Journal: Inflammation and Regeneration

Article Title: Imatinib inhibits pericyte-fibroblast transition and inflammation and promotes axon regeneration by blocking the PDGF-BB/PDGFRβ pathway in spinal cord injury

doi: 10.1186/s41232-022-00223-9

Figure Lengend Snippet: Microvascular endothelial cells induce pericyte-fibroblast transition via the PDGF-BB/PDGFRβ 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 PDGFRβ inhibitor imatinib (a selective PDGFRβ inhibitor) or Su16f (a specific PDGFRβ 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

Article Snippet: Primary pericytes were treated with culture medium containing 2% FBS, conditioned medium (EC-CM or Mye-CM) mixed in a 1:1 ratio to pericyte culture medium, or 250 ng/ml PDGF-BB (220-BB-050, R&D Systems) for 72 h. For PDGFRβ blocking experiments, pericytes were pretreated with imatinib (a selective inhibitor of PDGFRβ, 1 mg/ml, HY-590946, MCE) or Su16f (a special inhibitor of PDGFRβ, 10 μm, 3304, R&D Systems) for 12 h before treatment with Mye-CM. siRNA targeting mouse PDGF-BB and negative control siRNA were obtained from GenePharma (Shanghai, China). siRNA targeting PDGF-BB#1 was 5′-UCCGGAGUCGAGUUGGAAATT-3′, PDGF-BB#2 was 5′-GGUGAGAAAGAUUGAGAUUTT-3′, and PDGF-BB#3 was 5′-GCAAGCACCGAAAGUUUAATT-3′. jetPRIME transfection reagent (114–15, Polyplus Transfection) was used to transfect bEnd.3 cells according to the manufacturer’s protocols.

Techniques: In Vitro, Western Blot, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Immunostaining, Two Tailed Test