Skip to main content
. 2014 Dec 23;290(7):3910–3924. doi: 10.1074/jbc.M114.629899

FIGURE 11.

FIGURE 11.

Decreased proliferation, increased mineral apposition, and ALP activity and altered gene expression of menin-deficient calvarial osteoblasts. Primary osteoblasts were isolated from individual calvaria of 12-month-old Col1a1-Menin-Tg and control mice and cultured for 7–10 days in αMEM, and the following assays were then carried out. A: i, MTT dye viability assay. Absorbance was read at 595 nm. Values shown are mean ± S.E. (n = 3 independent experiments). *, p < 0.05. (ii) PrestoBlue cell viability assay. Absorbance was read at 570 nm and normalized to the 600-nm value. Values shown are the mean ± S.E. (n = 3 independent experiments). *, p < 0.05. B, Alizarin Red-S staining. Primary osteoblasts were plated in an osteogenic medium. i, cells were fixed at 14 days and stained with Alizarin Red-S. ii, quantitation was made by extraction and measurement of absorbance at 405 nm. Values are the mean ± S.E. (n = 3 independent experiments). *, p < 0.05. C, ALP. Primary osteoblasts were cultured in an osteogenic medium containing β-glycerophosphate and l-ascorbic acid for 7 and 14 days (shown). i, ALP staining. Washed, fixed cells were overlaid with nitro blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3′-indolyl phosphate p-toluidine salt) (NBT) and incubated at room temperature for 20 min in the dark. ii, ALP activity. p-Nitrophenyl phosphate substrate was added to lysed cell extracts, and the reaction was stopped with 3 n NaOH. Absorbance was read at 405 nm, and values were normalized to protein concentration. Values are the mean ± S.E. (n = 3 independent experiments). *, p < 0.05; **, p < 0.01. D, RNA was isolated, semi-quantitative RT-PCR and gel electrophoresis were performed, and densitometric analysis was made. Expression of osteoblast markers Runx2, osteocalcin, and Col1a1 was increased, OPG and RANKL were unaltered, the CDKIs p15 and p21 were increased, CDK4 was decreased, and the osteocyte marker Sclerostin, the pro-apoptotic Bax, and the pro-survival Bcl-2 were unaltered. *, p < 0.05; **, p < 0.01 Col1a1-Menin-Tg versus control.