Jaspamide induces increase in total cellular actin and de novo actin synthesis in HL-60 cells. (Aa) Coomassie-stained 12% SDS-PAGE gel showing total cellular protein of HL-60 cells exposed for 48 hours to DMSO 0.01% (lane 1) or jaspamide 10⁻⁷ mol/L (lane 2). (Ab) Dose-dependent actin accumulation: HL-60 cell extracts obtained from cells exposed for 24 hours to medium (lane 1), DMSO 0.01% (lane 2), jaspamide 10⁻⁷ mol/L (lane 3), jaspamide 10⁻⁸ mol/L (lane 4) were resolved on SDS-PAGE, and analyzed by Western blot using anti-actin MoAb. (B) Time course of actin accumulation. HL-60 cell extracts obtained from cells exposed to medium (lane 1), DMSO 0.01% (lanes 2, 4, and 6), or jaspamide 10⁻⁷ mol/L (lanes 3, 5, and 7) for 1 hour (lanes 2 and 3), 4 hours (lanes 4 and 5), or 24 hours (lanes 6 and 7) were resolved on SDS-PAGE, and analyzed by Western blot using anti-actin MoAb. (C) Effect of jaspamide on soluble and insoluble actin: Soluble, insoluble, and total actin fractions were prepared as described in Materials and Methods. (a) Coomassie blue and (b) immunoblot with anti-actin MoAb studies were performed (the lanes indicated are for both methods; ie, Fig 4Ca and Fig 4Cb). HL-60 cells treated with DMSO (lanes 1 through 3) or jaspamide 10⁻⁷mol/L for 24 hours (lanes 4 through 6) were examined for total (lanes 1 and 4) soluble (lanes 2 and 5) insoluble (lanes 3 and 6) actin. (D) De novo actin synthesis. (a) Effect of jaspamide on de novo actin synthesis. [S³⁵]-cystein-methionine-labeled HL-60 cells were exposed to medium (lane 1), DMSO (lane 2), or to jaspamide 10⁻⁷ mol/L (lane 3) for 1 hour, immunoprecipitated with rabbit anti-actin Ab, and the immunoprecipitates were resolved on SDS-PAGE and exposed to Fuji X-ray film. (b) Coomassie-stained SDS-PAGE of HL-60 cells treated for 1 hour with DMSO 0.01% (lanes 1 through 3) or jaspamide 10⁻⁷ mol/L (lanes 4 through 6) showing total (lanes 1 and 4) soluble (lanes 2 and 5) and insoluble (lanes 3 and 6) actin.