Xenbase Image ID: 146828

FIGURE 5. Lamin concentrations affect nuclear size in vivo in X. laevis embryos. A) Single-cell X. laevis embryos were microinjected with the indicated recombinant lamin proteins or lamin storage buffer as a control. The concentrations on the x-axis represent the final concentration of injected lamins in the embryo assuming an embryo volume of 1 μl. For the lamin combinations, the data point at 36 nM = 15 nM LB1 + 15 nM LB2 + 6 nM LA, and the data point at 55 nM = 30 nM LB1 + 25 nM LB2. Embryos were allowed to develop to early stage 8 and embryo extracts were prepared. Extracts containing endogenous embryonic nuclei were fixed, and nuclei were spun onto coverslips and visualized as described in Figure 1. For each data point, 50-100 embryos were microinjected and the cross-sectional areas of 460-2360 nuclei were measured from NPC-stained nuclei, averaged, and normalized to the buffer injected control (bold horizontal line set at 1.0). The error bars are SEM. * p<0.05, *** p<0.001, NS Not significant. B) Single-cell X. laevis embryos were microinjected with mRNA encoding the indicating lamins or water as a control. Embryos were allowed to develop to stage 11-12 and embryo extracts were prepared. Extracts containing endogenous embryonic nuclei were fixed, and nuclei were spun onto coverslips and visualized as described in Figure 1. Representative images are shown. For the control, nuclei stained for the NPC with mAb414 are shown, while in the other images fluorescently labeled lamins are visualized. C) Nuclear size data from the experimental approach described in B) are plotted as a function of the concentration of ectopically expressed lamins. For each data point, 50-100 embryos were microinjected and the cross-sectional areas of 70-2900 NPC-stained nuclei were measured, averaged, and normalized to the water injected control (bold horizontal line set at 1.0). On the x-axis, the concentrations of ectopically expressed lamin proteins were estimated from infrared fluorescence western blots by comparing the amounts of GFP- or mCherry-tagged lamins in extracts from microinjected embryos to known amounts of recombinant lamin proteins loaded on the same gel (data not shown). The error bars are SEM. All normalized nuclear area data points greater than 1.05 and less than 0.98 are statistically different from the 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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