Cytotoxic effect of FR on BM-MSCs. MSCs (5×10³, 4×10³, and 2×10³ cells/well) were treated with indicated concentrations (50, 100, 150, and 200 µM) of FR for 72 hours. Cell viability was measured by the tetrazolium-based colorimetric assay. Cell viability gradually decreased with increased FR dosage. The IC₅₀ of FR was 50 µM (A). DNA fragmentation assay was performed in MSCs after treatment with FR for 72 hours. There were no significant changes in the nuclear DNA in response to FR (B). Effect of FR in inducing apoptosis in MSCs. Cells (1×10⁶/plate) treated with 150 µM and 200 µM FR for 72 hours were co-stained with PI and FITC-conjugated annexin V and then examined by flow cytometry. Almost twice as many cells underwent apoptosis after FR treatment compared to control (C). Effect of FR exposure on mitochondria in MSCs. Loss of mitochondrial membrane potential was observed after FR treatment. MSCs were treated with FR for 72 hours, washed twice with phosphate buffer saline (PBS), and then treated with 100 nm MitoTracker Red CMXRos at 37℃ for 30 minutes. Fluorescence images were then recorded. Reduced fluorescence with increasing doses of FR is an indication of mitochondrial membrane potential loss, which is an indication of mitochondrial dysfunction as well as initiation of early apoptosis (D).