Fig. 3. Diffusion behavior of PML protein isoforms outside NBs. (A) U-2 OS cell expressing GFP–PML-IV (green) merged with the respective DIC image before FCS measurement. The inset shows only the GFP signals within the nucleus (indicated by dotted line) of this cell. +, position of the FCS laser beam. Scale bar, 5 µm. (B) Mean autocorrelation data obtained from FCS-count-rate traces for GFP–PML-IV-expressing cells (solid black line). Data were fitted using an anomalous diffusion model (dashed red line). The inset graph displays a residual plot from the fit. (C) Kinetics modeling of PML NB assembly according to a diffusion-binding model. Molecules with the potential to accumulate at PML NBs move by diffusion (D) in the nucleoplasm outside NBs. Upon stochastic encounter, molecules associate and dissociate from the periphery of the NB (k_on and k_off, respectively) and penetrate into and out of the core of the NB (k_in and k_out, respectively). Dashed circle, ROI for bleaching and recovery measurements employed in FRAP experiments.