared and subjected to Western blot analysis using antibodies as indicated.