Figure 7. Keratan sulfate secretion in spheroids as a function of culture conditions. Keratan sulfate was detected by immunoblotting directly from conditioned media after five days of culture of primary spheroids in protein-free media. The first 13 samples were cultured in protein-free DME/F12. Additions were: 1. None; 2. Insulin, transferrin, selenium (ITS); 3. FGF2, 10 ng/ml; 4. TGFβ1, 2 ng/ml; 5. PDGF BB, 10 ng/ml; 6. TGFβ1 and FG2; 7. ITS and FGF2; 8. ITS and TGFβ1; 9. ITS, TGFβ1, and FGF2; 10, ascorbate 2 phosphate 100 μM (A2P); 11. A2P and ITS; 12. A2P and TGFβ;13. A2P and FGF2; 14. keratinocyte serum free medium (KSFM); 15. KSFM with A2P; 16. KSFM with ITS; and 17. KSFM and TGFβ1. In B, the immunoblot shown in A was stripped and reprobed for lumican. Because of its modification with keratan sulfate, lumican appears as a heterogeneous smear and not as a sharp band. C: This panel shows quantitation of the keratan sulfate from A (gray bars) and the ratio between keratan sulfate and lumican from A and C (black bars).