*4.2. Ang II Infusion Model*

Twelve-week-old WT and TNFtg male mice were randomly divided into two groups that were infused with either water (H2O) or Ang II, which was dissolved in H2O. Ang II was administered by osmotic pumps to WT (*n* = 6) and TNFtg mice (*n* = 7) from 12 to 16 weeks of age. H2O was administered by osmotic pumps to WT (*n* = 6) and TNFtg mice (*n* = 4) as controls. The mice were anesthetized, and an osmotic pump containing 100 μL of either H2O or Ang II (Sigma-Aldrich, St. Louis, MO, USA) was implanted subcutaneously as previously described [22,25]. Ang II was continuously infused at a dose of 1.44 mg/kg/day from 12 to 16 weeks of age. Arterial blood pressure was measured by the tail-cuff method with a pulse transducer (BP98-A; Softron, Tokyo, Japan), as reported [26]. Mice were

monitored for signs of arthritis in a blinded manner, and each limb was individually scored on a scale of 0–4. Scores were assigned based on the extent of erythema or swelling present in each limb, assigning a maximum score of 16 per mouse, as described previously [27,28]. Mice were monitored until the age of 16 weeks, and then serum, hind limb, and spine (the fifth lumbar vertebra) samples were collected.

#### *4.3. Micro-Computed Tomography (CT) Analysis*

Bone samples were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline for 2 days, and PFA-fixed bone samples were immersed in 70% ethanol. Three-dimensional microarchitecture of the talus, tibia, and spine was evaluated by using a micro-CT system (Ele Scan mini; Nittetsu Elex, Tokyo, Japan) with an X-ray energy of 45 kVP (145 μA), as described previously [29,30]. The voxel resolution of all bone images was 15 μm. The bone properties of the tibia and the fifth lumbar vertebra, and bone erosion of the ankle (talus bones) were analyzed using analysis software (TRI/3D-BON; Ratoc System Engineering Co. Ltd., Tokyo, Japan). The analyzed region of the tibia trabecular bone comprised 67 slices of secondary spongiosa adjacent to the primary spongiosa (starting 0.5 mm from the distal border of the growth plate), that of the vertebra comprised the entire fifth lumbar vertebral body area (approximately 140 slices), and that of the tibia cortical bone comprised 33 slices of the midshaft (1 mm proximal region from the tibiofibular junction). The micro-CT parameters of the tibia and spine were described according to international guidelines [31]. The talus bones were evaluated using BV and Ev/Rpv for quantitative measurements of bone erosion [32]. Ev/Rpv on the whole talus was calculated automatically with the software according to the software program (TRI/3D-BON). We set the concave surface search range up to 0.15 mm, and the absorption surface extraction radius of curvature was 960 μm or less as described previously [33].

#### *4.4. Histological Analysis*

The hind limbs were decalcified in 10% EDTA (pH 7.2) at 4 ◦C for 4 weeks and subsequently embedded in paraffin. Sections (3 μm) were stained with hematoxylin and eosin (H&E) and Safranin O. The severity of inflammation and cartilage damage around the talus bone was scored on a scale of 0–4 under blinded conditions as described previously [27,28]. TRAP staining was performed to visualize osteoclast formation, and the sections were counterstained with methyl green. Histological analyses were performed using a BZ-X analyzer (Keyence, Osaka, Japan). The eroded surface per bone surface (ES/BS) and the number of osteoclasts per bone surface (N.Oc/BS) around the taluses were determined.

#### *4.5. Real-Time Quantitative Polymerase Chain Reaction (qPCR)*

qPCR was performed as described previously [30,34]. Total RNA was extracted from the right ankle joint using RNAiso Plus (Takara Bio, Shiga, Japan) and solubilized in ribonuclease (RNase)-free water. Complementary DNA (cDNA) was synthesized using the Prime Script RT reagent Kit (Takara Bio). qPCR reactions were performed using SYBR Green PCR Master Mix (Takara Bio) with the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression levels relative to *Gapdh* were calculated by the ΔΔCt method and normalized to control samples obtained from the WT mice. The qPCR analysis used the following primers: 5 -taccagctctgcggctct-3 and 5 -gccagccatttt ataccaatct-3 for *Agtr1a* (AT1R); 5 -atcaagaaggtggtgaagca-3 and 5 -gacaacctggtcctcagtgt-3 for *Gapdh*. All qPCR reactions yielded products with single peak dissociation curves.
