Intact ER stress response in S1Pcko chondrocytes.
(A) RNA from the chondroepiphyseal cartilage of E16.5 WT (lane 1) and S1Pcko (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 S1Pcko (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 S1Pcko 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 S1Pcko cartilage. BiP expression is seen in the ulna, carpal, and metacarpal regions in E16.5 WT (C) and S1Pcko (D) forelimbs. Sdf2l1 expression is seen in the femur of E15.5 WT (E) and S1Pcko (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 S1Pcko 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 S1Pcko 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 S1Pcko embryos were profiled.