FIGURE 8:

Inhibition of actin assembly by CK-636 and CK-548 decreases macrophage fusion and podosome formation. (A) Different concentrations of the Arp2/3 inhibitors CK-636 and CK-548 were added to macrophages at the onset of fusion induction with IL-4. Control cells were treated with DMSO. The fusion rates were determined after 72 h from confocal images of samples labeled with Alexa Fluor 488–conjugated phalloidin and DAPI. Results shown are mean ± SD from three independent experiments. Three to five random 20× fields per sample were used to count nuclei (250–300 nuclei/field; total ∼3600 nuclei). Fusion of control (DMSO-treated) cells was assigned a value of 100%. (B) Effect of inhibitors (each at 15 µM) on podosome formation. The fraction of cells with >10 podosomes was calculated and normalized to DMSO control. Results shown are mean ± SD from three independent experiments with three to five random 20× fields used per sample to count cells (100–150 cells/field). (C) Representative confocal micrographs of control (DMSO-treated) and CK 548-treated (15–60 µM) macrophages 72 h after incubation in the presence of IL-4. The cells were labeled with Alexa Fluor 488–conjugated phalloidin (white) and DAPI (teal). (D) Live imaging of macrophages treated with 15 µM Arp2/3 inhibitor CK-548. In each micrograph, time is shown in minutes:seconds. A single fusion event detected is shown.