Figure 2.

Epidermal attenuation of TH signaling in D2KO mice reduced VEGF-A-induced angiogenesis. (A) Western blot analysis of CD31 and VEGF-A expression in SCC skin lesions of mice treated with DMBA-TPA for the indicated times (n = 8). Quantification of the single protein versus tubulin levels is represented by diagrams (bottom). (B) Western blot analysis of CD31 and VEGF-A expression in skin lesions of D2WT and sD2KO mice (n = 15 for both groups) treated with DMBA/TPA for 20 weeks. Quantification of the single protein versus tubulin levels is represented by diagrams (bottom). (C) mRNA expression of VEGF-A, VEGFR1 and VEGFR2 in skin lesions of D2WT and D2KO mice (n = 15 for both groups). (D) CD31, VEGF-A, VEGFR2 and pVEGFR2 immunostaining were performed on paraffin-embedded sections of skin lesions from D2WT and sD2KO mice (n = 10 for both groups). Scale bars represent 50 μm. Quantification of single protein levels is represented by histograms (bottom). (E) CD31⁺ endothelial population and the CD45⁺ lymphocyte population were analyzed by flow cytometry in SCC lesions of D2WT (n = 6) and sD2KO (n = 6) mice (bottom). Relative quantification of CD31⁺ and CD45⁺ positive cells. Results are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. (F) Proteome profiler array of differentially regulated angiogenic proteins in D2WT and sD2KO skin lesions. Duplicate spots: 1, Amphiregulin; 2, Coagulation factor III; 3, Endostatin/Collagen XVIII; 4, IGFBP-2; 5, IL-1α, 6; CXCL1; 7, MIP-1α; 8, MMP-3; 9, CXCL4; 10, PIGF-2; 11, SDF-1; 12, Thrombospondin-2. One representative experiment of 4 replicates is shown. (G) Relative protein expression was obtained by comparing the mean pixel density of each factor in sD2KO group with each factor in the D2WT group (log₁₀ (D2KO-D2WT)); up-regulated factors are shown in red, down-regulated factors in green.