Immunofluorescence studies in human skin and in three-dimensional skin equivalents. (A) Skin sections obtained from a healthy individual were co-immunostained using anti- hemicentin-1 and anti-C17 antibodies (scale bars = 25 μm [left panel], 10 μm [middle panel], and 5 μm [right panel]; hemicentin-1 [HMCN1], green; C17, red; nuclei are stained in blue by DAPI). The experiment was repeated twice. (B–D) Human primary keratinocytes and fibroblasts transfected with HMCN1-specific siRNA (siHMCN1) or control siRNA (siControl) were used to generate three-dimensional organotypic cell cultures. (B) Punch biopsies were obtained from the skin equivalents at day 12, and co-stained for K14 and hemicentin-1 (HMCN1) (scale bar = 25 μm). (C) K14 and hemicentin-1 expression levels were quantified by ImageJ. Result represents mean ± SE of two independent experiments (one-way ANOVA; **P < 0.01, ***P < 0.005). (D) Human primary keratinocytes and fibroblasts transfected with HMCN1-specific siRNA (siHMCN1) or control siRNA (siControl) were used to generate three-dimensional organotypic cell cultures. Punch biopsies were obtained from the skin equivalents at day 12 and stained for C17 (scale bar = 25 μm). The experiment was repeated twice.