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Supplemental Figure S1. Unloading increased ROS production in erythroid cells, granulocytes and B lymphocytes. Bone marrow cells were separated using a flow cytometer and cell surface markers: anti-Ter119 (erythroid cells), CD3e (T lymphocytes), CD45R (B lymphocytes), Gr1 (granulocytes) and F4/80 (macrophages) antibodies. (A) Analyses of fluorescence intensity of the cells using a flow cytometer. (B) The relative fluorescence intensity of the cells. Normal loads are shown as 100% in the bar graphs. (C) The total percentage of the cell number with or without unloading. n = 5 each group. *P <0.05, ***P <0.001. The error bars indicate the SD.

Supplemental Figure S2. Real-time PCR analyses of the mRNA expression in bone marrow cells of mice that underwent normal loading and unloading for 3 and 7 days. (A) Alp: alkaline phosphatase, (B) Runx2: runt-related gene 2, (C) CatK: cathepsin K. n=6–8 each group. *P <0.05. The error bars indicate the SD.

Supplemental Figure S3. Morphological parameters of μCT scanning of the femora of 10-week-old Sod1+/+ and Sod1/ mice subjected to normal loading or unloading for 14 days: (A) bone volume fraction (BV/TV, %), (B) trabecular thickness (Tb.Th, μm), (C) trabecular number (Tb.N, 1/mm), (D) trabecular separation (Tb.Sp, μm). n = 8 each group. The error bars indicate the SD.

Supplemental Figure S4. Bone formation parameters in the hind-limbs of Sod1+/+ and Sod1/ mice that underwent unloading for 14 days compared with those observed in mice that underwent normal loading: (A) mineral apposition rate (MAR, μm/day), (B) mineralized surface per bone surface (MS/BS, %), (C) bone formation rate (BFR, μm3/μm2/day). n = 8 each group. The error bars indicate the SD.

Supplemental Figure S5. Bone resorption parameters in the hind-limbs of Sod1+/+ and Sod1/ mice that underwent unloading for 14 days compared with those observed in mice that underwent normal loading: (A) osteoclast surface per bone surface (Oc.S/BS, %) and (B) osteoclast number per bone surface (N.Oc/B.Pm, 1/mm). n = 8 each group. The error bars indicate the SD.

Supplemental Figure S6. Morphological parameters of μCT scanning of the femora of 10-week-old Sod1/ mice subjected to normal loading or unloading for 14 days with administration of vehicle and VC: (A) bone volume fraction (BV/TV, %), (B) trabecular thickness (Tb.Th, μm), (C) trabecular number (Tb.N, 1/mm), (D) trabecular separation (Tb.Sp, μm). n = 5 each group. The error bars indicate the SD.

Supplemental Figure S7. Morphological parameters of μCT scanning of the femora of 10-week-old wild-type mice subjected to normal loading or unloading for 14 days with administration of vehicle and VC: (A) bone volume fraction (BV/TV, %), (B) trabecular thickness (Tb.Th, μm), (C) trabecular number (Tb.N, 1/mm), (D) trabecular separation (Tb.Sp, μm). n = 5 each group. The error bars indicate the SD.

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