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

Figure 5. AKAP6 is required for the nuclear envelope localization of microtubule-organizing center (MTOC) proteins.

(A) C2C12 cells were differentiated for 2 days. Immunostaining shows that all PCM-1+ nuclei are also AKAP6+. (B) Quantification of AKAP6+ and PCM-1 nuclei in C2C12 cells treated with negative control (si-ctrl) or Akap6 (si-Akap6) siRNA after 2 days of differentiation indicates that AKAP6 is required for nuclear envelope localization of PCM-1. Data are represented as individual biological replicates (n = 3), together with mean ± SD. 95% CI = 6.21% to 8.74%; 95% CI = 4.63% to 9.43%. (C) MYOG-mScarlet cells were treated with si-ctrl or si-Akap6 and subsequently treated with doxycycline (Dox) for 3 days. Image analysis revealed that myogenin-induced localization of PCM-1 to the nuclear envelope is AKAP6-dependent. (D) Quantification of (C). (E, F) Quantification of PCNT (E) and AKAP9 (F) nuclear coverage in Dox-stimulated MYOG-mScarlet cells treated with si-ctrl or si-Akap6. (G) Co-immunoprecipitation (IP) of PCM-1 from MYOG-mScarlet but not from mScarlet lysate (L) using an anti-AKAP6 antibody. (H, I) Enriched C2C12 myotubes (troponin I) were transfected with si-ctrl or si-Akap6 and immunostaining demonstrates that AKAP6 is required for maintaining nuclear envelope localization of PCM-1 (H) as well as PCNT and AKAP9 (I). Scale bars (A, H) 20 µm, (C, I) 10 µm. Data (D–F) are shown as violin plots. The red line indicates the median, and dotted lines indicate the 25% and 75% percentile. N numbers indicate the total number of analyzed nuclei pooled from three biological replicates. *p<0.05.; **p<0.01; ***p<0.001.

Figure 5—source data 1. Underlying data for graphs in Figure 5B, D–F.
Figure 5—source data 2. Raw files and uncropped blots for Figure 5G.

Figure 5.

Figure 5—figure supplement 1. AKAP6 depletion does not affect nesprin-1α.

Figure 5—figure supplement 1.

Enriched C2C12 myotubes were transfected with non-targeting (si-ctrl) or Akap6 (si-Akap6) siRNA and immunostained for AKAP6 and nesprin-1α. Scale bars: 10 μm.
Figure 5—figure supplement 2. AKAP6 or nesprin-1α depletion does not prevent centrosome attenuation in MYOG-expressing cells.

Figure 5—figure supplement 2.

(A, B) mScarlet or MYOG-mScarlet transfected with the indicated siRNAs were stimulated with doxycycline (Dox) for 3 days. PCNT (A) and γ-tubulin (B) immunostaining intensities were quantified. Data are shown as violin plots. The red line indicates the median, and dotted lines indicate the 25% and 75% percentile. N numbers are the total number of analyzed centrioles pooled from three biological replicates. Note that here PCNT and γ-tubulin intensities were measured simultaneously.
Figure 5—figure supplement 3. MyoD induces nesprin-1α and AKAP6.

Figure 5—figure supplement 3.

(A) NIH3T3 cells were transfected with MyoD-GFP. Two days after transfection, nesprin-1α and AKAP6 were detected via immunostaining. Scale bar: 10 µm. (B) NIH3T3 cells were transfected with non-targeting siRNA (si-ctrl) or myogenin siRNA (si-Myog) and 1 day later transfected with MyoD-GFP. Indicated microtubule-organizing center (MTOC) components were detected using immunostaining and positive nuclei were quantified. Data are represented as mean ± SD. n = 3. ns: p>0.05; **p<0.01; ***p<0.001.