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. 2017 Oct 9;27(19):2999–3009.e9. doi: 10.1016/j.cub.2017.08.031

Figure 3.

Figure 3

MT Nucleation from the NE Requires Nesprin-1, Sun1/2, and Akap450

(A) 3D-SIM fluorescent image of the nucleus of a differentiated C2C12 cell, stained for Nesprin-1 (white, MANNES1E), Pericentrin (Pcnt, red), and microtubules (MTs, green) after 5 min nocodazole washout to allow MT regrowth. The scale bar represents 5 μm.

(B) 48 hr differentiated C2C12 cells, treated with the indicated siRNAs, were immunostained for Pericentrin (Pcnt, red), microtubules (MTs, green) and Myogenin (MYOG, gray) after nocodazole washout. The scale bar represents 10 μm. See also Figure S3A.

(C) Quantification of the mean percentage (%) of Myogenin-positive cells with MT nucleation from the NE as described in (B). Error bars ± SD; n represents total number of nuclei from at least three independent experiments. ∗∗∗p < 0.001; ∗∗p < 0.01; n.s., not statistically significant, t test.

(D) Differentiated human immortalized myotubes from a healthy control (wild-type) or from a patient carrying a nonsense mutation within the SYNE1 gene (23560 G>T) were immunostained for PCM1 (red), microtubules (MTs, green) and Myogenin (MYOG, gray) after nocodazole washout. Images represent maximum projections of confocal z sections. The scale bar represents 10 μm. See also Figures S3B and S3C.

(E) Quantification of the mean percentage (%) of Myogenin-positive cells with MT nucleation from the NE as described in (D). Error bars ± SD; n represents total number of nuclei from two independent experiments. ∗∗p < 0.01; t test.

(F) 48 hr differentiated C2C12 cells, treated with the indicated siRNAs were immunostained for Akap450 (red), microtubules (MTs, green) and Myogenin (MYOG, gray) after nocodazole washout. The scale bar represents 10 μm.

(G) Graph shows the mean percentage (%) of Myogenin-positive cells with MT nucleation from the NE as described in (F). Error bars ± SEM; n represents total number of nuclei from two independent experiments. ∗p < 0.05; n.s., not statistically significant, one-way ANOVA with Dunnett’s multiple comparisons test.