Additional file 3 of Hypoxic preconditioning induces epigenetic changes and modifies swine mesenchymal stem cell angiogenesis and senescence in experimental atherosclerotic renal artery stenosis

Additional file 3: Supplemental Figure 1. Schematic of the experimental protocol. RAS: renal artery stenosis, ARAS: atherosclerotic RAS, MSC: mesenchymal stem cells. Supplemental Figure 2. MSC Morphology and differentiation. (A) Adipose tissue-derived MSC harvested from healthy and ARAS pigs show typical morphological appearance under the microscope as spindle-shaped, fibroblast-like cells in culture.(B) Tri-lineage differentiation into adipocyte (FABP4), chondrocyte (Aggrecan), and osteocyte (Osteocalcin) lineages is achieved in both MSC groups (scale bar 200 μm). MSC: mesenchymal stem cells, ARAS: atherosclerotic renal artery stenosis, DAPI: nuclear stain, FABP4: positive staining represents adipocytes, chondrocytes, Osteocalcin: positive staining represents osteocytes, Aggrecan: positive staining represents. Supplemental Figure 3. Cell Migration. Cells were seeded in MSC growth medium (Advanced MEM) at 300,000 cells/cm2 in the top chamber of The MILLIPORE QCMTM 24-well Cell Migration Assay (ECM508) kit (Sigma-Aldrich, Billerica, MA, USA). After overnight culture either in normoxic or hypoxic conditions, the migrated cells were fixed with 1% paraformaldehyde and stained with the cell stain provided with the kit. Nonmigrated cells were removed from the upper side of the membrane. Migrated cells on the bottom were directly visualized under the microscope. Supplemental Figure 4. Apoptosis Assay. Representative flow cytometry scatterplots of MSC viability during normoxic and hypoxic conditions. MSC viability tested using Annexin V and Sytox shows MSC viability from healthy vs. ARAS pigs. This is a representative image where red panel represents live cells, orange panel represents dead cells, and green panel represents apoptotic cells. (A) The percentage of live and apoptotic cells were similar in both groups in either condition. MSC: mesenchymal stem cells, ARAS: atherosclerotic renal artery stenosis.