Fig. 3. Effects of acetaldehyde on OB differentiation. (A) Schematic representation of OB differentiation in ALDH2*1/*1-hiPSCs and ALDH2*1/*2-hiPSCs with or without 4 mM acetaldehyde. (B) Cell viability of the ALDH2*1/*1- and ALDH2*1/*2-hiPSCs derived OBs against acetaldehyde. (C) Measurement of ALDH2 activity in acetaldehyde-treated OBs. (D) Alizarin Red S staining confirming calcium deposition in OBs, measured by absorbance at 405 nm. (E) Quantitative data showing the expression of osteogenic markers, including RUNX2, COL1A1, OCN, and ALP, in OBs treated with or without 4 mM acetaldehyde for 3 days. (F) Protein expression of RUNX2, OCN, and ALDH2 in OBs with or without 4 mM acetaldehyde. GAPDH was used as a loading control. (G) Quantification of western blot results using the Adobe Photoshop. Data are presented as the mean±SEM. Statistical significance is indicated as ^#p<0.05, ^##p<0.01, ^###p<0.001 (t-test), *p<0.05, **p<0.01, ***p<0.001 (determined by two-way ANOVA). E8: Essential 8, ALDH2: acetaldehyde dehydrogenase 2, OBs: osteoblasts.