Anti runx2

After osteogenic differentiation, samples were fixed in paraformaldehyde (4%) for 15 min, permeabilized for 30 min with 0.1% Triton X-100, and then blocked using 5% bovine serum albumin (BSA) for 30 min. Subsequently, the samples were incubated with anti-RUNX2 (1:200; Huabio, China) and COL1A1 (1:200; Cell Signaling Technology, America) at 4 °C overnight and following a fluorescence-labeled secondary antibody for 2 h. Finally, the fluorescein isothiocyanate (FITC) (Solarbio, Beijing, China) stained cell body for 30 min and 4′,6-diamidino-2-phenylindole (DAPI) (Solarbio, Beijing, China) stained nucleus for 5 min. The results were recorded with a confocal microscope (Olympus FV3000, Japan).

BMSCs were fixed using a 4% paraformaldehyde solution (4 °C, 25 min), permeabilized with 0.5% Triton X-100 (room temperature, 20 min), blocked with 5% sheep serum (37 °C, 20 min), and washed with PBS at each step, except for the final step. They were then incubated with specific antibodies: anti-ALP (1:200, HUABio, Zhejiang, China), anti-RUNX2 (1:200, HUABio, Zhejiang, China), anti-OSX (1:200, HUABio, Zhejiang, China), anti-GPX4 (1:200, HUABio, Zhejiang, China), anti-ACSL4 (1:200, HUABio, Zhejiang, China), and anti-NRF2 (1:200, HUABio, Zhejiang, China) overnight at 4 °C. The next day, BMSCs were incubated with a secondary anti-rabbit antibody (1:200, Invitrogen, Carlsbad, USA) at 37 °C for 1 h. The cytoskeleton was stained with phalloidin (37 °C, 20 min), and the Nucleus was stained with DAPI (37 °C, 10 min), followed by cleaning with PBS at each step. Images were captured using a confocal microscope (Olympus, Tokyo, Japan).