Fig. S1. Atf4 regulates chondrocyte function during endochondral ossification. (A) Immunohistochemistry of growth plate sections of E16 wild-type (WT) and mutant (Atf4^−/−) humeri showing that Atf4 is only detected in WT and not mutant chondrocytes. Atf4-positive cells are brown. Sections are counterstained with Hematoxylin. n=3. Scale bars: 0.05 mm. (B) Alazarin Red S and Alcian Blue stained skeletons showing a delay in the formation of the primary ossification center in E16 Atf4^−/− cervical vertebrae (left; scale bars: 1 mm) and hindlimb digits (right; scale bars: 0.05 mm). (C) Magnified images of the chondrocyte reserve regions of BrdU immunohistochemistry and Hematoxylin-stained E16 and P0 WT and Atf4^−/− growth plate sections. BrdU-positive cells are brown. Scale bars: 0.02 mm. (D,E) Quantification of proliferation rate in reserve chondrocytes represented by the ratio of BrdU-positive cells normalized to total cells in WT and Atf4^−/− E16 (D) and P0 (E) humeri. The mean of the percentage of BrdU-positive cells ± s.e. is shown. The difference between WT and mutant is not statistically significant (P=0.199 and P=0.159 for E16 and P0, respectively).

Fig. S2. Atf4 is not required for PTHrP transcription and type II collagen and Hif1α synthesis. (A) qRT-PCR showing a slight, but not significant, decrease in mRNA levels of Fgf18 in P2 WT and Atf4^−/− cartilage. Data are normalized to expression levels in WT cartilage and 18S rRNA expression (n=3). (B) Atf4 fails to activate the PTHrP promoter in COS cells. DNA co-transfection was performed with a 1.1 kb PTHrP promoter-luciferase construct and an Atf4 expression vector. Statistical analysis by paired Student’s t-test. (C) Gli2 activates the PTHrP promoter in a dose-dependent manner in TMC23 cells. Cells were co-transfected with the same reporter construct as in Fig. S1A and the indicated amount of Gli2 expression vector. *Gli2 transactivated the PTHrP promoter significantly at lowest concentration. (D) Immunohistochemistry for type II collagen (Col2α1) protein on P0 WT and Atf4^−/− humerus growth plate sections. Representative images are displayed, showing similar type II collagen protein levels (brown) in the WT and mutants. Sections are counterstained with Hematoxylin. n=5. Scale bars: 0.2 mm. (E) Immunohistochemistry for Hif1α protein on P0 WT and Atf4^−/− humerus growth plate sections. (a,b) Magnified views of the boxed regions of reserve chondrocyte zones. HIF1α-positive cells are brown. Sections are counterstained with Hematoxylin. n=5. Scale bars: 0.1 mm.