Adiporon

FL83B, a hepatocyte cell line derived from a normal liver taken from 15 to 17 day old fetal mice, was purchased from ATCC (Manassas, VA). Cells were maintained in F‐12K medium containing 10% FBS supplemented with 1% penicillin/streptomycin at 37°C. Before treatments with various compounds, cells were washed with PBS, and the medium containing 2% FBS was replaced overnight. The purpose of using 2% FBS medium for the cell treatment was to reduce basal cellular activity and bring all cells to the phase of growth arrest thereby equalizing all cells into the same phase of cell cycle which enables pronounced effect following treatment with growth signals (Zetterberg and Larsson 1985; Van Rechem et al. 2010). It also provides more reproducible experimental conditions (Colzani et al. 2009). Recombinant mouse myostatin protein was obtained from R&D Systems (Minneapolis, MN), AdipoRon was purchased from Sigma‐Aldrich (St Louis, MO), human recombinant insulin is a product from MP Biomedicals (Santa Ana, CA).

Cell viability was assessed using CCK-8 (CK04; Dojindo, Kumamoto, Japan) to identify the cytotoxicity of AdipoRon to NP cells. NP cells were cultured in DMEM with 10% FBS and 1% penicillin for 72 h at 37 °C. After three days of preculture, cells were trypsinized and seeded into 96-well plates at a density of 5 × 10³ cells per well with 100 µL DMEM and incubated at 37 °C for 24 h. Cells were then treated with various concentrations (0–20 µM) of AdipoRon (SML0998; Sigma-Aldrich) at 37 °C for 24 h, and 10 µL CCK-8 solution was added to each well and incubated for another 24 h. The optical density (OD) was measured at 450 nm using a microplate reader (BIO-RAD model 680, Hercules, CA, USA). Cell viability was calculated from the absorbance values (Figure 9a).

For osteoclastogenesis, a total of 1 × 10⁴ RAW 264.7 (ATCC) cells were seeded per well on 24-well plates in 500 μl αMEM containing 5% FCS (Hyclone, GE Healthcare), 1% penicillin-streptomycin (Gibco) as well as 50 μM β-mercaptoethanol (Gibco) and 30 ng/ml murine RANKL (R&D Systems). To study the impact of AdipoRon on osteoclast differentiation, 10 μM AdipoRon (Sigma-Aldrich) were added to the culture medium. Tartrate-resistant acid phosphatase (TRAcP) activity was examined using the leucocyte acid phosphatase kit (Sigma-Aldrich) according to manufacturer’s specifications. TRAcP⁺ cells with ≥3 nuclei were considered as osteoclasts. In addition, 5 × 10³ RAW 264.7 cells were seeded per well in a Nunc Lab-Tek II 8-well Chamber Slide system (ThermoFisher Scientific) in a total of 300 μl osteoclast differentiation medium containing 30 ng/ml RANKL. Fully differentiated osteoclasts were treated with 10 μM AdipoRon for the indicated time points. FACS analysis as well as sorting of mature OCLs for subsequent Western-blot analyses was performed as described previously (Madel et al., 2018 ).

APNp (Sangon Biotech, China) was diluted with 0.9% saline solution to a concentration of 5 mg/mL and intraperitoneally injected at a dose of 20 μg/(g body weight) immediately after artery occlusion. The rats in the AdipoRon group received a regular diet containing AdipoRon. AdipoRon (30 mg/kg, Sigma) was mixed into the standard chow diet for 4 weeks ahead of establishing the MCAO/R model.

Chemicals: AdipoRon (#SML0998; Sigma-Aldrich), gemcitabine (#G6423, Sigma-Aldrich), trypan blue (#T8154; Sigma-Aldrich), propidium iodide (#P4864; Sigma-Aldrich), crystal violet (#C0775; Sigma-Aldrich), PD98059 (#P215; Sigma-Aldrich), dimethyl sulfoxide (DMSO) (A3672; AppliChem), and ethanol absolute anhydrous (308603; Carlo Erba). Antibodies: α-Tubulin (#3873; Cell Signaling Technology), cyclin E1 (#4129; Cell Signaling Technology), p44/42 MAPK (#9102; Cell Signaling Technology), phospho-p44/42 MAPK (#9101; Cell Signaling Technology), cyclin A1 (sc-751; Santa Cruz Biotechnology), vinculin (sc-73614; Santa Cruz Biotechnology), and p27^KIP1 (ab3203; Abcam).