A B C D 1,25(OH)2D2 1,25(OH)2D3 25(OH)D3 25(OH)D2 Vehicle MG-63 MG-63

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A B C D 1,25(OH)2D2 1,25(OH)2D3 25(OH)D3 25(OH)D2 Vehicle MG-63 MG-63 1,25(OH)2D3 25(OH)D3 25(OH)D2 Vehicle A B MG-63 MG-63 % 4MU (pM) / Protein (µg) % 4MU (pM) / Protein (µg) (nM) 0 100 200 (nM) 0 0.1 0.2 C D SaOS-2 SaOS-2 % 4MU (pM) / Protein (µg) % 4MU (pM) / Protein (µg) (nM) 0 100 200 (nM) 0 0.1 0.2 Comparative effects of 25(OH)D2, 25(OH)D3, 1,25(OH)2D2 and 1,25(OH)2D3 on ALP enzyme activity. MG-63 and SaOS-2 cells (1x104) were grown in DMEM-10% FBS culture medium untill confluency (48hr), then introduced to osteogenic differentiation medium and treated every other day with either (A&B) 100-200nM 25(OH)D2/D3 or (B&C) 0.1-0.2nM of 1,25(OH)2D2 and D3. Cultures were stopped on day 6 and ALP activity was measured. ALP activities were normalised to total protein content. Mean ± SEM enzymatic activity values from triplicate experiments have been presented as the amount of 4-methylumbelliferone (%4MU) generated after 45 min at 37C.

MG-63 SaOS-2 OSM FBS Representative images of MG-63 and SaOS-2 osteoblast – like cells in the presence (top row; OSM) or absence (bottom row; FBS) of osteogenic medium (OSM) containing 10mM β-glycerophosphate, and 50 µg/ml L-ascorbic acid over 28 days. Osteoblast-like cells (1x104) were grown to full confluency (48 hr) in DMEM-10%FBS, when media were replaced with OSM. Cultures were stopped on day 28, fixed in 70% ethanol, washed with deionised water and stained with Alizarin red staining solution. Plates were washed with deionised water and scanned with an Epson AX10 scanner.