Sc 377460

The HSkMSCs were seeded at 2 × 10⁴ cells/well into a 24-well plate on coverslips and were grown in a growth medium for 24 h; then, the culture medium was replaced with a fresh medium containing 0 and 25 μg/mL of exosomes. After 9 days, the cells were washed twice using cold PBS, fixed with 4% paraformaldehyde/PBS for 1 h at 4 °C, washed three times with PBS, and permeabilized using 0.3% Triton X-100 for 20 min at room temperature. Later, cells were blocked with 3% BSA/PBS for 1 h. The cells were incubated overnight at 4 °C with anti-myogenin (MYOG) (sc-12732, Santa Cruz, CA, USA) or anti-myod (MYOD) (sc-377460, Santa Cruz, USA) and were then incubated with anti-mouse IgG–FITC secondary antibody (Santa Cruz). The cells were counterstained using 1 μg/mL 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 1 min and were then observed with a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss Microimaging, Thornwood, NY, USA).

Primary antibodies used were anti-CD31 (CD31) (M0823; 1:30; Dako); anti-CD90 (CD90) (ab133350; 1:200; Abcam); anti-hDystrophin (DYSTROPHIN) (NCL-DYS3; 1:20; Leica); anti-hLamin A/C (LAMIN A/C) (ab108595; 1:200; Abcam); anti-Laminin (LAMININ) (L9393; 1:200; Sigma); anti-MYOD (MyoD1) (SC-377460; 1:50; Santa Cruz); anti-myosin heavy chain all fibers (MYHC) (A4-1025-c; 1:200; DSHB); anti-myosin heavy chain embryonic (MYH3) (F1.652; 1:50; DSHB); anti-myosin heavy chain fast fibers (MYHC II) (A4.74; 1:200; DSHB); anti-myosin heavy chain slow fibers (MYHC I) (A4.840; 1:200 M; DSHB); and anti-PAX7 (Pax7) (Pax7-c; 1:50; DSHB). Secondary antibodies used were donkey anti-mouse Alexa Fluor 488 (A21202; 1:500; Thermo Fisher), donkey anti-mouse Alexa Fluor 488 (A21042; 1:500; Thermo Fisher), donkey anti-mouse Alexa Fluor 555 (A31570; 1:500; Thermo Fisher), donkey anti-rabbit Alexa Fluor 488 (A21206; 1:500; Thermo Fisher), and donkey anti-rabbit Alexa Fluor 555 (A31572; 1:500; Thermo Fisher).

On day 8, cells were washed twice with PBS prior to being fixed in 4% paraformaldehyde (#09154‐85; Nacalai tesque) in PBS for 30 min at room temperature and then washed twice with PBS before being blocked with Blocking One histo (#06349‐64; Nacalai tesque) for 30 min at room temperature. Cells were then stained with primary antibodies at 4℃ overnight. Cells were then washed twice with PBS, followed by staining with corresponding fluorescently labelled secondary antibodies or rBC2LCN‐635 (Excitation =634 nm, Emission =654 nm, #185‐03161, Fujifilm Wako Pure Chemical Corp.,) and 4’,6‐diamidino‐2‐phenylindole: DAPI (D9542; Sigma‐Aldrich Corporation) for 2 h at room temperature. The following antibodies were used for immunostaining of dystrophin (ab15277, 1:500; Abcam) ‐myosin heavy chain: MHC (MAB4470, 1:500; R&D Systems, Inc., Mineapolis, MN), myoblast determination protein 1: MyoD (G‐1) (SC377460, 1:500; SantaCruz Biotechnology Inc.), myogenin (5FD) (SC52903, 1:500; Santa Cruz Biotechnology), SeV (MBLPD029, 1:500; Medical and Biological Laboratories Co, Ltd., Woburn, MA), goat anti‐mouse IgG (H+L) Alexa Fluor 488 conjugate (A11029, 1:1,000; LifeTechnologies, Thermo Fisher Scientific) and donkey anti‐rabbit IgG (H+L) Alexa Fluor 555 conjugate (A31572, 1:1,000; Invitrogen, Thermo Fisher Scientific) (Table S1).