Supplementary Material for: Osteogenic Effect of Inducible Nitric Oxide Synthase (iNOS)-Loaded Mineralized Nanoparticles on Embryonic Stem Cells

<b><i>Background/Aims:</i></b> This study investigated the effect of inducible nitric oxide synthase-loaded mineralized nanoparticles (iNOS-MNPs) on the osteogenic differentiation of mouse embryonic stem cells (ESCs). <b><i>Methods:</i></b> We prepared iNOS-MNPs using an anionic block copolymer template-mediated calcium carbonate (CaCO<sub>3</sub>) mineralization process in the presence of iNOS. iNOS-MNPs were spherical and had a narrow size distribution. iNOS was stably loaded within MNPs without denaturation. In order to confirm the successful introduction of iNOS-MNPs into the cytosol of ESCs, intracellular levels of nitric oxide (NO) was determined with a fluorometric analysis. A NO effector molecule, cyclic guanosine 3’,5’ monophosphate (cGMP) was also quantified with a competitive enzyme immunoassay. Cell viability in response to iNOS-MNP treatment was determined using the cell counting kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity assay, intracellular calcium quantification assay, and Alizarin red S staining for matrix mineralization were performed to investigate osteogenic differentiation of ESCs. The protein levels of Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osterix (OSX) as osteogenic-related factors were also assessed by immunofluorescence staining and Western blot analysis. The complex pathways associated with iNOS-MNP-derived osteogenic differentiation of ESCs were evaluated by network-based analysis. <b><i>Results:</i></b> Cells with iNOS-MNPs displayed a significant increase in NO and cGMP concentration compared with the control group. When cells were exposed to iNOS-MNPs, there were no adverse effects on cell viability. Importantly, iNOS-MNP uptake promoted the osteogenic differentiation of ESCs. Using transcriptome profiling, we obtained 1,836 differentially-induced genes and performed functional enrichment analysis with ClueGO and KEGG. These analyses identified significantly enriched and interconnected molecular pathways such as protein kinase activity, estrogen receptor activity, bone morphogenetic protein (BMP) receptor binding, ligand-gated ion channel activity, and phosphatidylinositol 3-phosphate binding. <b><i>Conclusion:</i></b> These findings suggest that iNOS-MNPs can induce osteogenic differentiation in ESCs by integrating complex signaling pathways.