Decreased markers of chondrocyte differentiation and increased apoptosis in the LY294002 treated tibiae. (A) E15.5 tibia organ cultures were analyzed by immunohistochemistry for collagen X expression. The hypertrophic cell area (marked by dashed blue circles), which showed positive collagen X stain, was smaller in the LY294002 treated bones. (B) Tibiae were also analyzed by immunohistochemistry for cyclin-dependent kinase inhibitor 1C (p57), a marker of cell cycle arrest in G1 and chondrocyte differentiation. There was a narrower area of p57 positive cells (showed by brackets) in the LY294002 treated bones. (C) E 15.5 tibiae treated for 6 days with LY294002 shoed an increased number of apoptotic cells in the hypertrophic and mineralized areas (black arrows) as shown by colorimetric TUNEL assay.