Induction of <i>Amelx</i> expression in MSCs-<i>TetR</i>/<i>Amelx</i> by Dox treatment.

<p>MSCs-<i>TetR</i>/<i>Amelx</i> were cultured in the growth medium in the presence (<b>+</b>) or absence (<b>-</b>) of Dox for 24–72 hours. Inducible expression of the <i>Amelx</i> gene was detected by RT-PCR (<b>a</b>) and western blot (<b>b</b>) analyses. GAPDH was used as a loading control. (<b>c</b>) MSCs-<i>TetR</i>/<i>Amelx</i> were cultured in the osteogenic induction medium in the presence (+) (black bars) or absence (-) (white bars) of Dox for 17 days in four different conditions. (Condition I: day 0–17 Dox-; Condition II: day 0–14 Dox-, day 15–17 Dox+; Condition III: day 0–14 Dox+, day 15–17 Dox-; Condition IV: day 0–17 Dox+). Expression of <i>Amelx</i> was determined by quantitative real-time RT-PCR analysis. Significant differences (*<i>P</i><0.01: ANOVA with Tukey’s multiple comparison test: n = 4) within each condition are shown.</p

Establishment of a tetracycline (Tet)-controlled <i>Amelx</i> expression system in MSCs.

<p>(<b>a</b>): Schematic diagram depicting the procedure to establish a Tet-controlled <i>Amelx</i> expression system in MSCs. TetR: Tet repressor, TO: Tet operator, Dox: doxycycline (tetracycline derivative). (<b>b</b>): MSC colony (MSC-<i>TetR</i>) in culture medium containing 500 μg/mL geneticin 10 days after transduction with the pLenti3.3/<i>TetR</i> expression vector (bar; 60 μm). (<b>c</b>): Expression of TetR repressor gene in MSCs (without transduction) and MSCs-<i>TetR</i> was determined by RT-PCR. HEK293 cells subjected to the same transduction procedure (HEK-<i>TetR</i>) were used as a positive control. (<b>d, e</b>): MSCs-<i>TetR</i> were lentivirally transduced with the expression vector pLenti6.3/TO/V5/<i>Amelx</i> or plenti6.3/V5-GW/<i>GFP</i>. MSCs-<i>TetR</i>/<i>Amelx</i> (<b>d</b>) and MSCs-<i>TetR</i>/<i>GFP</i> (<b>e</b>) were selected by 10 μg/mL blastcidin S (bar; 200 μm).</p

Effects of forced expression of <i>Amelx</i> on ALP activity and mineralized nodule formation.

<p>MSCs-<i>TetR</i>/<i>Amelx</i> at 3 passages were cultured in the growth medium (Con) or osteogenic induction medium (Os) in the presence (<b>+</b>) or absence (-) of Dox in 24-well plates. (<b>a</b>) ALP activity on day 7 and 14 was examined by ALP staining (bars: 100 μm). (<b>b</b>) Mineralized nodule formation on day 21, 28 and 35 was detected by von Kossa staining. (<b>c, d</b>): Calcium deposition was determined by Alizarin Red S staining on day 7, 14 and 21 (<b>c</b>) and the staining intensity was quantitatively analyzed (<b>d</b>). Significant differences (*<i>P</i><0.01: ANOVA with Tukey’s multiple comparison test: n = 9) are shown.</p

Effects of forced expression of <i>Amelx</i> at different osteogenic differentiation stages on matrix calcification of MSCs.

<p>MSCs-<i>TetR</i>/<i>Amelx</i> at 8 passages were cultured in the growth medium (Con) or osteogenic induction medium (Os). Dox was applied to MSCs-<i>TetR</i>/<i>Amelx</i> on day 0–10 (<b>a</b>: early stage), day 10–20 (<b>b</b>: intermediate stage), or day 20–30 (<b>c</b>: late stage) of osteogenic differentiation, and Alizarin Red S staining was performed at day 10, day 20, or day 30, respectively. The staining intensity was also quantitatively analyzed. Significant differences (*<i>P</i><0.01: ANOVA with Tukey’s multiple comparison test: n = 9) are shown.</p

Controllable expression of <i>osterix</i>, <i>BSP</i> and <i>osteocalcin</i> in MSCs-<i>TetR</i>/<i>Amelx</i> by Dox treatment.

<p>MSCs-<i>TetR</i>/<i>Amelx</i> were cultured in the osteogenic induction medium in the presence (+) (black bars) or absence (-) (white bars) of Dox for 17 days in four different conditions. (Condition I: day 0–17 Dox-; Condition II: day 0–14 Dox-, day 15–17 Dox+; Condition III: day 0–14 Dox+, day 15–17 Dox-; Condition IV: day 0–17 Dox+). Expression of <i>Runx2</i> (<b>a</b>), <i>osterix</i> (<b>b</b>), <i>BSP</i> (<b>c</b>), <i>osteopontin</i> (<b>d</b>) and <i>osteocalcin</i> (<b>e</b>) was determined by a quantitative real-time RT-PCR analysis. Significant differences (**<i>P</i><0.01, *<i>P</i><0.05: ANOVA with Tukey’s multiple comparison test: n = 3) within each condition are shown.</p

Effects of forced expression of <i>Amelx</i> in MSCs-<i>tetR</i>/<i>Amelx</i> on expression of osteogenic marker genes.

<p>MSCs-<i>TetR</i>/<i>Amelx</i> were cultured in growth medium (Con) or osteogenic induction medium (Os) in the presence (<b>+</b>) or absence (-) of Dox for 21 days. (<b>a</b>) The expression of osteogenic marker genes (<i>Runx2</i>, <i>osterix</i>, <i>osteocalcin</i>, <i>BSP</i>, and <i>osteopontin</i>) was examined by RT-PCR analysis. <i>GAPDH</i> was used as an internal control. Quantitative real-time RT-PCR analysis was performed to examine expression of <i>osterix</i> (<b>b</b>), <i>type I collagen</i> (<b>c</b>), <i>BSP</i> (<b>d</b>), and <i>DMP1</i> (<b>e</b>) genes. <i>GAPDH</i> was used as an internal control. Significant differences (*<i>P</i><0.01: ANOVA with Tukey’s multiple comparison test: n = 3) are shown.</p