CA-WASp requires Arp2/3 activity to increase F-actin production. (A) Percentage increase in total cellular F-actin because of CA-WASp, measured by flow cytometry in HT1080 cells (blue) and U937 cells (red) cultured with DMSO, 20μM or 40μM CK666; n = 3, mean ± SD. (B-C) Confocal images of (B) interphase HT1080 and (C) prometaphase U937 cells showing DNA (DAPI, blue), GFP-CA-WASp (green), and F-actin (red). Dashed white circles show individual ∼ 10 μm² areas used to measure F-actin density. Bar = 10 μm. (D-E) Nuclear F-actin density in prometaphase (D) HT1080 and (E) U937 cells with and without CA-WASp expression cultured in the conditions shown; n > 10 for each condition. Confocal microscopy was performed with a Zeiss LSM 710 inverted confocal microscope with a 40× C-Apochromat NA 1.2 WD 280-mm objective. Image analysis used ImageJ software. Fluorochromes were DAPI, eGFP-WASp, and Alexa Fluor 647–phalloidin.