**Appendix A**

**Figure A1.** The protein expression of angiotensin II type 1 receptor (AT1R) in the joint tissues was increased in tumor necrosis factor-transgenic (TNFtg) mice. (**A**) Western blot analysis of AT1R expression in the fore paw tissues. The fore paw samples were obtained from 16-week-old wild-type (WT) and TNFtg mice. (**B**) Immunohistochemistry staining images of AT1R. The expression of AT1R in the left ankle joint specimens was determined. Original magnification ×40. A tissue specimen processed without the primary antibody is presented as a negative control. Detailed methods are described in the Supporting Information.

**Figure A2.** Systolic blood pressure in wild-type (WT) and tumor necrosis factor-transgenic (TNFtg) mice administered angiotensin II (Ang II) and water (H2O). Ang II was administered by osmotic pumps to WT and TNFtg mice from 12 to 16 weeks of age. Values are the mean ± SEM. \*, *p* < 0.05 (WT (Ang II) vs. WT (H2O)). n.s., not significant (TNFtg (Ang II) vs. TNFtg (H2O)).

**Figure A3.** Trabecular and cortical bone parameters in wild-type (WT) and tumor necrosis factor-transgenic (TNFtg) mice administered angiotensin II (Ang II) and water (H2O). Ang II was administered by osmotic pumps to WT (*n* = 6) and TNFtg (*n* = 7) male mice from 12 to 16 weeks of age. H2O was administered by osmotic pumps to WT (*n* = 6) and TNFtg (*n* = 4) male mice as controls. (**A**) Trabecular bone parameters of the tibia. (**B**) Cortical bone parameters of the tibia midshaft. (**C**) Trabecular bone parameters of the spine (fifth lumbar vertebra). Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tt.Ar, total cross-sectional area inside the periosteal envelope; Ct.Ar, cortical cross-sectional area. Values are the mean ± SEM. n.s., not significant. \*, *p* < 0.05.

**Figure A4.** Trabecular and cortical bone parameters of the tibia and spine. Tumor necrosis factor-transgenic (TNFtg) mice were crossed with AT1R-deficient (AT1R−/−) mice. Wild-type (WT; *n* = 9), AT1R−/<sup>−</sup> (*n* = 7), TNFtg (*n* = 12), and TNFtg AT1R−/<sup>−</sup> (*n* = 8) male mice were analyzed at the age of 16 weeks. (**A**) Trabecular bone parameters of the tibia. (**B**) Cortical bone parameters of the tibia midshaft. (**C**) Trabecular bone parameters of the spine (fifth lumbar vertebra). Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tt.Ar, total cross-sectional area inside the periosteal envelope; Ct.Ar, cortical cross-sectional area. Values are the mean ± SEM. n.s., not significant. \*, *p* < 0.05.

**Figure A5.** Effect of angiotensin (Ang II) on osteoclast differentiation. Primary mouse bone marrow cells were isolated from the long bones of 10-week-old wild-type mice. Non-adherent bone marrow cells were seeded on 48-well plates and incubated for 2 days with macrophage colony-stimulating factor (M-CSF; 25 ng/mL). After pre-culture for 2 days, yielded bone marrow-derived macrophages were stimulated with RANKL (50 ng/mL) and Ang II at the indicated concentrations for an additional 3 days in the presence of M-CSF. The formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (TRAP + MNCs) was visualized by TRAP staining. (**A**) Representative TRAP staining images. (**B**) The number of osteoclasts. TRAP + MNCs with three or more nuclei were counted as osteoclasts. Bone marrow-derived macrophages were co-cultured with neonatal calvarial osteoblasts in the presence of 10 nM 1α, 25-dihydroxyvitamin D3, and 1 μM prostaglandin E2. The cells were stimulated with Ang II (0.01–10 μM) for 7 days. TRAP + MNCs were visualized by TRAP staining. (**C**) Representative TRAP staining images. (**D**) The number of osteoclasts. Values are the mean ± SEM. n.s., not significant. \*, *p* < 0.05 vs. non-treated control. Detailed methods are described in the Supporting Information.

#### **References**


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