*3.3. Evaluation of Alkaline Phosphatase Activity Assay with the Addition of BMP-4*

The results of the alkaline phosphatase activity assay at Days 1, 3, 5, and 7 are presented in Figure 5. In general, there were increases in the alkaline phosphatase activity with longer incubation time up to Day 7. Notably, the group treated with 2 ng/mL BMP-4 at Day 3 had a significantly higher activity compared with that of the control group at Day 3 *(p* < 0.05).

## *3.4. Gene Ontology*

A total of 25,737 mRNAs were differentially expressed. Scatter plots of the differentially expressed mRNAs are shown in Figure 6. A Venn diagram of the gene ontology analysis of differentially expressed mRNAs is shown in Figure 7. When compared with the 0 ng/mL control group, 1270 mRNAs were upregulated and 1070 mRNAs were downregulated in the 2 ng/mL group. In the 6 ng/mL group, 1536 mRNAs were upregulated and 1889 mRNAs were downregulated compared to controls. In the 10 ng/mL group, 1525 mRNAs were upregulated and 1533 mRNAs were downregulated compared to controls. A clustering analysis of differentially expressed mRNAs related to osteoblast differentiation is shown in Figure 8. The changes in expression of RUNX2 and CTNNB1 are shown in Figure 9. While the expression of CTNNB1 was increased dose-dependently, the expression of RUNX2 was highest at 6 ng/mL and the expression decreased at the higher dose of 10 ng/mL (Figure 9A,B). The phosphoinositide-3-kinase–protein kinase B/Akt (PI3K/AKT) signaling pathway was involved in the target genes chosen for stem cell differentiation (Figure 10).

**Figure 5.** Alkaline phosphatase activity on Days 1, 3, 5, and 7. \* Statistically significant differences were noted when compared with the 0 ng/mL group on Day 3 (*p* < 0.05).

**Figure 6.** Scatter plots showing the expression of BMP-4 at 0, 2, 6, and 10 ng/mL (x, y-axis: Relative expression level; red indicates that the expression level of the y-value is higher than that of the x-value and green indicates that the expression level of the y-value is lower than that of the x-value). (**A**) 2/0, (**B**) 6/0, (**C**) 10/0, (**D**) 6/2, (**E**) 10/2, and (**F**) 10/6 ng/mL.

**Figure 7.** Venn diagram analysis (fold change, 1.3, log2-normalized read counts of 4 were selected).

**Figure 8.** The results of clustering analysis of differentially expressed mRNAs related to osteoblast differentiation (fold change 1.3, log2-normalized read counts of 4 were selected).

**Figure 9.** (**A**) Log2-normalized read counts regarding the expression of RUNX2 and CTNNB1. (**B**) Log2 fold change regarding the expression of RUNX2 and CTNNB1.

**Figure 10.** PI3K-AKT signaling pathway.
