Edu incorporation assay

BMSCs viability was evaluated using the CCK‑8 assay and EdU incorporation assay (Both Beyotime Institute of Biotechnology, Shanghai, China). For the CCK‑8 assay, BMSCs were seeded on 96‑well plates at a density of 3×10³ cells/well. Following cell adhesion to the plates, wells were randomly treated with different reagents. At predetermined time points (3 days or 5 days), the culture media was removed and cells were washed with PBS. After that, 100 μL fresh culture media and 10 μL CCK‑8 solution were added to each well. Subsequently, the plates were incubated at 37°C for 30 min. Then, absorbance was detected at 450 nm by a microplate reader (Thermo, MA, USA). For the EdU assay, BMSCs were seeded on 24‑well plates at a density of 2.5×10⁴ cells/well and randomly treated with different reagents for 3 days. After 60% confluence, cells were incubated with 50 μM EdU media for 2 h in dark, fixed in 4% paraformaldehyde for 30 min, and then stained by DAPI (Beyotime) for 30 min. The EdU-stained cells were photoed by a fluorescence microscope (Carl Zeiss Meditec, Jena, Germany). The cell positive rate of each well was calculated by counting the EdU-positive nuclei (red) and blue fluorescent nuclei in five random microscopic fields.

Colony forming unit (CFU) assay and 5-ethynyl-2′-deoxyuridine (p;[) incorporation assay were employed to assess the proliferation of TMPCs. For the CFU assay, TMPCs were plated at a density of 1 ​× ​10⁴ ​cells/well in 6-well tissue culture plates for 10 days without changing the standard growth medium. DAPT/DMSO-treated cultures were grown in the standard growth medium for 3 days and then supplemented with DAPT (10 ​μM) or DMSO vehicle for an additional 7 days. On day 10 after plating, cells were fixed for crystal violet staining. Type Ι colonies (CFU–F) were counted as previously described [8 (link)].
EdU incorporation assay was performed according to the manufacturer's instructions (Beyotime Biotechnology, Beijing, China). Briefly, cells were seeded in a 6-well plate at a density of 1 ​× ​10⁴ ​cells/well and incubated with DAPT (10 ​μM) or DMSO vehicle. After culture for 48 ​h, cells were exposed to 10 ​μM EdU reagent for 2 ​h, then fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with a Click-iT reaction cocktail. The cell nuclei were marked with Hoechst at a concentration of 5 ​μg/mL for 30 ​min. The ratio of EdU-positive cells was calculated as the positive staining cells/the total cells ​× ​100% using the ImageJ software 1.8.

Cell proliferation was evaluated using an EDU incorporation assay (Beyotime, Shanghai, China). Cells were treated with 100 μg/mL ox- LDL for 24 h and grouped as described earlier. The cells were then digested and seeded into 96-well plates at a density of 2×10³ cells/well. Cells were cultured with 20 μM EdU diluent at 37°C and 5% CO^{2 (link)} for 2 h. The cells were then fixed with 4% paraformaldehyde for 15 min. After washing the cells with phosphate-buffered saline (PBS), 1 mL of permeation solution was added to each well and incubated at room temperature for 15 min. Cells were incubated with endogenous peroxidase blocking solution at room temperature for 20 min and washed thrice with PBS. Click reaction solution (50 μL) was added to each well and incubated in darkness at room temperature for 30 min. The cells were then washed thrice with PBS. Streptavidin-hrp (STREptavin-HRP) working solution was added, incubated at room temperature for 30 min, and then washed thrice with PBS. Subsequently, 0.1 mL TMB colour solution was added and incubated for 30 min at room temperature. The absorbance was measured at 370 nm.