Fig. 4

Regulatory effects of repeated hAMSC treatment on DMD-associated inflammation in skeletal muscle. A, B Horseradish peroxidase (HRP)-diaminobenzidine (DAB) immunostaining for F4/80 and CD206 in the diaphragm (A) and tibialis anterior (TA) muscle (B) of 18-week-old WT, control mdx, and four-time hAMSC-treated mdx mice (hAMSC-treated mdx-4). Original magnification, × 50. Scale bars, 1 mm. C Quantification of F4/80-positive area in the cross-section (% of total area) of the diaphragm of control mdx (n = 3) and four-time hAMSC-treated mdx (n = 4) mice. D Quantification of the ratio of CD206-positive area in the cross-section (% of F4/80 staining area) of the TA muscle of control mdx (n = 3), and four-time hAMSC-treated mdx (n = 4) mice. E Horseradish peroxidase (HRP)-diaminobenzidine (DAB) immunostaining for human Ku80 in the tibialis anterior (TA) muscle of 12-week-old WT, control mdx, and four-time hAMSC-treated mdx mice (hAMSC-treated mdx-4). Original magnification, × 80. Scale bars, 500 μm. High-magnification images represent the boxed regions in the hAMSC-mdx (left panel). Original magnification, × 200. Scale bar, 250 μm. F Quantification of the ratio of Ku80-positive area in the cross-section area of the control mdx (n = 8) TA muscle, and four-time hAMSC-treated mdx (n = 7) mice. G, H Cytokine and chemokine expression in the diaphragm (G) and TA muscle (H) of 12-week-old WT, control mdx, and four-time hAMSC-treated mdx mice were analyzed using the Proteome Profiler™ Array. The signals cropped from original images in Additional file 1: Figure S7 show changes in the expression levels of interleukin-1 receptor antagonist (IL-1ra), and tissue inhibitor of metalloproteinases-1 (TIMP-1) relative to those of positive control (PC) signals. Signal intensity in the regions of interest (ROIs) was quantified using array images (left panels), and representative data (graph) are presented. All data are presented as the mean ± SD, statistical differences are expressed relative to control mdx mice (^#P < 0.05, ^##P < 0.01 and ^####P < 0.0001, t-test)