Type I collagen synthesis by human gingival fibroblasts (hGF) treated with or without 1 µM shikonin. Immunostaining for type I collagen using an anti-type I collagen monoclonal antibody. (A) Intensity of fluorescence immunostaining without (control) or with shikonin (** p < 0.01 vs. control). (B) Cells were incubated with fluorescently labeled secondary antibody and stained with DAPI (scale bar= 500 μm). (C) After incubation of the cells with the secondary antibody, chromogenic immunostaining was performed, and images were obtained by using a single-lens reflex camera. (D) Type I collagen in the cell culture supernatant after treatment for 1 week was measured by using ELISA. (* p < 0.05, control vs. 1 µM shikonin). (E,F) Effects of shikonin on the gene expression of VEGF and FN in stimulated hGFs incubated with 1 µM shikonin for 24 h prior to cell harvest. The gene expression of VEGF and FN was determined by using PCR. (** p < 0.01, control vs. 1 µM shikonin). (G) Protein expression of FN was evaluated by immunoblotting analysis. (H) The quantification was performed using image analysis software. (** p < 0.01 vs. control).