Journal: Nature communications
Article Title: Depletion of CD206 + M2-like macrophages induces fibro-adipogenic progenitors activation and muscle regeneration.
doi: 10.1038/s41467-022-34191-y
Figure Lengend Snippet: Fig. 5 | Impact of TGF-β signaling on C2C12 myoblast differentiation. a Relative mRNA expression levels of myogenesis-related marker genes in C2C12 myoblast co- cultured with M2-induced BMDM treated with recombinant TGF-β1 and TGF-β receptor I/II inhibitor (Ly21) (n = 4 wells per group). Data are shown as the means ± SEM. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc tests. *p < 0.05 was considered as significant. b Relative mRNA expression levels of myogenesis-related marker genes. M2-induced BMDM inhibi- tory effect on primary myoblast differentiation was released by the depletion of CD206+ M2-like MΦ upon diphtheria toxin (DT) treatment to the BMDM obtained from Tg mice (n = 3 wells per group). Data are shown as the means ± SEM. Statistical significance wasdeterminedby one-wayANOVAfollowed by Tukey’s post hoc tests. *p < 0.05, and **p < 0.01 were considered as significant. c Representative images of primary myoblast co-cultured with M2-induced BMDM with or without DT, stained with anti-MyoG antibody (n = 3 wells per group). d Representative images of C2C12 myoblast co-cultured with M2-induced BMDM (obtained from uninjured Tg mice) with or without DT, stained with embryonic myosin heavy chain (eMyH3) antibody, and DAPI (n = 4 wells per group). The images were taken using Keyence micro- scope. Scale bar 200 μm. e, f Relative myotube area and fusion index were quan- tified (n = 4 wells per group). Data are expressed as mean ± SEM. Statistical significance for e was determined by one-way ANOVA followed by Tukey’s post hoc tests, and for f was determined using the two-tailed Student t-test (*p < 0.05, and ns non-significant). AU arbitrary units, Con control, BMDM bone marrow-derived macrophages, and Ly21 Ly2109761 (TGF-β receptor I/II inhibitor).
Article Snippet: For the in vitro experiments, Dulbecco’s modified Eagle’smedium (DMEM) low glucose (Cat#041-29775) andDMEMhigh glucose (Cat#08459-64) were purchased from GibcoTM Life Technologies, Japan (Tokyo, Japan); horse serum (Hyclone) (Cat#SH30074.03) was purchased fromUSDonor Equine Serum andmurine interleukin-4 (IL-4) (Cat#021249) was purchased from Peprotech Inc; recombinant mouse TGF-β1 (Cat#7666-MB, 5 ng/mL), recombinant mouse macrophage colony-stimulating factor (M-CSF) (Cat#416-ML, 100ng/mL), andmonoclonalmouse anti-TGF-β1, 2, and 3 (1D11) (Cat#MAB1835, 0.5 μg/mL) antibodies were purchased from R&D Systems; Ly2109761 (TGF-βRI/II inhibitor) (Cat#CS-057, 5 ng/mL) was purchased from Chem Scene.
Techniques: Expressing, Marker, Cell Culture, Recombinant, Staining, Two Tailed Test, Control, Derivative Assay
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