Journal: Advanced Science

Article Title: Targeting Fibrotic Scarring by Mechanoregulation of Il11ra1 + /Itga11 + Fibroblast Patterning Promotes Axon Growth after Spinal Cord Injury

doi: 10.1002/advs.202513476

Figure Lengend Snippet: Mechanotransduction induced the Itga11 + /Il11ra1 + fibroblast subset via LRP6‐Wnt/β‐Catenin‐MMP7 signaling axis. A–C) Representative chopped Western blot images A) and quantitation of the expression of phosphorylated β‐catenin B) and β‐catenin C) in vivo. N = 3 samples, respectively. D–F) Representative chopped Western blot images D) and quantitation of the expression of phosphorylated β‐catenin E), and β‐catenin F) in vitro. N = 3 samples, respectively. G) Representative multichannel immunostaining images revealing the expression active β‐catenin and phalloidin in the migrating fibroblasts in different groups. The white boxed regions were the high magnifications of the active β‐catenin signal and DAPI, which were presented in separate channels. H)The quantifications of the percentage of active β‐catenin entering nucleus. N = 9 cells. I) Heatmap showing the expression levels of LRP5 and LRP6 from scRNA‐seq. J,K) The representative chopped Western blot images J) and quantitation K) showing the expression of LRP6 and phosphorylated LRP6 proteins in vivo. N = 3 animals. L) Schematic of the experimental design for the in vitro validation. M) Western blot results of related proteins expressions in vitro following intervention with HLY78 and Salinomycin respectively. N) Representative multichannel immunostaining images showing the expression active β‐catenin and phalloidin in the migrating fibroblasts in Medium hydrogel group following intervention with HLY78. The white boxed regions were higher magnifications of the active β‐catenin signal and DAPI, which were presented in separate channels. The quantifications of the proportion of active β‐catenin entering nucleus following the intervention with HLY78 were listed on the right. N = 9 cells. O,P) Representative multichannel immunostaining images showing the expression active β‐catenin and phalloidin in the migrating fibroblasts in High hydrogel group O) and Low hydrogel group P) following intervention with Salinomycin. The white boxed regions were higher magnifications of the active β‐catenin signal and DAPI, which were presented in separate channels. The quantifications of the proportion of active β‐catenin entering nucleus following the intervention with HLY78 were listed on the right. N = 9 cells, respectively. Q) Flow cytometric analysis of Itgall + Il11ra1 + fibroblasts in vitro in Medium hydrogel group following intervention with HLY78. N = 3 samples. R,S) Flow cytometric analysis of Itgall + Il11ra1 + fibroblasts in vitro in High hydrogel group R) and Low hydrogel group S) following intervention with Salinomycin. N = 3 samples, respectively. T) Representative chopped Western blot images showing the expression of the related proteins in vivo following intervention with HLY78 and Salinomycin respectively. U) Schematic representation of the LRP6‐Wnt/β‐Catenin‐MMP7 pathway axis. Data were shown as the mean ± SD; p values were determined by two‐tailed one‐way ANOVA with Tukey's multiple‐comparisons test or two‐tailed paired t ‐tests.

Article Snippet: For the in vitro studies, fibroblasts seeded onto HADA/HRR hydrogels were treated with LRP6 phosphorylation enhancer HLY78 (20 μ m , HY‐122816, MedChem‐Express, USA) or LRP6 phosphorylation inhibitor Salinomycin (1 μ m , HY‐15597, MedChem‐Express, USA) in the medium.

Techniques: Western Blot, Quantitation Assay, Expressing, In Vivo, In Vitro, Immunostaining, Biomarker Discovery, Two Tailed Test