Neither SDX-101 nor SDX-308 shows toxic effects on hematopoietic progenitors or affects osteoblast differentiation. (A-B) CD34⁺ cells were used for standard colony-formation assays using Methocult GF H4434 (Stem Cell Technologies, Vancouver, BC, Canada). The assay was performed in the presence of SDX-101 (75 μM) (⊡, top), SDX-308 (7.5 μM) (⊡, bottom), or DMSO (0.1%) (▪) as control. Numbers of CFU colonies formed were quantified under an inverted microscope after 14 days. Formation and relative distribution of BFU-E, CFU-M, and CFU-GM colonies were evaluated. Images were obtained using an Olympus microscope (numeric aperture 0.40, 10× magnification), and software (MagnaFire 4.1, Optronics). Data shown are the mean ± SD of triplicates of colony-formation assay. (C) ALP activity and (D) Og2 promoter activity: MC-42 cells were plated at a density of 5 × 10⁴ cells/cm² in 35-mm plates and were cultured in ascorbic acid (50 μg/mL) containing α-MEM for 15 days and treated with the indicated concentration of SDX-101, SDX-308, or volume-matched vehicle for 24 hours. Cells were then harvested for ALP assay and luciferase assay. ALP activity and luciferase activity were normalized into total protein. (E) Mineralization. MC-42 cells were grown as described in Figure 4C-D for 15 days. Inorganic phosphate was then added to a final concentration of 5.0 mM in the presence or absence of SDX-101, SDX-308, or vehicle for 48 hours. Samples were then stained using the von Kossa method. Images were obtained by direct scanning of the mineralization dish using the ScanMaker 9800Xl (Microtek International), and the randomly selected representative areas were shown.