Fig 1: The expression of PDGFB is significantly increased after SCI or macrophages M2 polarization. (A) Western blot was used to detect the expression levels of PDGFB, PDGFD and PDGFRß before (Pre) and at 3, 7, and 14 dpi in the injury core of mice (n = 5 per group). (B) Quantitative analysis of PDGFB in (A). The blots were quantified as previously described. **p < 0.01, *p < 0.05 (3 and 7 dpi vs. pre); ### p < 0.001, ## p < 0.01 (3 and 7 dpi vs. 14 dpi). (C) Quantitative analysis of PDGFD in (A). ****p < 0.0001 (3, 7, and 14 dpi vs. pre). (D) Quantitative analysis of PDGFRß in (A). ***p < 0.001 (7, 14 dpi vs. pre and 3 dpi). (E) Western blot was used to detect the expression levels of PDGFB in M0, M1 or M2 macrophages (n = 5 per group). (F) Quantitative analysis of PDGFB in (E), the blots were quantified as previously described, **p < 0.01 (M2 vs. M0), ### p < 0.001 (M2 vs. M1). (G) ELISA was used to detect the concentration of PDGFB in CM0, CM1 or CM2 (n = 5 per group), The results were expressed as mean ± SD. **p < 0.01 (CM2 vs. other groups).
Fig 2: M2 macrophages secrete PDGFB acting on PDGFRß to promote PDGFRß+ pericytes migration in vitro. (A) Scratch test was used to detect the migration of PDGFRß+ pericytes after being treated with DMEM (Sham), conditioned medium of M2 macrophages (CM2), 10 ng/ml recombinant mouse PDGFB protein (PDGFB), CM2 plus 10 µm PDGFRß inhibitor (CM2+SU16f) or PDGFB plus 10 µM PDGFRß inhibitor (PDGFB+SU16f) for 72 h (n = 3 per group). (B) Transwell test was used to further detect the migration of PDGFRß+ pericytes after being treated in Sham, CM2, PDGFB, CM2 + SU16f or PDGFB+SU16f groups for 20 h (n = 3 per group). (C) Quantitative analysis of the wound closure rate in (A), the results were quantified as previously described, ****p < 0.0001, ***p < 0.001 (groups vs. Sham), #### p < 0.0001 (groups vs. CM2 and PDGFB). (D) Quantitative analysis of the number of transmembrane cells in (B), the results were quantified as previously described, ****p < 0.0001 (CM2 and PDGFB vs. other groups). ND, not determined. Scale bars, 100 µm. (E) Schematic representation. M2 macrophages could secrete PDGFB, acting on PDGFRß of PDGFRß+ pericytes, which promote the formation of fibrotic scar, corral macrophages and limit inflammation after SCI.
Fig 3: Microvascular endothelial cells induce pericyte-fibroblast transition via the PDGF-BB/PDGFRß signaling pathway in vitro. a, b Western blot analysis (a) and quantification (b) of PDGF-BB in bEnd.3 cells transfected with siNC or siRNAs targeting Pdgfb. c The expression levels of PDGF-BB in bEnd.3 cells transfected with siNC or siPdgfb#2 followed by myelin debris treatment were detected by ELISA. d, e Western blot analysis (d) and quantification (e) of PDGF-BB in bEnd.3 cells treated as described above in c. f Experimental schematic diagram of pericyte phenotypic transition transfected with siNC or siPdgfb#2 followed by myelin debris treatment. g Immunostaining of NG2 (green, upper panel), FSP1 (green, middle panel), and vimentin (green, lower panel) in primary pericytes treated as described above in f. h Quantification of the percentage of NG2+, FSP1+, and vimentin+ pericytes in g. i Experimental schematic diagram of pericyte phenotypic transition blocked with the PDGFRß inhibitor imatinib (a selective PDGFRß inhibitor) or Su16f (a specific PDGFRß inhibitor) followed by Mye-CM. j Immunostaining of NG2 (green, upper panel), FSP1 (green, middle panel), and vimentin (green, lower panel) in primary pericytes treated as described in i. k Quantification of the percentage of NG2+, FSP1+, and vimentin+ pericytes in j. Scale bars: 25 µm (g and j). Data are expressed as mean ± s.e.m. n = 3 independent cultures. **p < 0.01 and ***p < 0.001 by one-way ANOVA followed by Tukey’s post hoc test in b versus siNC, and k. *p < 0.05, **p < 0.01, and ***p < 0.001 versus siNC by unpaired two-tailed Student’s t test in c, e, and h
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