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jbmr1971-sm-0001-SuppFig-S1.tif1780K

Figure S1. Differentiation stages during the primary calvarial osteoblast culture. Primary calvarial osteoblasts were isolated from neonatal mice and cultured in alpha-MEM containing 10% Heat-inactivated FBS. (A) Alkaline phosphatase staining was performed at indicated time points. (B) RNA were extracted and qPCR was performed to analyze the mRNA expression level of alkaline phosphatase. *p<0.05, n=3. Data are mean + SD.

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Figure S2. Body weight of control and Osx-CKO mice. (A) Representative images of control and Osx-CKO mice at birth (P0). (B) Body weight of female mice at indicated time points. (C) Body weight of male mice at indicated time points. *p<0.05, n= 5–41 per group. Data are mean ± SD.

jbmr1971-sm-0003-SuppFig-S3.tif297K

Figure S3. FIP200 deletion in Osx-CKO mice. Lysates were extracted from heart, liver, calvaria, and growth plate (distal femur and proximal tibia) of newborn control and FIP200 Osx-CKO mice. Lysates were subjected to immunoblot analysis with indicated antibodies. Cell lysates extracted from the FIP200+/+ and FIP200-/- MEF cells were used as positive and negative control respectively.

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Figure S4. FIP200 deletion does not affect endochondral bone development at birth. Skeleton preparation of control (upper panel) and Osx-CKO (lower panel) mice at birth. (A) There was comparable arm skeletal development (a: humerus b: radius c: ulna). (B) There was comparable leg skeletal development (d: femur e: fibula f: tibia). (C) There was no obvious abnormality in thoracic cage development (arrows point to vertebrae and * indicates rib). Scale bar = 3mm.

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Figure S5. FIP200 deletion in osteoblasts leads to decreased bone mass in vertebrae. Trabecular parameters were determined by MicroCT for the third lumbar vertebra of one month old Osx-CKO and control female mice. (A) Bone volume/tissue volume (BV/TV); (B) Trabebular number (TbN); (C) Trabecular thickness (TbTh); and (D Trabebular spacing (TbSp). *p<0.05, n= 8 per group. Data are mean + SD.

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Figure S6. FIP200 deletion in osteoblasts leads to osteopenia and compromised bone mechanical properties in adult male mice. (A-H) Trabecular and cortical parameters were determined by MicroCT for the femurs from one month (1M) to one year (1Y) old Osx-CKO male mice: (A) Bone volume/tissue volume (BV/TV); (B) Trabecular number (TbN); (C) Trabecular thickness (TbTh); (D) Trabecular spacing (TbSp); (E) Cortical bone thickness; (F) Cortical bone area; (G) Cortical bone outer perimeter; (H) Cortical bone inner perimeter. (I-L) Four-point bending test with femurs: (I) Yield load; (J) Ultimate load; (K) Stiffness; (L) Energy to failure. For each group, n=5-7, *p<0.05. Data are mean + SD.

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Figure S7. MicroCT analysis of femur and third lumbar vertebra in one month old male wild type and Osx-Cre mice. (A-H) Trabecular and cortical parameters for the femurs: (A) Bone volume/tissue volume (BV/TV); (B) Trabebular number (TbN); (C) Trabecular thickness (TbTh); (D) Trabebular spacing (TbSp); (E) Cortical bone thickness; (F) Cortical area; (G) Cortical bone outer perimeter; (H) Cortical bone inner perimeter. (I-L) Trabecular parameters for the third lumbar vertebra: (I) Bone volume/tissue volume (BV/TV); (J) Trabebular number (TbN); (K) Trabecular thickness (TbTh); (L) Trabebular spacing (TbSp). n=6-7, *p<0.05. Data are mean + SD.

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Figure S8. FIP200 deletion in osteoblasts by Col2.3-Cre leads to decreased bone mass and deficient autophagy. (A-D) Trabecular parameters were determined by MicroCT for the femurs of one month old Col2.3-CKO and control female mice: (A) Bone volume/tissue volume (BV/TV); (B) Trabebular number (TbN); (C) Trabecular thickness (TbTh); and (D Trabebular spacing (TbSp).*p<0.05, n= 5–8 per group. Data are mean + SD. (E) Lysates were extracted from heart, liver, and femur of one month old control and FIP200 Col2.3-CKO mice. Lysates were subjected to immunoblot analysis with indicated antibodies. Cell lysates extracted from the FIP200+/+ and FIP200-/- MEF cells were used as positive and negative control respectively. (F) Primary calvarial osteoblasts were isolated from control or Col2.3-CKO neonatal mice and cultured in complete medium for 3 days. Cell lysates were subjected to immunoblot analysis with indicated antibodies. (G) Control and FIP200 null primary calvarial osteoblasts were cultured in the complete or starvation medium for 3h with or without 100 µM Chloroquine. The cell lysates were subjected to immunoblot analysis with indicated antibodies. (H) Control and FIP200 null primary calvarial osteoblasts were cultured towards terminal differentiation. Early osteoblast differentiation was evaluated by Alkaline phosphatase staining at day 7 and terminal differentiation was evaluated by Alizarin red staining at day 21.

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Figure S9. FAK inhibitor and FAK/Pyk2 inhibitor can not rescue the deficient osteoblast differentiation due to FIP200 deletion. Bone marrow cells were collected from 6-8 weeks old control or Osx-CKO mice and subject to osteoblast differentiation. Starting from day 7, vehicle (DMSO) or FAK inhibitor (FAKi), or FAK/Pyk2 dual inhibitor (FAKi/Pyk2i) at indicated concentrations was added to the culture till end of the experiment. Alkaline phosphatase staining and Alizarin red staining were performed at day 14 and day 21 respectively.

jbmr1971-sm-0010-SuppData.docx34KSupporting Information

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