Figure S2.
Establishment and validation of a Nesprin-2 mutant mouse line. (A) Annotation of Syne2 gene (NM_001005510.2) from NCBI RefSeq. The deletion site in the mutant mouse line is indicated. Exons are shown as green bars. (B) An example of genotyping results. PCR products from WT and mutant were distinguished by agarose gel electrophoresis. (C) Western blot of the lysates from P9 WT and mutant cerebella. (D) Immunofluorescence of Nesprin-2 in the cerebella from P9 WT and Nesprin2 mutant mice. The zoom-in views of the selected regions are shown. (E) Immunocytochemistry of CGC reaggregate cultures with Nesprin-2 (green) and Lamin B1 (magenta). The fluorescence intensity was analyzed along the dashed yellow lines. (F) Top: Nissl staining of mid-sagittal cryosections of P9 cerebella. Bottom: Area quantification of midsagittal sections of P9, P12, and P30 samples. n = 5, 7, and 10 mice (WT), n = 7, 5, and 9 mice (mutant) from three to four independent experiments. Unpaired t test. (G) Left: Immunocytochemistry of CGC reaggregate cultures stained with β-tubulin (green) and γ-tubulin (magenta). The zoom-in views of the selected regions are shown. Right: Quantification of the nuclear-centrosome distance. n = 205 (WT) and 148 (mutant) cells. Unpaired t test with Welch’s correction. (H) Quantification of nuclear rotation in CGC reaggregate cultures. During time-lapse imaging, duration of the nuclear rotation over 10° was counted. n = 40 (WT) and 38 (mutant) cells from four independent experiments. Unpaired t test. Bars show mean ± SEM. Scale bars, 20 μm in D upper panels; 5 μm in D lower panels, E and G lower panels; 500 μm in F; 50 μm in G upper panels. Source data are available for this figure: SourceData FS2.

Establishment and validation of a Nesprin-2 mutant mouse line. (A) Annotation of Syne2 gene (NM_001005510.2) from NCBI RefSeq. The deletion site in the mutant mouse line is indicated. Exons are shown as green bars. (B) An example of genotyping results. PCR products from WT and mutant were distinguished by agarose gel electrophoresis. (C) Western blot of the lysates from P9 WT and mutant cerebella. (D) Immunofluorescence of Nesprin-2 in the cerebella from P9 WT and Nesprin2 mutant mice. The zoom-in views of the selected regions are shown. (E) Immunocytochemistry of CGC reaggregate cultures with Nesprin-2 (green) and Lamin B1 (magenta). The fluorescence intensity was analyzed along the dashed yellow lines. (F) Top: Nissl staining of mid-sagittal cryosections of P9 cerebella. Bottom: Area quantification of midsagittal sections of P9, P12, and P30 samples. n = 5, 7, and 10 mice (WT), n = 7, 5, and 9 mice (mutant) from three to four independent experiments. Unpaired t test. (G) Left: Immunocytochemistry of CGC reaggregate cultures stained with β-tubulin (green) and γ-tubulin (magenta). The zoom-in views of the selected regions are shown. Right: Quantification of the nuclear-centrosome distance. n = 205 (WT) and 148 (mutant) cells. Unpaired t test with Welch’s correction. (H) Quantification of nuclear rotation in CGC reaggregate cultures. During time-lapse imaging, duration of the nuclear rotation over 10° was counted. n = 40 (WT) and 38 (mutant) cells from four independent experiments. Unpaired t test. Bars show mean ± SEM. Scale bars, 20 μm in D upper panels; 5 μm in D lower panels, E and G lower panels; 500 μm in F; 50 μm in G upper panels. Source data are available for this figure: SourceData FS2.

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