4 6 diamidino 2 phenylindole dapi dye

The subcellular distribution of proteins was examined by immunofluorescence staining. Specifically, the cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 20 min, and incubated with 3% bovine serum albumin (BSA) for 1 h. Then, the cells were stained with the primary antibodies of interest, such as anti-FMRP and anti-BEX1 (Abcam, Cambridge, MA, USA) overnight at 4 . On the next day, the cells were washed thrice with phosphate-buffered saline (PBS) and incubated with secondary antibodies Alexa Fluor® 594-conjugated goat anti-mouse IgG or goat anti-rabbit IgG for 2 h at room temperature (Santa Cruz, Dallas, Texas, USA). The cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) dye for 5 min (Beyotime, Shanghai, China). Finally, we observed cells under an FV1000 laser-scanning confocal microscope (Olympus, Tokyo, Japan).

The myotube diameter was measured by using myosin heavy chain (MHC) staining [37] . Brie y, myotubes were grown and differentiated on glass coverslips. After supplemented with different treatments as mentioned above, the chicken myotubes were then xed, permeabilized, and incubated overnight with anti-MHC (1:1000) (Abcam, US). After antibody removal and washing, the slips were incubated for 4 h with 1:1000 a nity-puri ed Alexa Fluor 488 dye-conjugated goat anti-mouse antibody (Beyotime, Shanghai, China). Antibody was again removed with washing, then the slips were incubated for 3 min with 4',6-diamidino-2-phenylindole (DAPI) dye (Beyotime, Shanghai, China). The slips were observed under a confocal uorescence microscope (Dragon y, Andor, Belfast, UK) and photographed. Average myotube diameter was calculated using Image J from 10 myotubes per visual eld and 10 visual elds were recorded from each of the different sample. Myotubes were de ned as all multinucleated cells positive for the MHC stain and containing at least three nuclei [37] .