Intact ER stress response in S1P^cko chondrocytes.

(A) RNA from the chondroepiphyseal cartilage of E16.5 WT (lane 1) and S1P^cko (lane 2) used in microarray analysis were converted to cDNA and amplified by XBP-1 PCR primers to identify spliced (s) XBP-1 mRNA in a 4.8% polyacrylamide gel. (B) XBP-1 was amplified by PCR using XBP-1 PCR primers 3S and 12AS with cDNA derived from E16.5 WT (lanes 1, 3, 5) and S1P^cko (lanes 2, 4, 6) epiphyseal cartilage RNA and the PCR product restriction digested with PstI which selectively cuts the un-spliced (u) XBP-1 mRNA, and the resulting products visualized in a 2% agarose gel. In lanes 1 and 2, the cDNA used are from the same embryos used in (A) and for genome-wide expression profiling. Each lane in lanes 3–6 show analyses from RNA pooled from the chondroepiphysis of two different embryos. Thus a total of five WT and five S1P^cko embryos were analyzed. The inverse of the gels are shown in both (A) and (B) to enhance visualization of spliced XBP-1 mRNA. (C–F) Expression signaling for two ATF6-driven genes, BiP and Sdf2l1, as seen by in situ hybridization analyses in WT and S1P^cko cartilage. BiP expression is seen in the ulna, carpal, and metacarpal regions in E16.5 WT (C) and S1P^cko (D) forelimbs. Sdf2l1 expression is seen in the femur of E15.5 WT (E) and S1P^cko (F). Bar: 10 µm. (G) A scatter plot generated from quantitative real-time PCR analysis in the murine UPR RT² Profiler PCR Array system comparing the relative expression of 84 genes between WT and S1P^cko chondrocytes. A log transformation plot is shown in which the relative gene expression level of each gene (2^-ΔCt) in WT is plotted against the corresponding value in S1P^cko to indicate fold changes (2^-ΔΔCt). The black line indicates no fold change (fold change of 1). The pink lines indicate a fold change of 2 (gene expression threshold). All genes within these two lines are considered to be similar in expression to WT. Only Ddit3 or Insig-1 were significantly differentially expressed among the 84 genes profiled. A total of four WT and four S1P^cko embryos were profiled.