The effect of COMP and TSP-4 on collagen and proteoglycan production and deposition. Chondrocytes were stimulated with COMP (10 µg/mL), TSP-4 (10 µg/mL) alone or in combination with TGF-β1 (0.5 ng/mL) for seven days. (a) Immunofluorescence staining of collagen (Col) I, II, IX, X and XII as well as Safranin-O staining of proteoglycans at day 10. (b) Immunoblot assays of chondrocyte supernatants at day 10. Roti®blue staining of proteins was used as a loading control (LC). (c) Table summarizing the effect of COMP, TSP-4 and TGF-β1 on the total amount of different collagen types (matrix-associated and soluble). The amount of matrix-associated collagens was visually scored from IF staining and combined with band intensities for soluble proteins derived from immunoblots. The number of arrows indicates a strong, moderate or mild effect compared to the control. The direction of the arrows indicates an increase (↑) or decrease (↓) compared to control. No changes are indicated by a minus. The number of arrows indicates strong (three arrows), moderate (two arrows) or mild (one arrow) changes compared to control. (Immunofluorescence staining, n = 3; immunoblot n ≥ 2 per group); unstimulated cells were used as a control; scale bar (collagens) = 100 µm; and (proteoglycans) = 200 µm.