Thermo 1500

In total, 2 × 10⁴ cells were added to each well of a 96‐well culture plate and incubated overnight. The medium was then removed, and the cells were fixed with 4% PFA and blocked with 1% BSA. The fixed cells were incubated with an anti‐ATAP2 antibody (1:200, Santa Cruz Biotechnology, Dallas, Texas, USA) and an anti‐WEDE15 antibody (1:100, Beckman Coulter, Marseille, France) for 1 hour at room temperature and then washed three times with PBS containing 0.1% BSA followed by incubation with the secondary antibodies for 1 hour. Then, 100 µL of 3,3',5,5'‐tetramethylbenzidine (TMB, Cell Signaling Technology, Inc, Danvers, MA, USA) substrate solution was added to each well and incubated for 20 minutes. The enzymatic reaction was stopped by the addition of 2 M sulphuric acid. The optical density of each well was measured at 450 nm with a multidetector microplate reader (Thermo 1500; Thermo Fisher Scientific, Inc).

The NR8383 cell line was purchased from Shanghai Institute for Biological Sciences, Chinese Academy of Science. NR8383 cells were cultured at 37 °C in F12 K medium (Sigma-Aldrich, St. Louis, MO) supplemented with 15% FBS (PAA Laboratories GmbH, Austria), 2 mM L-glutamine (Amresco Inc., USA), and 1% penicillin/ streptomycin (Haoyang Biological Manufacture Co., Tianjin, China) in an incubator with 5% CO₂ [23 (link)]. MC3T3-E1 cells (iCell Bioscience Inc., Shanghai, China) were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS in an incubator with 5% CO₂.
Cell viability was determined by Cell Counting Kit-8 (CCK-8, Beyotime Institute of Biotechnology, China). Briefly, NR8383 (1 × 10⁷ cells/mL) or MC3T3-E1 cells (1 × 10⁵ cells/mL) in 96-well plates were exposed to different concentrations of AG-QDs (0, 10, 25, 50, 100, 200, and 500 μg/mL) at 37 °C for 12, 24, 48, 72 and 96 h, and were then treated with CCK-8 probes to determine cell viability with a microplate reader (Thermo-1500, USA).