As1008

The cells were lysed on ice in buffer containing protease inhibitors (AS1008, Aspen). The protein fractions were collected and separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE). Then, the proteins were electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore). The membranes were blocked with 5% skim milk. Then, primary antibodies were added and incubated overnight at 4 °C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The antibodies used in this study included anti-Nrf2 (1:1000, PA5-27882, Thermo Fisher Scientific), anti-β-Actin (1:10,000, TDY051, TDY Biotech), anti-Histone H3 (1:10,000, #4499, Cell Signaling Technology), anti-Drp1 (1:1000, #8570, Cell Signaling Technology), anti-p-Drp1 (Ser616) (1:500, PA5-106169, Thermo Fisher Scientific), anti-VDAC1 (1:3000, ab15895, Abcam), anti-NOQ1 (1:3000, ab80588, Abcam), anti-HO-1 (1:5000, 10701-1-AP, Proteintech Group), anti-SOD2 (1:3000, ab68155, Abcam), anti-cleaved Caspase-3 (1:1000, AF7022, Affinity Biosciences), anti-Bax (1:2000, #2772, Cell Signaling Technology) and anti-Bcl-2 (1:1000, ab19649, Abcam).

In a solution containing a protease inhibitor (AS1008; Aspen), PC12 cells were dissolved on ice for 30 min. BCA protein assay kit was used to measure the protein concentration (PC0020; Solarbio). The protein samples were heated at 100°C for 10 min to completely denature the proteins. The protein samples were then separated by SDS‐PAGE electrophoresis and transferred to PVDF membranes (IPVH00010; Millipore). After being blocked for an hour with 5 percent skim milk, the membrane was incubated with the primary antibody at 4°C overnight. The antibodies used in this study consisted of anti‐DHODH (1:1000, 14877‐1‐AP; Proteintech), anti‐ACSL4 (1:1000, 22401‐1‐AP; Proteintech), anti‐COX2 (1:1000, 66351‐1‐Ig; Proteintech), anti‐GPX4 (1:1000, 67763‐1‐Ig; Proteintech), anti‐ALOX15 (1:1000, ab244205; Abcam), anti‐P53 (1:1000, AB_297667; Abcam). After being washed three times with TBST, the membrane was incubated for 10 min at room temperature with a secondary antibody that had been enzyme‐labeled. Imaging was carried out using an ECL chemiluminescence substrate (BL520B; Biosharp). Band intensities were quantified using Image J software.

Lysis buffer (#AS1004, Aspen, South Africa) containing 1% protease inhibitor (#AS1008, Aspen) was used to lyse cells or callus samples, after which protein was separated via SDS-PAGE and transferred to NC membranes (#IPVH00010, Millipore, USA) that were blocked with 5% nonfat milk and stained overnight at 4 °C overnight with antibodies specific for collagen I (1:500, Sigma, USA,#ab34710), ALP (1:1000, Sigma, USA,#ab95462), Osteocalcin (1:500, Sigma, USA, #ab93876), RunX2 (1:500, Sigma, USA, #ab23981), SIK2 (1:1000, Sigma, USA, #SAB1302059), SIK3 (1:1000, Sigma, USA, #SAB3500695), and GAPDH (1:10,000, Sigma, USA, #ab37168). Blots were then stained with appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) (#AS1058, Aspen), and proteins were detected with a chemiluminescence detection system. Each experiment was repeated three times.