Cx3cl1

TA muscles were harvested and fixed with 4% paraformaldehyde (PFA) for 1 h at room temperature and then immersed in 30% sucrose overnight at 4 °C, as described in our previous study [14 (link)]. On day 2, the TA muscles were cryopreserved in an optical cutting temperature (OCT) compound (Tissue Tek) at −80 °C. TA muscle samples were sliced into 5-μm-thick frozen cross-sections using a Leica CM3050 cryostat. The muscle sections were incubated with primary antibodies including human-specific dystrophin (NBP2-79783, 1:200, Novus Biologicals, Centennial, CO, USA), human nuclear antigen (NBP2–34342, 1:100, Novus Biologicals, Centennial, CO, USA), CX3CL1(1:100, ABclonal, Woburn, MA, USA), VCAM-1(1:100, ABclonal, Woburn, MA, USA) and Myh3 (DSHP, 1:100) at 4 °C overnight, respectively, and anti-rabbit/mouse/rat secondary antibodies conjugated to Alexa Fluor 594 (Life Technologies) at room temperature for 1 h. Images were taken using a confocal microscope (FV1000, Olympus, Tokyo, Japan). For cell-engraftment quantification, four sections at 150 μm intervals in each TA muscle were analyzed. The number of muscle fibers in a cross-section area was measured using ImageJ with the colocalization plugin (NIH).

Total tissue lysates were separated on SDS-PAGE and electrotransferred onto polyvinylidene fluoride membranes (0.22 μm) by wet blotting (Bio-Rad). Primary antibodies detected the following proteins: β-actin (1:100,000, Cat No. AC026, Abclonal), α-tubulin (1:50,000, Cat No. 66031-1-Ig, Proteintech), CX3CL1 (1:1000, Cat No. A14198, Abclonal), GNB5 (1:1000, Cat No. A4447, Abclonal), AKT2 (1:5000, Cat No. ab131168, Abcam), p-AKT (1:1000, Cat No. YP0006, Immunoway), NF-κB (1:2000, Cat No. 66535-1-Ig, Proteintech), p-NF-κB (1:1000, Cat No. 93H1, Cell Signaling Technology), Bad (1:1000, Cat No. ab32445, Abcam), Bcl-2 (1:2000, Cat No. ab182858, Abcam), occludin (1:3500, Cat No. 13409-1-AP, Proteintech), claudin-1 (1:4500, Cat No. 13050-1-AP, Proteintech), which were then incubated with goat anti-rabbit IgG and goat anti-mouse IgG and HRP-conjugated(1:5000, Cat No. CW0103S, CW0102S, CWBIO). Then, stripping buffer (CW0056M, CWBIO) was used for the replacement of antibodies. The density of each protein was detected by an ECL detection kit (Solarbio).