Competitive binding studies between jaspamide and Rh-phalloidin. Jaspamide induces F-actin aggregation in HL-60 cells and in human monocytes. Competitive studies (A through C). HL-60 cells were stained with Rh-phalloidin in the presence of (A) medium (control). (B) 10−8 mol/L jaspamide and (C) 10−7 mol/L jaspamide; analyzed by CLSM. No actin aggregation is seen. Aggregation of F-actin in HL-60 cells (D through F). HL-60 cells were incubated for 24 hours with (D) medium. (E) 10−7 mol/L jaspamide subsequently washed and stained with Rh-phalloidin (no jaspamide added during staining with Rh-phalloidin). Aggregation of F-actin is seen in (E). (F) HL-60 cells grown for 24 hours in the presence of 3.94 μmol/L CD were stained with Rh-phalloidin (arrows indicate cytochalasin-induced small focal F-actin clusters). Costaining of HL-60 cells with Rh-phalloidin and anti-actin MoAb (G through I). HL-60 cells grown for 24 hours in the presence of 10−7 mol/L jaspamide were costained for F-actin (Rh-phalloidin) and total actin (anti-actin MoAb labeled with anti-mouse–FITC). Colocalization analysis was performed: (G) Actin labeled with anti-actin MoAb (green), (H) F-actin labeled with Rh-phalloidin (red), (I) colocalization of the total actin and F-actin staining is observed (yellow). Effect of jaspamide on monocytes (J through Q). Freshly isolated monocytes were grown for 24 hours in IMDM containing 10% FBS. The cells were incubated for additional 24 hours with the following: (J through L, P) medium; (M through O, Q) 10−7 mol/L jaspamide. (J, M, P, and Q) Monocytes stained with Rh-phalloidin to show the distribution of F-actin. In (P) and (Q) the actin staining (red) is overlaid on the Nomarski image (gray) phase microscopy of the same cells. (L and O) Monocytes stained with Giemsa to show the changes in cell morphology. (A through F: original magnification × 250; G through H: original magnification × 750; J through O: original magnification × 250; P and Q: original magnification × 2,000).
