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. 2021 Oct 4;10:e65672. doi: 10.7554/eLife.65672

Figure 4. The nesprin-1α interaction partner AKAP6 is a potential mediator of myogenin-induced nuclear envelope microtubule-organizing center (NE-MTOC) formation.

(A) Scheme illustrating the bioinformatics workflow used to identify potential myogenin downstream candidates. (B) Venn diagram depicting the numbers of genes matching criteria for the individual data sets and for intersection of data sets. Criteria for myogenin ChIP-seq data (red): Genes where myogenin binding was detected at the promoter region; criteria for C2C12 RNA-seq data (green) and for microarray data of rat heart development (blue): upregulated genes. (C) ChIP-PCR analysis of doxycycline (Dox)-treated MYOG-mScarlet cells using an anti-myogenin antibody or an IgG1 control showing that myogenin binds an E-box in the Akap6β promoter region. (D) RT-PCR analysis of MYOG-mScarlet cells in the absence of Dox (-Dox) or treated with Dox for the indicated time points demonstrating that Akap6β is upregulated upon myogenin expression. The two bands for Akap6β derive from alternative splicing of the first exon of Akap6β, which results in an ~200 bp insertion in the 5’ untranslated region. Gapdh was used as equal input control. Please note that the same samples and Gapdh control were used as in Figure 2J. (E) C2C12 cells were differentiated for 2 days, and immunostaining shows that all AKAP6+ nuclei are also nesprin-1α+. Scale bar: 20 µm. (F) High-resolution Airyscan image of (E). Arrowhead indicates AKAP6 localized at the cytoplasmic side of nesprin-1α signal. Arrow marks nesprin-1α that is localized at the nuclear side of AKAP6 signal. Scale bar: 0.5 µm. (G) Myoblasts from healthy donors (wt) and from patients carrying a mutation in the SYNE1 gene (SYNE1-/-) were differentiated for 4 days. Immunostaining analysis showed that loss of nesprin-1α is associated with loss of AKAP6 from the nuclear envelope in differentiated myotubes (troponin I). Scale bars: 10 µm.

Figure 4—source data 1. Raw files and uncropped gels for Figure 4C.
Figure 4—source data 2. Raw files and uncropped gels for Figure 4D.

Figure 4.

Figure 4—figure supplement 1. The myogenin antibody specifically precipitates the Akap6β promoter region in induced MYOG-mScarlet cells.

Figure 4—figure supplement 1.

Indicated lysates were probed using the anti-myogenin antibody or an isotype control (IgG). The relative abundance of (A) the Akap6β promoter region or (B) an intronic region of Akap6 was determined by qPCR. Data are represented as individual biological replicates (n = 3), together with mean ± SD. ns: p>0.05; ****p<0.001.
Figure 4—figure supplement 2. Quantification of nesprin-1α and AKAP6 signal at the nuclear envelope.

Figure 4—figure supplement 2.

Differentiated C2C12 cells were immunostained for nesprin-1α and AKAP6. Signal intensity was quantified as line scan profiles across the nuclear envelope. 10 line scan profiles were obtained in regular intervals along the nuclear periphery and pooled from 10 nuclei. Data are shown as mean ± SD.