Fig. 6. Activated HIF1 pathway in Pten^Co/Co;Col2a1-Cre growth plates. (A-D,F-M,O-V) Sections of proximal tibial growth plates from E16 mouse embryos (A,B,F,G,J,K,O,P,S,T) and P5 femoral growth plates (C,D,H,I,L,M,Q,R,U,V) were subjected to IHC for HIF1 protein expression (A-D) or in situ hybridization with RNA probes for Vegf (F-I), Pgk (J-M), p21^Cip1 (O-R) or p57^Kip2 (S-V). Note the ectopic activation of HIF1 protein within mutant growth plates (B,D, red arrowheads). (E) Western blot analysis of HIF1 protein accumulation in the nuclei of control or mutant chondrocytes isolated from knee joints. (N) mRNA expression of total Vegf, Vegf isoforms (Vegf₁₂₀, Vegf₁₆₄) and Pgk within control or mutant cartilage from knee joints as revealed by real-time RT-PCR. Error bars represent means ± s.d. from six replicate PCR reactions of a single experiment.^*P<0.05, ^**P<0.01. Scale bar: 200 µm in A,B,F,G,J,K,O,P,S,T; 470 µm in C,D,H,I,L,M,Q,R,U,V.