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Supplementary Material

DEV057802 Supplementary Material

Files in this Data Supplement:

  • Supplemental Table S1 -
  • Supplemental Table S2 -
  • Supplemental Table S3 -
  • Supplemental Figure S1 -

    Fig. S1. 11Enh-Cre; Sox9flox/+ mice. Sox9flox/flox mice were mated with 11Enh-Cre transgenic mice to generate 11Enh-Cre; Sox9flox/+. Then, 11Enh-Cre; Sox9flox/+ and Sox9flox/flox mice were intercrossed. Pregnant mice were sacrificed and embryos were processed for histological analysis. Sox9flox/+ embryos were used as controls. (A) The Sox9flox/+ heterozygotes that harbor 11Enh-Cre were recovered with the expected Mendelian frequency. (B) 11Enh-Cre; Sox9flox/+ mice developed dwarfism. Scale bar: 3 cm. (C) Eighty-five percent of 11Enh-Cre; Sox9flox/+ mice were viable beyond 6 months of age. (D) 11Enh-Cre; Sox9flox/flox conditional knockout mice were recovered from 12.5 to 16.5 dpc with the expected Mendelian frequency. (E) Skeleton of Sox9flox/+, 11Enh-Cre; Sox9flox/+ and 11Enh-Cre; Sox9flox/flox embryos at 16.5 dpc. The skeletons of 11Enh-Cre; Sox9flox/+ embryos contained straight long bones and appeared to be almost normal. Scale bar: 2 mm. (F) Forelimb skeleton and calvariae of Sox9flox/+, 11Enh-Cre; Sox9flox/+ and 11Enh-Cre; Sox9flox/flox embryos at 16.5 dpc. Scale bar: 2 mm. (G) Relative length of humerus, radius, femur and tibia of Sox9flox/+, 11Enh-Cre; Sox9flox/+ and 11Enh-Cre; Sox9flox/flox embryos at 16.5 dpc. The average length of each skeletal component of Sox9flox/+ embryos was set at 1. Data (B,G) are mean + s.d. **, P<0.01.

  • Supplemental Figure S2 -

    Fig. S2. Apoptosis and differentiation of 11Enh-Cre; Sox9flox/flox humeral chondrocytes. (A) Semi-serial sections of the central region of humeral cartilage at 12.5 dpc were stained with Safranin O, subjected to the TUNEL assay, and immunostained with anti-cleaved caspase 3. Scale bar: 100 µm. (B) Positive controls in TUNEL assay. TUNEL-positive cells were detected in the interdigital regions of paws of both the control Sox9flox/+ mice and the 11Enh-Cre; Sox9flox/flox conditional knockout mice at 13.5 dpc. Scale bar: 100 µm. (C) Semi-serial sections of proximal humerus at 15.5 dpc were stained with HE and hybridized with Col2a1, Ihh, Col10a1 and Mmp13 cRNA probes. Scale bar: 200 µm.

  • Supplemental Figure S3 -

    Fig. S3. 11Prom-Cre; Sox9flox/+ mice. Sox9flox/flox mice were mated with 11Prom-Cre transgenic mice to generate 11Prom-Cre; Sox9flox/+. Then, 11Prom-Cre; Sox9flox/+ and Sox9flox/flox mice were intercrossed. Pregnant mice were sacrificed and embryos were processed for histological analysis. Sox9flox/+ embryos were used as controls. (A) The Sox9flox/+ heterozygotes that harbor 11Prom-Cre were recovered with the expected Mendelian frequency. (B) Three-week-old 11Prom; Sox9flox/+ mice developed normally. Scale bar: 3 cm. (C) 11Prom-Cre; Sox9flox/flox conditional knockout mice were recovered from 12.5 to 16.5 dpc with the expected Mendelian frequency. (D) Skeleton of Sox9flox/+, 11Prom-Cre; Sox9flox/+ and 11Prom-Cre; Sox9flox/flox embryos at 16.5 dpc. Scale bar: 2 mm. (E) Forelimb skeleton and calvariae of Sox9flox/+, 11Prom-Cre; Sox9flox/+ and 11Prom-Cre; Sox9flox/flox embryos at 16.5 dpc. Scale bar: 2 mm. (F) Relative length of humerus, radius, femur and tibia of Sox9flox/+, 11Prom-Cre; Sox9flox/+ and 11Prom-Cre; Sox9flox/flox embryos at 16.5 dpc. The average length of each skeletal component of Sox9flox/+ embryos was set at 1. **, P<0.01. (G) Vertebral bodies of Sox9flox/+ and 11Prom-Cre; Sox9flox/flox embryos at 16.5 dpc. Frontal view. Scale bar: 1 mm. Data (B,F) are mean + s.d.

  • Supplemental Figure S4 -

    Fig. S4. Marker gene expression and proliferation of chondrocytes in 11Prom-Cre; Sox9flox/flox mice. (A) Semi-serial sections of proximal humerus at 14.5 dpc were stained with Hematoxylin and Eosin (HE), immunostained with anti-Sox9 antibodies, and hybridized with Col2a1, Col11a2, Ppr and Col10a1 cRNA probes, as indicated. Metaphyseal flat proliferative chondrocytes in Sox9flox/+ humerus are outlined (red line, left panels). Metaphyseal proliferative chondrocytes outlined in 11Prom-Cre; Sox9flox/flox humerus (red line, right panels) did not express Sox9, indicating that they are Sox9floxdel/floxdel proliferative chondrocytes. 11Prom-Cre; Sox9flox/flox humerus was characterized by a reduced population of metaphyseal proliferative chondrocytes (red outlined areas) and by the absence of Col10a1 expression. Scale bar: 200 µm. (B) Semi-serial sections of the central region of humeral cartilage at 13.5 dpc were stained with HE., subjected to the TUNEL assay, and immunostained with anti-cleaved caspase 3. Scale bar: 100 µm. (C) 11Prom-Cre; Sox9flox/flox humerus showed a significantly increased number of BrdU-positive cells in the bone collar at 13.5 dpc (P<0.01) and a significantly decreased number of BrdU-positive cells in chondrocytes at 14.5 dpc (P<0.01). Magnifications of boxed regions in the top panels are shown in the second and third rows, respectively. Scale bar: 100 µm. Error bars indicate mean + s.d. (n=4).

  • Supplemental Figure S5 -

    Fig. S5. SOX9 knockdown in non-chondrocytic cell lines and signaling molecules in Sox9 conditional knockout mice. (A) The transfection efficiencies of control GFP plasmid were 60-70% for SW1353 cells, Hela cells and Saos2 cells. Scale bar: 200 µm. (B) Western blots showed that SW1353 cells expressed SOX9 proteins, whereas Hela cells and osteosarcoma Saos2 cells did not express SOX9. (C) Transfection with SOX9-b or SOX9-c siRNA did not affect the caspase 3/7 activities in Hela cells and Saos2 cells. (D) Transfection with SOX9-b or SOX9-c siRNA did not affect the amount of cleaved DNA-histone complexes in Hela cells and Saos2 cells. (E) Semi-serial sections of Sox9flox/+ and 11Enh-Cre; Sox9flox/flox samples in Fig. 1B immunostained with anti-phospho-Akt antibody. Scale bar: 50 µm. (F) Semi-serial sections were stained with Safranin O-Fast Green-Iron Hematoxylin (SO) and immunostained with the antibodies indicated. In Sox9flox/+ cartilage flat proliferative chondrocytes are outlined (red line, left panels). In 11Prom-Cre; Sox9flox/flox cartilage, Sox9floxdel/floxdel proliferative chondrocytes are outlined (red line, right panels), as suggested in Fig. S3A. There were no obvious differences in the immunoreactivity against anti-phospho-ATF2 (activating transcription factor 2), anti-phospho-SAPK (stress-activated protein kinase)/JNK (Jun N-terminal kinase) and anti-phospho-p38 MAPK (p38 mitogen-activated protein kinase) antibodies between 11Prom-Cre; Sox9flox/flox mice and Sox9flox/+ mice. Scale bar: 100 µm. Data (C,D) are mean + s.d.

  • Supplemental Figure S6 -

    Fig. S6. Inactivation of Pten partially recovers the phosphorylation of Akt in Sox9floxdel/floxdel chondrocytes. Sox9flox/flox were mated with Ptenflox/flox to generate Sox9flox/+; Ptenflox/+ mice. Sox9flox/+; Ptenflox/+ were mated with Sox9flox/+; Ptenflox/+ to generate Sox9flox/flox; Ptenflox/flox and Sox9flox/flox; Ptenflox/+ mice. 11Prom-Cre were mated with Sox9flox/flox; Ptenflox/flox to generate 11Prom-Cre; Sox9flox/+; Ptenflox/+ mice. 11Prom-Cre; Sox9flox/+; Ptenflox/+ were mated with Sox9flox/flox; Ptenflox/+ to generate 11Prom-Cre; Sox9flox/flox; Ptenflox/flox mice. Semi-serial sections of proximal humerus at 14.5 dpc were stained with HE, immunostained with anti-Sox9, anti-Pten, anti-phospho-Akt and anti-Akt antibodies, and hybridized with Col2a1 and Col10a1 cRNA probes, as indicated. Patterns of Sox9 expression were similar between 11Prom-Cre; Sox9flox/flox; Pten+/+ mice and 11Prom-Cre; Sox9flox/flox; Ptenflox/flox mice (second row). Loss of Sox9 expression in proliferative chondrocytes indicates deletion of the Sox9 gene (Sox9floxdel/floxdel proliferative chondrocytes). The extent of zones of flat proliferative chondrocytes in Sox9flox/+; Ptenflox/+ mice or Sox9floxdel/floxdel proliferative chondrocytes are indicated by red lines. Pten proteins were reduced in Sox9floxdel/floxdel proliferative chondrocytes of 11Prom-Cre; Sox9flox/flox; Ptenflox/flox mice, suggesting the deletion of Sox9 and Pten genes. Col2a1, the target gene of Sox9, was also decreased in this region. This suggests that the mild phenotype of 11Prom-Cre; Sox9flox/flox; Ptenflox/flox mice, as compared with that of 11Prom-Cre; Sox9flox/flox mice, was not due to the reduced recombination frequency in Sox9flox/flox when four alleles of loxP sites are present, but was due to rescue caused by Pten inactivation. Phosphorylation of Akt was reduced in Sox9floxdel/floxdel; Pten+/+ proliferative chondrocytes and recovered in Sox9floxdel/floxdel; Ptenfloxdel/floxdel proliferative chondrocytes. Panels in Fig. 5D are magnifications of zones of these flat proliferative chondrocytes or Sox9floxdel/floxdel proliferative chondrocytes. Scale bar: 100 µm.