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. 2017 Oct 9;114(43):E8996–E9005. doi: 10.1073/pnas.1708725114

Fig. 2.

Fig. 2.

Localization of MyoD transcript to Staufen1 foci. (A) qRT-PCR analysis of the Staufen1 (Stau1) transcript relative to Gapdh in freshly sorted MuSCs and after 24 and 48 h in culture. Pax7 was used as a control. (B) RNA-IP using an anti-Staufen1 antibody or IgG control was carried out using C2C12 cells. RNA immunoprecipitates and input lysate RNA were reverse transcribed, amplified using PCR with primer pairs for intron1 or the ORF in MyoD, and plotted relative to IgG control. (C) RNA-IP using an anti-Staufen1 antibody was carried out on freshly isolated quiescent MuSCs. RNA immunoprecipitates and input lysate RNA were reverse transcribed and amplified using PCR with primer pairs for intron1, the ORF, or the 3′-UTR in MyoD. Arf1 was used as a positive control. Values were normalized to IgG control and plotted relative to Gapdh. (D) qRT-PCR on input material for the Staufen1 IPs with primer pairs for intron1, the ORF, or the 3′-UTR in MyoD. Values are standardized to Gapdh. (E–G) Staufen1 protein and MyoD mRNA localization was visualized in quiescent MuSCs by combining smFISH for MyoD (red) with immunofluorescence for Staufen1 (green). Cells were counterstained with DAPI (blue) and imaged by confocal microscopy. (E) Representative photographs of colocalization analysis of Staufen1 protein and MyoD1 transcripts. (Scale bar, 5 μm.) (F) Quantification of MyoD mRNA smFISH staining at Staufen1 foci. Each triangle represents a Staufen1 focus for which the MyoD and Staufen1 staining intensities are plotted. Solid lines represent thresholds that were determined by staining knockout cells (MyoD) or by using secondary antibodies only (Staufen1). The number of foci above or below the MyoD threshold are given as a percentage. (G) A 3D confocal image (Left) rendered as a 2D image in the XY plane (Top Middle) with orthogonal views of the XZ (Bottom) and ZY (Right) planes. White lines denote the location of the 2D image in the orthogonal planes. White arrowheads denote the localization of Staufen1 and MyoD mRNA outside the nucleus. Data are reported as mean ± SEM. *P < 0.05; ns, not significant.