PTEN deficiency causes dyschondroplasia in mice by enhanced hypoxia-inducible factor 1 signaling and endoplasmic reticulum stress

DEV028118 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Histological characterization of chondrodysplasia in Pten^Co/Co;Col2a1-Cre mice. (A,B) H&E-stained sections of normal growth plate cartilage (A) or cartilaginous nodule (B). Chondrocytes within the nodule were disorganized (B, arrowheads). (C,D) Alcian Blue and Sirius Red double-stained sections of cartilaginous nodules. Cartilaginous neoplasms (blue) were limited at their periphery by a lamella of well-mineralized trabecular bone (rose). Myxoid changes manifested as fraying of the matrix were also seen (D, asterisk). Scale bar: 100 µm

• Supplemental Figure S2 -

  Fig. S2. Normal proliferation in embryonic growth plate cartilages of Pten^Co/Co;Col2a1-Cre mice. (A) BrdU detection was performed on sections of control or mutant growth plates from E13.5, E15.5 and E17.5 embryos. No significant differences were observed between mutant and control growth plates except for a small number of BrdU-negative chondrocytes at the lower zone of proliferation (A, red arrowheads). (B) The percentage of BrdU-positive chondrocytes in the whole proliferating zone was determined. Mean ± s.d. of four sections from two mice of each genotype is shown. Hours for BrdU labeling: 1.5. Scale bar: 114 µm (E13.5); 130 µm (E15.5); 200 µm (E17.5).

• Supplemental Figure S3 -

  Fig. S3. Progressively delayed chondrocyte differentiation within Pten^Co/Co;Col2a1-Cre growth plates. Control or mutant tibial growth plate sections from E13.5 (A-D), E16 (E-J) embryos or P1 (K-P) newborn mice were hybridized in situ with RNA probes for Ppr (A,B,E,F,K,L), Col10a1 (C,D,G,H,M,N), or subjected to von-Kossa staining (I,J,O,P). Red bars indicate layers of terminal hypertrophic chondrocytes. Scale bar: 350 µm in A-D; 240 µm in E-H; 200 µm in I,J,O,P; 333 µm in K-N.

• Supplemental Figure S4 -

  Fig. S4. Remarkable ER stress in Pten^Co/Co;Col2a1-Cre chondrocytes cultured under hypoxia. Electron microscopy of control (A,C) or mutant (B,D) chondrocytes cultured under normoxic (A,B) or hypoxic (C,D) conditions for 5 days. Note that only mutant chondrocytes cultured under hypoxia suffered greatly from distended and fragmented ERs (D). Scale bar: 4 µm.

• Supplemental Figure S5 -

  Fig. S5. No increase in apoptosis in Pten^Co/Co;Col2a1-Cre growth plates. (A,B) TUNEL assay was performed on P5 proximal humeral growth plate sections. Apoptotic cells were found at the chondro-osseous junction (A,B, black arrowheads) and in the bone marrow cavity (A,B, red arrowheads) of control (A) and mutant (B) mice. Scale bar: 200 µm.

• Supplemental Figure S6 -

  Fig. S6. Elevated HIF1α pathway and ER stress in Pten^Co/Co;Col2a1-Cre growth plates. Sections of P1 costal growth plates were subjected to IHC for HIF1α protein expression (A,B), or to ISH for mRNA expression with probes for Vegf (C,D), Pgk (E,F), p21^Cip1 (G,H), p57^Kip2 (I,J) or BiP (K,L). Growth plate sections in A and B were from the sixth ribs; the other sections were from the sixth to seventh ribs. Note the ectopic activation of HIF1α protein (B, red arrowhead) and increased mRNA of Vegf, Pgk, p21^Cip1 and BiP (D,F,H,L, yellow arrowheads) right above the hypertrophic zone of mutant growth plates. Scale bar: 400 µm.

• Supplemental Figure S7 -

  Fig. S7. Abundant angiogenesis in postnatal growth plate cartilages of Pten^Co/Co;Col2a1-Cre mice. (A,B) Whole-mount tissue immunostaining for vascular network upon the epiphyseal condyles of P3 distal femurs using anti-CD31 antibody. The density of the vessel network represented as a percentage of the vascularized surface (C) as well as the total length of vessels (D) laying over the lateral femoral condyles (A,B, boxed area) were increased in mutant mice. Mean ± s.d. of eight samples from four mice of each genotype. **P<0.01. (E,F) H&E staining of P25 growth plates. Blood vessels invaded into mutant cartilage at the region between the resting and hypertrophic zone (F, arrowheads). The inset shows a higher magnification view of the indicated blood vessel (F, red arrowhead) containing blood cells and endothelial cells. Scale bar: 220 µm in A,B; 85 µm in E,F; 30 µm in inset.