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FIGURE 4. Lamin concentrations affect nuclear size in X. laevis embryo extracts. A) Embryo extracts containing endogenous embryonic nuclei were prepared from X. laevis embryos at stage 7 to early stage 8. Recombinant lamins were added to the indicated final concentrations, and the same total volume was added to each reaction. For the control reaction, an equal volume of lamin storage buffer was added. Nuclei were incubated for 60 minutes at 20°C. Nuclei were then fixed, spun onto coverslips, processed for immunofluorescence, and stained with mAb414 to visualize the NPC (red) and Hoechst to visualize the DNA (blue). Representative images are shown. White arrows denote intranuclear lamin puncta that form upon addition of high concentrations of recombinant lamin proteins. B) Nuclear size data from one representative experiment are shown. For each bar, the cross-sectional areas of 370-1242 nuclei were measured from NPC-stained nuclei and averaged. The error bars are SD. * p<0.05, ** p<0.01, *** p<0.001, NS Not significant. From left to right, the number of nuclei quantified/p-values are: 674/not applicable, 459/10-23, 734/10-12, 513/0.03, 1242/10-18, 704/10-9, 616/0.12, 456/10-31, 763/10-23, 882/10-10, 569/10-45, 370/10-20, 605/0.009. C) Cumulative data from 2 different embryo extracts are shown. For each data point, the average cross-sectional nuclear area was measured as in B) and normalized to the buffer addition control (bold horizontal line set at 1.0). The error bars are SEM. All normalized nuclear area data points greater than 1.05 are statistically different from the buffer control (1.0) by at least p<0.05. Image published in: Jevtić P et al. (2015) Copyright © 2015. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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