Figure 1

SHIP1 inhibition reduces viable cell numbers and either G2/M or G0/G1 cell cycle arrest. (A) U266 MM cells were treated for 10 min with increasing concentrations of PtdInd(3,4)P₂ or PtdIns(3,4,5)P₃, using shuttle PIP kit as described. Cells were subsequently lysed and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blots were probed with antibodies against phosphorylated Akt. Equal loading was confirmed by reprobing the blots with antibodies against actin. (B) OPM2, U266 and RPMI8226 cells were treated for 48 h with increasing concentrations of 3AC, and viable cell numbers were determined by a cell-counting kit. The means of at least three independent experiments are shown. Asterisks indicate significant differences between OPM2 cells and U266 and RPMI8226 cells. —●—, OPM2; —■—, RPMI8226; —▲—, U266. (C) MM cell lines were cultured in the presence or absence of 12.5 µmol/L 3AC of 48 h and 10 µmol/L BrdU for 14 h. Cells were subsequently labeled with APC-anti-BrdU and 7AAD, and cell cycle status was analyzed by flow cytometry. A representative example of two independent experiments is shown. conc, concentration. ▦, sub G0/G1; ▩, G0/G1; ▤, G2/M; ▥, S.