Fig. 2. Microstructures form as a result of fission events from PML bodies. (A) HEp-2 cells stably expressing GFP-PML were used to measure the mean fluorescence signal intensity (arbitrary units) of GFP in PML bodies prior to heat shock (pre), and in PML body remnants immediately following heat shock at 43°C for 30 minutes (post), and up to 30 minutes into the recovery phase following the heat shock. Measurements from single optical slices (not deconvolved) for five PML bodies and nucleoplasmic background (x) are shown. Error bars indicate the measurement error, defined as the range obtained from three separate measurements of each object after subtraction of its corresponding background. Between 30% and 70% of the GFP-PML signal is lost as a result of the stress. Recovery of the signal begins to occur following the stress. (B) 3-D reconstruction of a PML-containing microstructure budding from a parental PML body. U2OS cells stably expressing GFP-PML IV were heat shocked in an environmental chamber on the stage of the microscope. Z stacks of images were collected consecutively (10 seconds/stack capture time). I is a low magnification image of the entire nucleus prior to budding of a microstructure (full field width corresponds to 12 µm). The PML body that will bud is indicated (arrow). Panels II-IV are high magnification images of the PML body shown in I, at time points through the budding process (see Movie 1, http://jcs.biologists.org/supplemental/).

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