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. 2016 Jan 1;27(1):35–47. doi: 10.1091/mbc.E15-05-0307

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© 2016 Giacomini et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 6: — Lmnb1 deficiency alters the distribution of NPCs in mouse cortical neurons. NPCs were analyzed in 7 DIV Lmnb1^+/+ and Lmnb1^Δ/Δ primary cortical neurons as described in Materials and Methods. (A, B) Representative maximal projections of z-stack confocal images of Lmnb1^+/+ and Lmnb1^Δ/Δ neurons stained for NPCs (A; green) and Nup153 (B; green). (C–E) TEM images of Lmnb1^+/+ (C) and Lmnb1^Δ/Δ (D, E) neurons showing NPCs (arrows) in transversal (C, D) and paratangential (E) sections. Asterisks indicate mitochondria. n, nucleus. Scale bars, 200 nm. (F) Top, 3D models of a Lmnb1^Δ/Δ nuclear membrane fragment representing the reconstruction of an 800-nm tomogram acquired in HAADF STEM from three semithin serial sections (Supplemental Movie S1). Scale bars, 200 nm. Bottom, single tomogram slices corresponding to sections S1–S3 in the three-dimensional model. White arrows point to NPCs; asterisks indicate mitochondria. n, nucleus. (G) Frequency distribution of interpore distances in nuclei of Lmnb1^+/+ (n = 198) and Lmnb1^Δ/Δ (n = 557) neurons. The frequency of interpore distances was determined using electron micrographs from >30 transversal sections. (H) Representative Western blot of NPCs and actin in lysates from Lmnb1^+/+ and Lmnb1^Δ/Δ cultured neurons. (I) Quantitative analysis of NPC expression. The data are normalized to actin. Bars represent the average ratio ± SEM. n = 4 per genotype. *p < 0.05, Student’s t test. (J, K) Lmnb1^Δ/Δ neurons were infected with LV syn-tagged-V5-LMNB1 and analyzed by immunocytochemistry. (J) Representative maximal projection of confocal z-stack images of V5 (green) and LMNB1 (red) immunoreactivity in Lmnb1^Δ/Δ neurons infected with LV syn-tagged-V5-LMNB1. (K) Representative maximal projection of confocal z-stack images of Tpr (green) and V5 (red) immunoreactivity in Lmnb1^Δ/Δ neurons infected with LV syn-tagged-V5-LMNB1. In all fluorescence images, nuclei are counterstained with DAPI (blue). Scale bars, 5 μm.