Reduced sIL-1ra in fibroblasts potentiates epidermal keratinocyte proliferation. (A) Immunoblot analysis of epidermis from indicated OTCs treated with either vehicle (PBS) or 50 ng/ml exogenous IL-1ra. Cell proliferation was measured using PCNA and cyclin D1. Values below each band were derived as described in Fig. 3 C. (B) Specific knockdown of sIL-1ra in human fibroblasts. (left) Knockdown efficiency was monitored by qPCR and normalized with control ribosomal protein P0. Specificity of knockdown was assessed by the relative expression level of icIL-1ra. (right) Protein expression of sIL-1ra and icIL-1ra as determined by ELISA. (C) Reduced fibroblasts sIL-1ra increase epidermal proliferation. OTCs were constructed using K_CTRL with either control (F_CTRL) or sIL-1ra knockdown (F_sIL-1ra) fibroblasts. H&E, hematoxylin and eosin staining. (top) Ki67, cell proliferation (white arrows); DAPI, nuclear staining. Bars, 40 µm. (bottom) Bars, 20 µm. (left) Mean numbers of proliferating cells were derived as described in Fig. S1. Broken lines denote epidermal–dermal junction. (right) Immunoblot analysis of keratinocytes and fibroblasts extracted from two independent indicated OTCs. Equal amounts of total protein (50 µg) were resolved, electrotransferred, and probed for the indicated proteins. Values below each band represent the mean fold differences in expression level with respect to K_CTRL or F_CTRL extracted from K_CTRL/F_CTRL OTC. **, P < 0.01; ***, P < 0.001. Data are mean ± SD, n = 3.