Generation of Sam68-Deficient Mice

<div><p>(A) The genomic organizations of the wild-type and targeted <i>sam68</i> alleles after homologous recombination are depicted. The location of the DNA fragment used as a probe for the Southern blot analysis is shown, as well as the sizes of the two BglII fragments detected for wild-type and targeted <i>sam68</i> alleles. The targeted allele replaces exon 4 and part of exon 5 of <i>sam68</i> with a PGK-neomycin cassette.</p><p>(B) Southern-blot analysis of genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. DNA fragments corresponding to wild-type (4.5 kb) and the targeted (5.5 kb) alleles are illustrated.</p><p>(C) Western blot analysis of Sam68 expression. Protein extracts from wild-type, heterozygous, and homozygous cells subjected to immunoblot analyses using normal rabbit serum, anti-Sam68 AD1 antibody, the peptide antibody AD1 preabsorbed with the immunogenic peptide corresponding to amino acids 330–348 of mouse Sam68, anti-Sam68 Sc333 antibody that recognizes the C-terminal 20 amino acids of Sam68, and anti-actin antibodies as loading control. The migration of the molecular mass markers is known on the left in kDa.</p></div

Ex Vivo Activity of Sam68^+/+ and Sam68^−/− Osteoblasts and Osteoclasts

<div><p>Marrow stromal cells were isolated from the long bones of juvenile mice and maintained under conditions that promote osteoblast differentiation.</p><p>(A) Cultures were fixed in 4% paraformaldehyde after 6 or 18 days and stained in situ for ALP activity and with silver nitrate (von Kossa) to detect mineralized nodules. Sam68^−/− cultures stained more intensely for ALP at early and late time points and produced significantly more mineralized nodules after 18 days. Asterisks represent <i>p</i> < 0.01.</p><p>(B) Primary osteoclasts were isolated from the crushed long bones of the same mice and plated on glass coverslips or on dentin slices to quantify numbers and activity, respectively. Osteoclasts were identified as cells with three or more nuclei that stained positive for TRAP activity (upper) and excavated pits in dentin slices, as demonstrated by SEM (lower, bar = 20 μm). No statistical differences were observed either in the number of TRAP-positive cells or in their resorptive activity.</p></div

Ex Vivo Adipogenesis Analysis of Sam68^−/− Mouse Embryonic Fibroblasts

<div><p>MEFs were isolated from mouse embryos at embryonic day 14.5. Equal number of MEFs from Sam68^+/+ and Sam68^−/− was plated on glass cover slips in 24 well-plates. Adipocyte differentiation was carried out at indicated times by the addition of complete media containing the pioglitazone.</p><p>(A) Cultures were fixed in 4% paraformaldehyde and stained with Oil Red O to detect the fat droplets stored in adipocytes and photographed (top). The cell images were magnified ×10 and ×20 as indicated.</p><p>(B) RT-PCR was carried out on total cellular RNA isolated after differentiation of the MEFs for day 0, 2, 4, 6, and 12. The DNA fragments were visualized on agarose gels stained with ethidium bromide. The expression of adipogenic markers C/EBPβ, C/EBPδ, PPARα, and KLF5 was examined as well as the expression of controls including Sam68, β-actin, and GAPDH.</p></div

Histologic Analysis of Undecalcified Bone from Sam68<b>^+/+</b> and Sam68^−/− Mice

<p>Sections of tibia fixed in 4% paraformaldehyde and embedded in plastic were stained for ALP (A–C, E–G) activity to identify osteoblasts or for TRAP (B–D, F–H) activity to identify osteoclasts. Staining patterns were similar in 4-month-old Sam68^+/+ (A and B), 4-month-old Sam68^−/− (C and D), and 12-month-old Sam68^−/− (G and H) mice compared with 12-month-old Sam68^+/+ mice (E and F). Magnification at source, left panels ×10 and right panels ×40. Micrographs are representative of those taken from five to seven sections in each group of animals.</p

Quantitative Micro-CT of Trabecular Bone Composition and Architecture

<div><p>(A) Bone volume/tissue volume (BV/TV) and structure model index (SMI) were calculated on the femur and fourth lumbar vertebra of six or seven mice in each group using 3D Creator software supplied with the Skyscan instrument. Results expressed as the mean ± SD showed significant differences (<i>p</i> < 0.01) between 4-month-old Sam68^+/+ mice (solid black) and 12-month-old Sam68^+/+ mice (hatched black) but not between 4-month-old Sam68^−/− mice (solid white) and 12-month-old Sam68^−/− mice (stippled white).</p><p>(B) The distance between trabeculae was reflected in a shift to the right of the distribution curves for 12-month-old Sam68^+/+. Solid black = 4-month-old Sam68^+/+; hatched black = 12-month-old Sam68^+/+; solid red = 4-month-old Sam68^−/−; stippled red = 12-month-old Sam68^−/−. The asterisks denote <i>p</i> < 0.01.</p></div

Old Sam68^−/− Mice Are Protected from the Development of Fatty Bone Marrow

<p>Sections of undecalcified bone were stained with von Kossa and toluidine blue and images captured at original magnifications of ×2 (A), ×40 (B and C) to evaluate mineralized tissue (A, black), marrow adipocytes (B, white), and the mineralization fronts (C, yellow and green). The 12-month-old Sam68^+/+ bone demonstrated a significant reduction in bone (A) and increase in marrow adipocytes (B) and a decrease in the distance between two consecutive fluorochrome labels (C). Magnification at source was ×40. Micrographs are representative of four to six screened in each group of animals.</p

Enhanced Osteogenic Differentiation of the C3HT101/2 Embryonic Cell Line Depleted of Endogenous Sam68

<div><p>(A) C3HT101/2 cells transfected with an empty vector (pSuper-retro) or a vector containing an shRNA (Sam68 shRNA) were selected with puromycin, and knockdown populations depleted of Sam68 were identified. The reduction in Sam68 protein was analyzed by immunoblotting with anti-Sam68 (AD1) antibody and anti–β-actin antibodies as loading controls.</p><p>(B) Osteogenic differentiation was carried out with conditioned medium containing BMP-2 for the indicated times. To assess the level of osteogenic differentiation in these cells, expression of late osteoblast marker, osteocalcin (OCN), was analyzed by RT-PCR and compared with β-actin and GAPDH controls. The DNA fragments were visualized by agarose gel stained with ethidium bromide.</p></div

Immunohistochemical Localization of Sam68 in Embryonic Mice

<div><p>(A) Embryonic mice were removed from pregnant dams at E14.5 and E16.5, fixed in 4% paraformaldehyde, and embedded in paraffin. The entire embryo was immunostained with the AD1 anti-Sam68 antibody and counterstained with methyl green, and the image was captured at ×1.2 magnification.</p><p>(B) Embryonic soft tissues from the brain, heart and gut were stained with hematoxylin (left) and immunostained with anti-Sam68 antibody (right), and images were captured at ×20 magnification.</p><p>(C) Intense anti-Sam68 immunoreactivity was seen in chondrocytes in the nasal septum (panels A–D), in developing vertebra (panels E–H), and in the femoral epiphysis (panels I–K), as well as in diaphyseal osteoblasts (panel L). Adjacent sections were stained with hematoxylin and eosin (panels A, E, and I) or with antibody preadsorbed with the immunizing peptide (panels B, F, and J). Sam68 was localized primarily in the nucleus of cells in a variety of tissues but was also found occasionally in the cytoplasm. Magnification at source ×20, except for panels D, H, and L, which were ×40. Staining patterns are representative of three to five embryos.</p></div