Fig 2: KCs express elevated levels of EMT markers during co-culture with WNT5A-activated HDFs or IL-6 stimulation. (a) Illustration of the co-culture system involving KCs and HDFs. (b) Increased levels of slug, vimentin and N-cadherin following co-culture of KCs with WNT5A-activated HDFs. (c) IL-6-stimulated KCs as well as patient-derived keloid keratinocytes from two different keloid tissues (KK1, KK2) show elevated gene expression levels of vimentin compared to the normal keratinocytes. (d) Western blot analysis of KCs co-cultured for 24 h with WNT5A-treated HDFs shows significantly increased levels of p-STAT3, slug and vimentin and decreased level of E-cadherin. Data are from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.005; Mann–Whitney U test. KC keratinocytes, KK keloid keratinocytes, HDF human dermal fibroblasts, EMT epithelial-mesenchymal transitio, WNT5A Wnt family member 5A, IL interleukin

Fig 3: KFs express higher levels of WNT5A and pro-inflammatory cytokines relative to normal fibroblasts. (a) Double-immunofluorescence staining of keloid tissues show elevated WNT5A expression in α-SMA-positive cells in the dermis relative to normal tissues (scale bars, 50 μm). In vitro analysis shows (b) increased gene and protein expression of WNT5A in KFs relative to HDFs (scale bars, 20 μm). *p < 0.01, ***p < 0.005; Mann–Whitney U test. Data are from three independent experiments. Human cytokine profile array shows (c) increased CCL-2/MCP-1, IL-6, IL-8 and CXCL-1 production from KFs relative to HDFs. KF keloid fibroblasts, HDF human dermal fibroblasts, α-SMA α-smooth muscle actin, CCL2 C–C motif chemokine ligand 2, CXCL-1 C–X–C chemokine ligand 1, IL interleukin, WNT5A Wnt family member 5A

Fig 4: Human keloid tissues show increased expression of WNT5A and EMT markers. (a) Bulk RNA-seq shows upregulation of WNT5A and EMT phenotype markers in keloid samples. GSEA of RNA-seq reveals significantly enriched gene sets in the (b) EMT and (c) IL-6/JAK/STAT3 pathways in keloid samples. Keloid tissues express (d) increased N-cadherin and decreased E-cadherin in the epidermis [scale bars, 100 μm (low-power field, LPF) and 50 μm (high-power field, HPF)]. Expression of (e) vimentin and α-SMA mRNA increases in keloid tissues including the reticular dermis [scale bars, 2 mm (LPF) and 250 μm (HPF)]. Expression of (f) WNT5A is significantly increased in keloid tissues [scale bars, 200 μm (LPF) and 50 μm (HPF)]. The tissue analyses were performed from three different keloid and normal tissues, with three independent experiments performed as technical replicates. *p < 0.05, **p < 0.01, ***p < 0.005, Mann–Whitney U test. EMT epithelial-mesenchymal transition, GSEA gene set enrichment analysis, LPF low-power field, HPF high-power field

Fig 5: Effect of WNT5A silencing on IL-6 production and levels of EMT markers. (a) Control KFs produce significantly increased levels of IL-6 compared to control HDFs, while KFs treated with siWNT5A produce significantly decreased levels of IL-6 compared to siWNT5A-treated KFs. mRNA levels of (b) α-SMA, (c) vimentin, and (d) N-cadherin and (e) Western blot analysis of KCs co-cultured with HDFs, KFs, KFs treated with control siRNA and KFs treated with siWNT5A. (f) Western blot analysis of KCs co-cultured with KFs treated with or without IL-6 neutralizing antibody (αIL-6). Data are from three independent experiments. *p < 0.05, ***p < 0.05, ***p < 0.005; Mann–Whitney U test. HDF human dermal fibroblasts, KF keloid fibroblasts, KC keratinocytes, EMT epithelial–mesenchymal transition, IL interleukin, α-SMA α-smooth muscle actin