Fig. 5
Effects of SMS1 knock-out on osteoblast differentiation. a Schematic outline of the experimental design. b Representative pictures of ALP-staining of primary calvarial osteoblast cultures in the absence or presence of 1 μM 4-OHT at day 6 after BMP2 stimulation (left). Quantification of the staining is shown (right). Inactivation of SMS1 resulted in reduced ALP activity of calvarial osteoblasts. *p < 0.005 versus SMS1f/f;SMS2−/− (4-OHT+, BMP2+) and ERT2-Cre;SMS1f/f;SMS2−/−(4-OHT−, BMP2+); Values are expressed as the mean ± SD, n = 8. c Gla-Osteocalcin contents of primary calvarial osteoblast cultures in the absence and presence of 4-OHT at day 6 after BMP2 stimulation. *p < 0.005 versus SMS1f/f;SMS2−/− (4-OHT+, BMP2+) and ERT2-Cre;SMS1f/f;SMS2−/− (4-OHT−, BMP2+); Values are expressed as the mean ± SD, n = 6. d After treatment with and without 1 μM 4-OHT for 1 day, SMS1f/f;SMS2−/− and ERT2-Cre;SMS1 f/f;SMS2−/− mice calvarial osteoblasts were cultured with and without 100 ng/ml BMP2 in α-MEM with 10% FBS containing 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate for 4 days. Col1a1, BSP and OPN mRNA levels were normalized to GAPDH mRNA. Treatment with 4-OHT does not affect the differentiation of primary SMS1f/f;SMS2−/− calvarial osteoblasts. *p < 0.005 versus SMS1f/f;SMS2−/− (4-OHT+, BMP2+) and ERT2-Cre;SMS1f/f;SMS2−/− (4-OHT−, BMP2+); Values are expressed as the mean ± SD, n = 8. e Decreased bone nodule formation in the 4-OHT-induced SMS1 inactivation. Representative images of nodule mineralization stained with Alizarin red. SMS1f/f;SMS2−/− and ERT2-Cre;SMS1f/f;SMS2−/− mice calvarial osteoblasts were treated with and without 1 μM 4-OHT for 1 day, and cultured with and without 100 ng/ml BMP2. On day 17, the cells were stained with Alizarin red (upper). Quantification of the staining is shown (lower). *p < 0.005 versus SMS1f/f;SMS2−/− (4-OHT+, BMP2+) and ERT2-Cre;SMS1f/f;SMS2−/− (4-OHT−, BMP2+); Values are expressed as the mean ± SD, n = 8