Runx2 and Sp7 (Osterix) levels in RA and RAR antagonist treated MC3T3-E1 cells.

<p>(<b>A</b>) QRT-PCR analysis of <i>Runx2</i> and Sp7 (<i>Osterix)</i> expression at day 3, 7 and 14 of cells treated with 400 nM RA or 1 µM AGN. (<b>B</b>) Representative Western blot analysis of Runx2 and Osterix at day 3, 7 and 14 of cells treated as in (<b>A</b>) and quantification of the Western bands relative to Actb (relative ratio). Means +/– SD, ** p<0.01 and *** p<0.001, RA vs Control, and ^# p<0.05, ^## p<0.01 AGN vs Control.</p

Osteocyte markers in RA and RAR antagonist treated MC3T3-E1 cells and periosteal and serum phenotype in vitamin A treated rats.

<p>QRT-PCR analysis of <i>Tnfsf11 (RANKL)</i> and <i>Dmp1,</i> (<b>A</b>) <i>Phex, Sost</i> and <i>Fgf23</i> (<b>B</b>) expression at day 14 and 21 during a mineralization experiment of MC3T3-E1 cells treated with 400 nM RA or 1 µM AGN. (<b>C</b>) Representative Western blot analysis of Phex, Dmp1 and full length RANKL at day 14 and 21 of MC3T3-E1 cells treated as in (A) and quantification of Western bands relative to Actb (relative ratio). (<b>D</b>) Dmp1 and Cathepsin K (CatK) immunohistochemical staining at the diaphyseal periosteal site in rats suffering from hypervitaminosis A and in control rats. Upper panel: Arrow heads indicate Dmp1 negative osteocytes close to the periosteum (Ps) in control rat bone and arrows indicate Dmp1 positive osteocytes close to the periosteum in hypervitaminosis A rat bone. Lower panel: only Vitamin A animals show clear CatK staining at the Ps site. (<b>E</b>) Serum Fgf23 and phosphate levels in rats from (D). Means +/– SD, * p<0.05, ** p<0.01 and *** p<0.001 RA vs Control. ^# p<0.05, ^## p<0.01 AGN vs Control.</p

RA and RAR-dependent effects on osteoblast proliferation and on treatment start during <i>in vitro</i> mineralization.

<p>(<b>A</b>) Cell proliferation of MC3T3-E1 cells, treated with 400 nM RA or 1 µM AGN during the first 14 days of a mineralization experiment. (<b>B</b>) Cell number of viable and non-viable MC3T3-E1 cells after 10 days, with or without 400 nM RA. (<b>C</b>) Alizarin Red stain quantification of MC3T3-E1 cells, treated with 400 nM RA or 1 µM AGN from day 0, 10, 14 or 18 followed by analysis at day 25. Control mineralization level is set at 1 and dotted line represent background (no osteogenic induction). Means +/– SD, * p<0.05, ** p<0.01 and *** p<0.001 vs Control.</p

The effect of RA and RAR signaling on osteoblast mineralization <i>in vitro</i>.

<p>(<b>A</b>) Representative diagram of quantification of Alizarin Red stain in primary human osteoblasts (from a single individual) treated with RA at 4 and 400 nM, a Cyp26 inhibitor (R115866, 5 µM) and a pan-RAR antagonist (AGN, 1 µM) and 400 nM RA + AGN for 25 days. Below are representative photographs of the cultures. (<b>B</b>) Alizarin Red stain quantification of the mouse preosteoblast cell line MC3T3-E1 treated with RA at 400 nM, a Cyp26 inhibitor (R115866, 5 µM) and a pan-RAR antagonist (AGN, 1 µM) and 400 nM RA + AGN for 25 days. Below are representative photographs of the cultures. Means +/– SD, not significant (ns), * p<0.05 and *** p<0.001, vs Control, and ### p<0.001 vs RA.</p

QRT-PCR analysis of genes associated with osteoblast differentiation and endogenous RA degradation.

<p>(<b>A</b>) Expression levels of mRNA for <i>Alpl (alkaline phosphatase, liver/bone/kidney)</i> and <i>Bglap</i> (<i>Osteocalcin)</i> during a mineralization experiment of MC3T3-E1 cells treated with 400 nM RA or 1 µM AGN. (<b>B</b>) mRNA expression of <i>Cyp26b1</i> at day 1, treated as in (A). (<b>C</b>) mRNA expression of <i>Cyp26b1</i> at day 1, 3, 7, 14 and 21 of a mineralization experiment of MC3T3-E1 cells +/– 1 µM AGN. Means +/– SD, * p<0.05, ** p<0.01 and *** p<0.001 RA vs Control. # p<0.05, ## p<0.01 and ### p<0.001 AGN vs Control. ¤ p<0.05 and ¤¤¤ p<0.001 vs Control day 1.</p

Effects of a high vitamin A intake on serum levels, bone and body weight.

<p>(<b>A</b>) Comparison of serum vitamin A levels, body weight, femur length and cross sectional area of mid diaphysis of femur, and (<b>B</b>) periosteal mineralizing surface, bone formation rate and mineral apposition rate as determined by histomorphometric analysis of calcein double-labeled bones in rats with a high vitamin A intake and pair-fed controls. Means +/– SEM, * p<0.05 vs Control.</p