WASp^EMM is unable to reconstitute podosomes as effectively as WASp^WT, even at equivalent levels of WASp expression.^WASBMDCs were transduced on day 2 of culture with EGFP-WASp by lentiviral infection (multiplicity of infection, 30 and 100). On day 7 of culture, cells were either lysed for Western blot analysis or harvested and plated on fibronectin-coated glass coverslips or chamber slides for 4 hours. Cells were then fixed with 4% paraformaldehyde, permeabilized with Triton X-100, stained with rabbit anti-EGFP and anti–rabbit Alexa Fluor 488, Alex Fluor 568 phalloidin, mouse anti-vinculin, and goat anti–mouse Cy5, and mounted in ProFade Gold. Slides were imaged using a Zeiss LSM710 laser scanning spectral confocal microscope and a 63× Plan-Apochromat NA 1.4 WD 190-mm oil immersion lens, and images were acquired with Zen 2009 software (Zeiss). All image analysis was performed using ImageJ Version 1.45d software. (A) Western blot of transduced ^WASBMDCs immunoblotted for WASp and GAPDH (loading control). (B) Confocal images of transduced cells were blindly analyzed to determine the proportion of cells that produce podosomes. The percentage of transduced cells producing podosomes for each construct was calculated from 3 independent experiments, with a total of 194-391 transduced cells analyzed for each condition. Differences between constructs were analyzed by ANOVA, with comparisons made with EGFP-only–transduced cells. (C) Likelihood of transduced cells forming podosomes increases with increasing expression of WASp protein. WASp expression within individual cells was estimated by calculating the integrated density of the EGFP signal within the cells outline. This value was converted to the number of SEMs of the EGFP signal in untransduced cells (Z score of negative population). Cells were grouped for EGFP intensity as follows: Z score < 2 (EGFP negative cells), 2-3, 3-4, 4-5, > 5, and the percentage of podosome-positive cells within each banding was calculated for each WASp construct. Data from all 4 EVH1 missense mutant WASp-transduced cells were combined and analyzed together (labeled EMM) to avoid statistical errors associated with small numbers of cells expressing high levels of EVH1 missense mutant WASp. The numbers of cells analyzed for each construct at each expression band is shown in supplemental Table 1. Results presented represent combined data from 2 independent experiments (total of 3094 cells analyzed). (D) Logistical regression to determine the size of the impact of EGFP-WASp expression on the likelihood of an individual cell forming podosomes for each individual construct (compensating for difference between experiments). EGFP-WASp expression was expressed as Z scores of untransduced EGFP signal and error bars represent 95% confidence intervals for the likelihood ratio. (E) Among cells that formed podosomes, the number of podosomes produced per cell was counted and the median was calculated for each construct. The average median podosome number per cell from 3 independent experiments is represented, with differences between constructs analyzed using ANOVA. ***P < .005, **P < .01, *P < .05, and ns indicates P > .05.