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

Figure 2. Myogenin expression is sufficient to induce nuclear envelope microtubule-organizing center (NE-MTOC) formation in non-muscle cells.

(A) NIH3T3 fibroblasts were transfected with constructs encoding GFP, MyoD-GFP or myogenin-GFP (Myog-GFP). After three days, PCM-1 localization was assessed by immunostaining. Arrows indicate nuclei of transfected cells which have recruited PCM‑1. Scale bars: 10 µm. (B) Quantification of (A) demonstrating that myogenin induces nuclear envelope localization of PCM‑1 more efficiently than MyoD. Data are represented as individual biological replicates (n = 3), together with mean ± SD. ***: p < 0.001; 95% CI of difference Myog-GFP vs. MyoD-GFP = 30.99% to 49.22%. n = 3. (C-H) NIH3T3 Tet-ON mScarlet or MYOG-2A-mScarlet (MYOG-mScarlet) cells were treated with doxycycline (Dox) for three days. After immunostaining, nuclear envelope localization of PCM-1 (C-D), PCNT (E-F), and AKAP9 (G-H) was analyzed and quantified. Data are depicted as violin plots. Red line indicates the median, dotted lines indicate the 25% and 75% percentile. ***: p < 0.001. Scale bars: 20 µm (I) Immunostaining of MYOG-mScarlet cells treated with Dox for three days showing the presence of nesprin‑1α+ nuclei. Scale bars: 20 µm (J) RT-PCR analysis of MYOG-mScarlet cells in the absence of Dox (-Dox) or treated with Dox for the indicated time points demonstrating that nesprin‑1α is upregulated upon myogenin expression. Gapdh was used as equal input control. (K) ChIP-PCR analysis of Dox-treated MYOG-mScarlet cells using an anti-myogenin antibody or an IgG1 control showing that myogenin binds an E-box in the nesprin-1α promoter region. (L-M) Immunostaining of α-tubulin and subsequent quantification of nuclear envelope coverage after 30s of microtubule regrowth following cold-induced microtubule depolymerization in mScarlet or MYOG-mScarlet cells treated with Dox for three days. Data are depicted as violin plots. Red line indicates the median, dotted lines indicate the 25% and 75% percentile. ***: p < 0.001. Scale bars: 20 µm. N numbers indicate total number of analyzed nuclei pooled from three biological replicates.

Figure 2—source data 1. Underlying data for graphs in Figure 2B, D, F and H.
Figure 2—source data 2. Raw files and uncropped gels for Figure 2J.
Figure 2—source data 3. Raw files and uncropped gels for Figure 2K.

Figure 2.

Figure 2—figure supplement 1. Myogenin is required for MyoD-induced nuclear envelope recruitment of PCM-1.

Figure 2—figure supplement 1.

(A) Three days after ectopic expression of MyoD-GFP in NIH3T3 fibroblasts, immunostaining revealed that nuclear PCM-1 localization is associated with high myogenin (Myog) expression (yellow arrowhead). (B) NIH3T3 cells were treated with negative control (si-ctrl) or myogenin (si-Myog) siRNA and 24 hr later transfected with a construct encoding MyoD-GFP. Immunofluorescence staining indicates that myogenin is required for MyoD-induced PCM-1 nuclear recruitment. (C) C2C12 cells were transfected with the indicated siRNAs and differentiated for 2 days. RT-PCR analysis reveals that depletion of MyoD reduces myogenin levels in differentiating myoblasts but not vice versa. Scale bars (A, B): 10 µm.
Figure 2—figure supplement 1—source data 1. Raw files and uncropped gels for Figure 2—figure supplement 1C.
Figure 2—figure supplement 2. Myogenin targets are specifically precipitated in induced MYOG-mScarlet cells.

Figure 2—figure supplement 2.

Chromatin immunoprecipitation (ChIP) was performed on the indicated lysates using the anti-myogenin antibody or an isotype control (IgG). The relative abundance of (A) the Syne1 α-isoform promoter region, (B) an intronic region of Syne1 (negative control), or (C) the desmin promoter region (positive control) were determined by qPCR. Data are represented as individual biological replicates (n = 3), together with mean ± SD. ns: p>0.05; ***p<0.01; ****p<0.001.
Figure 2—figure supplement 3. Microtubule depolymerization in mScarlet and MYOG-mScarlet cells.

Figure 2—figure supplement 3.

Immunostaining of α-tubulin in mScarlet or MYOG-mScarlet cells treated with doxycycline (Dox) for 3 days following cold-induced microtubule depolymerization without regrowth. Scale bar = 20 µm.