SK1-I alters survival signaling. (A-C) U937 cells were cultured in medium containing 2% serum in the presence of 10 μM SK1-I for the indicated times (A); in medium containing 10% serum in the presence of 20 μM SK1-I for the indicated times (B); or with the indicated concentrations of SK1-I for 5 minutes (C). Equal amounts of lysate proteins were resolved by SDS-PAGE and analyzed by Western blotting with antibodies against pAkt, Akt, p-ERK1/2, ERK1/2, p-p38, p-ATF-2, p-JNK, and p-cJun. Blots were stripped and reprobed with antitubulin (A), anti-Akt (B), or anti-ERK1/2 (C) to demonstrate equal loading. (D) Enforced expression of myristoylated Akt protects cells from SK1-I–mediated lethality. U937 cells stably expressing empty vector or constitutively active Akt (myristoylated Akt) were treated with the indicated concentrations of SK1-I for 24 hours. Cells were stained with annexin V/PI and apoptosis was determined by flow cytometry. (Inset) Immunoblot of lysate proteins from duplicate cultures with anti-Akt and antitubulin antibodies.