Journal: Signal Transduction and Targeted Therapy

Article Title: Wnt-associated DKK3 in keratinocytes mediates radiation-induced hyperplasia, dermatitis and skin fibrosis

doi: 10.1038/s41392-025-02541-z

Figure Lengend Snippet: Radiation increases DKK3 expression and canonical Wnt activity in vivo and in vitro via ROS. a Representative in situ hybridization images of DKK3-positive cells in thoracic skin sections of irradiated (20 Gy) and nonirradiated (control) wild-type mice at 4 weeks post-irradiation ( n = 5 skin sections/mouse from n = 5 mice/group). Scale bars: 25 μm. b Representative immunofluorescence images of thoracic skin sections from DKK3/canonical Wnt dual reporter mice at 0 days (control, n = 2), 6 days ( n = 2), and 14 days ( n = 2) postirradiation with 20 Gy. Canonical Wnt activity is represented by GFP (green), and DKK3 expression is represented by the m-Cherry signal (red). Nuclei are stained with DAPI (blue). Scale bars: 50 μm. N/TERT-1 keratinocytes with the Wnt reporter were irradiated in vitro with 4 Gy. c DKK3 levels (ELISA) and d canonical Wnt activity ( Gaussia luciferase assay) in culture media at different time points postirradiation. RLU: relative light units. e Canonical Wnt activity in the culture media of Wnt-reporter keratinocytes transfected with DKK3 siRNA, nontargeting (NT) siRNA or mock-transfected controls at 24 h postirradiation with 4 Gy. f ROS levels (H2DCFDA assay) in Wnt-reporter keratinocytes at 2 and 24 h postirradiation with 4 Gy. MFI: mean fluorescence intensity. Wnt-reporter keratinocytes were treated with different concentrations of the chemical ROS inducer rotenone. g DKK3 levels (ELISA) and h canonical Wnt activity ( Gaussia luciferase assay) in the media at 12 h after treatment. i – l 3D skin model of Dox-induced DKK3-overexpressing (DKK3 ↑ ) Wnt-reporter keratinocytes and unstimulated controls. I DKK3 levels (ELISA) and j canonical Wnt activity ( Gaussia luciferase assay ) in culture media at 72 h after Dox induction. k Representative image of H&E-stained 3D skin sections and quantification of epidermal (ED) thickness. l Representative image and quantification of Ki67-stained sections. The data are presented as the means ± SEMs. Statistical analysis was performed via Student’s t test ( a - d , f , i - l ), one-way ANOVA with Tukey’s multiple comparisons test ( g , h ) or two-way ANOVA with Tukey’s multiple comparisons test ( e ), * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the control or as indicated

Article Snippet: A Gaussia luciferase assay kit (GeneCopoeia, MD, USA) was used for quantification of canonical Wnt (β-catenin) activity in N/TERT-1 keratinocytes and TGF-β–SMAD pathway activity in K4 fibroblasts with the respective reporters.

Techniques: Expressing, Activity Assay, In Vivo, In Vitro, In Situ Hybridization, Irradiation, Control, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Luciferase, Transfection, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Wnt-associated DKK3 in keratinocytes mediates radiation-induced hyperplasia, dermatitis and skin fibrosis

doi: 10.1038/s41392-025-02541-z

Figure Lengend Snippet: DKK3 activates canonical Wnt activity via TGF-β1 in keratinocytes. a – f N/TERT-1 keratinocytes with Dox-inducible DKK3 overexpression and a canonical Wnt reporter were stimulated with Dox (DKK3 ↑ ) for 24 h and compared with unstimulated controls. a Heatmap showing gene expression analysis (nCounter) of substantially and significantly differentially expressed genes (orange indicates upregulation, and blue indicates downregulation by DKK3↑ versus the control; n = 3 biological replicates, >1.4-fold change, p < 0.05). b TGF-β1 mRNA levels (RT‒qPCR). c TGF-β1 protein levels (western blot). d Cells were treated with TGF-β1 protein (rTGF-β1), a small molecule inhibitor of the TGF-β receptor 1 and 2 kinase (TGF-β RKI, LY2109761), or a TGF-β antibody (TGF-β ab) 24 h after Dox-induced DKK3 overexpression. Canonical Wnt activity was measured 24 h later in the culture medium ( Gaussia luciferase assay). RLU: relative light units. e , f 3D-skin models of N/TERT-1 keratinocytes with Dox-induced or unstimulated DKK3 overexpression were treated with TGF-β RKI or TGF-β. e Quantification of Ki67-stained sections and f epidermal thickness in H&E-stained sections at 72 h after TGF-β inhibition. The data are presented as the means ± SEMs. Statistical analysis was performed via Student’s t test ( b , c ) or one-way ANOVA with Tukey’s multiple comparisons test ( d – f ), * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 compared with the control or as indicated

Article Snippet: A Gaussia luciferase assay kit (GeneCopoeia, MD, USA) was used for quantification of canonical Wnt (β-catenin) activity in N/TERT-1 keratinocytes and TGF-β–SMAD pathway activity in K4 fibroblasts with the respective reporters.

Techniques: Activity Assay, Over Expression, Gene Expression, Control, Western Blot, Luciferase, Staining, Inhibition

Journal: Signal Transduction and Targeted Therapy

Article Title: Wnt-associated DKK3 in keratinocytes mediates radiation-induced hyperplasia, dermatitis and skin fibrosis

doi: 10.1038/s41392-025-02541-z

Figure Lengend Snippet: DKK3 induction in keratinocytes triggers a broad profibrotic signaling cascade leading to macrophage polarization and myofibroblast activation in vitro. a DKK3 was knocked down (DKK3 siRNA, NT siRNA refers to nontargeting siRNA controls), overexpressed (DKK3 ↑ ) or left unmodulated (control) in N/TERT keratinocytes. After 7 days, cytokine and growth factor expression in the cells was quantified via RT‒qPCR. The conditioned media were harvested for multiplex antibody-based protein profiling and for the maturation of macrophages from freshly isolated monocytes from human PBMCs for another 7 days. Keratinocyte-conditioned macrophages (KcMф) were characterized on the basis of surface marker expression. K4 fibroblasts were either cocultured with KcMф or cultured in KcMф supernatants for only 2 days. The expression of fibrosis-related genes was evaluated in these conditioned fibroblasts. b Cytokine and growth factor expression (RT‒qPCR) and c multiplex antibody-based protein secretion in DKK3-modulated keratinocytes. d The M2-associated surface markers CD206 and CD163 (FCM) were increased in macrophages conditioned with media from DKK3↑ keratinocytes (KcMф DKK3 ↑ ) and decreased in macrophages conditioned with media from DKK3-knockdown keratinocytes (KcMф DKK3 siRNA). e TGF-β–SMAD pathway activity ( Gaussia luciferase assay) in K4 fibroblasts after coculture with conditioned macrophages. f Fibrosis-related gene expression (RT‒qPCR) in K4 fibroblasts directly cocultured (cell‒cell contact) with conditioned macrophages. Only macrophages cultured with DKK3-overexpressing keratinocytes (KcMф DKK3 ↑ ), but not those cultured with DKK3-knockdown keratinocytes (KcMф DKK3 siRNA), exhibited increased expression of profibrotic genes in K4 fibroblasts. g Fibrosis-related gene expression (RT‒qPCR) in K4 fibroblasts was unaffected by KcMф supernatants (no cell‒cell contact). The data are presented as the means ± SEMs. Statistical analysis was performed via one-way ANOVA with Tukey’s multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 compared with the control or as indicated. Figure 6 was partially created in BioRender. Huber, P. (2025) https://BioRender.com/w3iggj5

Article Snippet: A Gaussia luciferase assay kit (GeneCopoeia, MD, USA) was used for quantification of canonical Wnt (β-catenin) activity in N/TERT-1 keratinocytes and TGF-β–SMAD pathway activity in K4 fibroblasts with the respective reporters.

Techniques: Activation Assay, In Vitro, Control, Expressing, Multiplex Assay, Isolation, Marker, Cell Culture, Knockdown, Activity Assay, Luciferase, Gene Expression