Anti foxo3a

HOB and MC3 T3‐E1 cells were cultured on a coverslip. When cell confluence reached 95%–100%, the cells were washed with PBS thrice (10 min each time). After fixation in 4% formaldehyde (Aladdin) for nearly 20 min at room temperature, the cells were washed with PBS again. Then, the cells were permeabilized with 0.4% Triton X‐100 (Thermo Fisher) for nearly 1 h and blocked in BSA buffer for about 1 h at room temperature. Following this, the cells were incubated with the primary antibodies anti‐FOXO3a (Abcam, 5 µg/ml) and anti‐TRIM33 (Abcam, 1 µg/ml) for nearly 24 h at 4 °C and then incubated with FITC‐conjugated secondary antibody for about 1 h in the dark. The cells were then washed with PBS and stained with DAPI. The coverslips were sealed with glycerin and observed under a fluorescence microscope (Olympus, Japan).

Cell lysates were collected in cell lysis buffer (Beyotime, Shanghai, China) containing a complete protease inhibitor tablet (Roche, Basel, Switzerland) and 1 mM phenylmethanesulfonylfluoride. The protein levels were quantified with a Lowry kit (Bio-Rad, Berkeley, CA, USA) according to the manufacturer’s instructions. PVDF membranes (Millipore, Billerica, MA, USA) were incubated with the following antibodies: anti-PKM2 (SAB, Washington, MD, USA); anti-PKM1 (Proteintech); anti-HIF-1α (Cell Signaling Technology (CST), MA, USA); anti-FoxO3a (Abcam, Cambridge, UK); anti-procaspase-8, anti-procaspase-3, anti-PARP and anti-Beclin-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-cyclin D1 (CST); anti-actin (CWBIO, Beijing, China); and anti-LC3 and anti-p62 (Sigma, St. Louis, MO, USA). All HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. Protein detection was performed using either Pierce ECL (CWBIO, Beijing, China) or Pierce SuperSignal Pico (Thermo Fisher Scientific, USA) reagents.

Immunoblots were probed with anti‐PrP^C (Millipore), anti‐FOXO3a (Abcam, Melbourne, Australia), anti‐phospho‐FOXO3a (Abcam), anti‐AKT (Cell Signaling), anti‐phospho‐AKT (Cell Signaling, Arundel, Australia), anti‐p38 MAPK (Cell Signaling), anti‐phospho‐p38 MAPK (Cell Signaling), and anti‐KLF5 (R&D Systems, Noble Park, Australia). Histone H3 (Cell Signaling) or α‐tubulin (Sigma) was used as loading control. Membrane fraction of cell lysates (100 μg) were incubated in the presence of 2 μg of either anti‐PrP^C (Millipore), anti‐EGFR (Cell Signaling), or IgG isotype control antibody overnight at 4 °C with mixing. To each immunoprecipitation 60 μL of Protein A/G agarose bead slurry (Millipore) was added and incubated with mixing for 2 h at room temperature. The beads were spun down and washed five times with ice cold PBS. The final spin was resuspended in SDS/PAGE loading buffer and 5 μL loaded per well. Anti‐HSP70 was used as a loading control for 1% input.

Cells were lysed and protein extracted with RIPA buffer (Thermo Fisher Scientific). Then, 30 μg of total proteins was separated by 10%‐15% SDS‐polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (EMD Millipore). After blocking with 5% fat‐free milk solution, the protein of interest was detected by using a primary antibody and a corresponding HRP‐linked secondary antibody. The primary antibodies used were as follows: anti‐Runx2 (0.4 μg/mL), anti‐eNOS (0.5 μg/mL) from Santa Cruz Biotechnology; anti‐β‐actin (0.4 μg/mL) from Sigma‐Aldrich; anti‐osteopontin (OPN) (1 μg/mL) from Bioworld Technology; anti‐pAktS473 (1:2000), anti‐pAktT308 (1:1000), anti‐Akt (1:1000), anti‐Rictor (1:1000), anti‐RheB (1:1000), anti‐p85α (1:1000), anti‐Calponin‐1(1:1000), anti‐PARP‐1 (1:1000), anti‐Histone H3(1:2000), anti‐Bcl‐XL(1:1000), anti‐Bim(1:1000), anti‐Bax(1:1000), anti‐pS6K(1:1000), anti‐S6K(1:1000) from Cell Signaling Technology; anti‐pFOXO3a (1:2000), anti‐FOXO3a (1:1000), anti‐α‐SMA (0.5 μg/mL) from Abcam, anti‐caspase 3 (1:2000) from Cell Signaling Technology. and anti‐GAPDH (1 μg/mL) from GeneTex. The blots were developed with an enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific) and exposed to X‐ray film to obtain optimal results in the dark room. The density of bands was quantified by Image‐Pro Plus 6.0 software.

Astragaloside IV (Solarbio, Science&Technology, Beijing, China; cat:SA8640; HPLC ≥ 98%). Dulbecco’s Modified Eagle Medium (DMEM; cat:12320032), fetal bovine serum (FBS; cat:26170043), and penicillin–streptomycin (PS; cat:10378016) were purchased from Gibco (Waltham, MA, USA). Dimethyl sulfoxide (DMSO; cat:D4540) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Estradiol benzoate injection (Jinke, Sichuan, China); progesterone injection (Xianju, Zhejiang, China); mifepristone tablets (Resources Zizhu, Beijing, China). Anti-IDO1(cat: ab211017), anti-PI3KCA (cat: ab40776), anti-AKT1(cat: ab179463), anti-LC-3(cat: ab48394), anti-FOXO3A (cat: ab154786), anti-PCNA (cat: ab92552), and anti-βactin (cat:ab8226) antibodies were purchased from Abcam (Cambridge, UK). Anti-caspase-9 (cat:10380-1-AP), anti-FADD (cat:14906-1-AP), and anti-PTEN (cat:22034-1-AP) antibodies were obtained from Proteintech (Wuhan, China). PerCP/Cyanine5.5 anti-rat CD3 (cat: 201417), Alexa Fluor^® 488 anti-rat CD4 (cat: 201511), and APC anti-rat CD25 (cat: 202113) were purchased from Biolegend (San Diego, CA, USA). FOXP3 Monoclonal Antibody (150D/E4)-PE (eBioscience, San Diego, CA, USA; cat:12477441).

Total proteins were extracted from HOB and MC3 T3‐E1 cells, the primary osteoblasts isolated from the bone tissues of patients with osteoporosis and osteoarthritis or the osteoblasts isolated from the mouse bone marrow. The protein concentrations were tested using the BCA Protein Assay Kit (Beyotime). In total, 30 μg of protein from each sample was applied for SDS‐PAGE. After separation, the proteins were transferred into PVDF membranes (Roche, Basel, Switzerland). The PVDF membranes were blocked for nearly 1 h and then incubated with the primary antibodies anti‐FOXO3a (Abcam, 1/2500), anti‐TRIM33 (Abcam, 1/1000), anti‐Lamin B (Abcam, 1/2000), anti‐GAPDH (Abcam, 1/2500), anti‐CBP (Abcam, 1/1000), anti‐Gadd45a (Abcam, 1/1000), anti‐FOXO1 (Abcam, 1/2000), anti‐FOXO4 (Abcam, 1/2000) and anti‐catalase (Abcam, 1/2000) at 4°C for about 24 h. This was followed by the incubation with the corresponding secondary antibodies (Abcam, 1/2000) at room temperature for nearly 1 h. Protein bands were visualized using BeyoECL Moon (Beyotime). LaminB and GAPDH were applied as the references for nuclear protein and total protein, respectively.