ATO prevents the transcription of GLI target genes.

<p>Human osteosarcoma cells were cultured with or without 1 µM ATO. An equivalent volume of vehicle was used as the control. Total RNA collected from osteosarcoma cell lines was examined by real-time polymerase chain reaction (PCR). A comparative Ct (ΔΔCt) analysis was performed to examine fold changes in mRNA expression compared with <i>β-actin</i>. Real-time PCR showed that ATO decreased the transcription of GLI target genes, including <i>PTCH1</i>, <i>GLI1</i>, and <i>GLI2</i>, in 143B, Saos2, HsOs1, and U2OS cells. The experiment was performed in triplicate with similar results (error bars represent mean [SD]) (*P < 0.01, **P < 0.05).</p

ATO elicits DNA damage in human osteosarcoma.

<p>COMET assay was performed to detect DNA damage in single cells after ATO treatment. 143B cells were treated with ATO (3 µM) or an equivalent volume of control vehicle for up to 48 h and analyzed by performing the COMET assay. Graphs represent DNA damage by tail length and tail moment, evaluated as described in the Materials and Methods section. These experiments were performed in triplicate with similar results (*P < 0.01).</p

ATO elicits DNA damage and apoptosis.

<p>Human osteosarcoma cells were cultured with or without 3 µM ATO. An equivalent volume of vehicle was used as the control. Western blot analysis was performed 48 h and 72 h after ATO treatment. (A) Western blot analysis revealed that ATO treatment increased the protein levels of γH2AX, cleaved PARP, and cleaved caspase-3. ATO treatment decreased the protein levels of Bcl-2 and Bcl-xL. (B) Western blot analysis performed after cisplatin (CDDP) and recombinant human Sonic Hedgehog (rSHH) treatment showed that CDDP treatment upregulated the expression of γH2AX. Addition of Sonic Hedgehog decreased the expression level of γH2AX protein, which was upregulated by CDDP treatment. (C) Western blot analysis was performed following CDDP and recombinant human Sonic Hedgehog (rSHH) or ATO treatment. Addition of Sonic Hedgehog decreased the expression level of γH2AX protein, which was upregulated by CDDP treatment. Addition of ATO restored the γH2AX expression attenuated by rSHH treatment. These experiments were performed in triplicate with similar results.</p

ATO inhibits anchorage-independent osteosarcoma growth.

<p>Treatment of 143B and Saos2 cells with 3 µM ATO reduced the number of colonies in soft agar at 14 days. An equivalent volume of vehicle was used as the control. These experiments were performed in triplicate with similar results (*P < 0.01) (error bars represent mean [SD]).</p

ATO prevents osteosarcoma growth in vivo.

<p>143B cells (1 × 10⁶) were subcutaneously inoculated into nude mice. Tumor volume was calculated weekly using the formula LW² /2 (where L and W represent the length and width of tumors). Seven days after inoculation, the tumor volume was set as 1 and was evaluated at different time points. (A) ATO treatment inhibited tumor growth as compared with control (<b>*</b>P < 0.05 or **P < 0.01) (error bars represent mean [SD]). Kaplan-Meier analysis revealed that ATO treatment provided a significant survival benefit (**P < 0.01). (B) Apoptotic cell death in the tumors was analyzed by TUNEL staining, which showed that ATO treatment increased apoptotic cell death in vivo (<b>*</b>P < 0.05 or **P < 0.05) (error bar indicates SD).</p

ATO prevents human osteosarcoma cell proliferation.

<p>WST assay showed that the growth of 143B, Saos-2, HsOs1, and U2OS cells was prevented by 1 µM or 3 µM ATO treatment for 96 h. An equivalent volume of vehicle was used as the control. The experiment was performed in triplicate with similar results (*P < 0.05, **P < 0.01) (error bars represent mean [SD]).</p

Effects of overexpression of <i>Bmi1</i> on HSCs <i>in vitro.</i>

<p>(A) Colony formation by HSCs isolated from <i>Tie2-Cre</i> (Control) and <i>Tie2-Cre</i>;<i>R26Stop^FLBmi1</i> (Bmi1) mice. Single CD34^-LSK cells were sorted into 96-well microtiter plates containing the SF-O3 medium supplemented with 10% FBS and multiple cytokines (10 ng/ml SCF, 10 ng/ml TPO, 10 ng/ml IL-3, and 3 u/ml EPO) and allowed to form colonies. At day 14 of culture, the colonies were counted and individually collected for morphological examination. Absolute numbers of LPP and HPP-CFCs which gave rise to colonies with a diameter less and greater than 1 mm, respectively are shown as the mean ± S.D. for triplicate cultures (left panel). Absolute numbers of each colony types were defined by the composition of colonies (right panel). Colonies were recovered and examined by microscopy to determine colony types. Composition of colonies is depicted as n, neutrophils; m, macrophages; E, erythroblasts; and M, megakaryocytes. (B) Colony formation by HSCs cultured for 10 days. CD34^-LSK cells from <i>Tie2-Cre</i> (Control) and <i>Tie2-Cre;R26Stop^FLBmi1</i> (Bmi1) mice were cultured in the SF-O3 serum-free medium supplemented with 50 ng/ml of SCF and TPO. At day 10 of culture, the cells were counted (left panel) and plated in methylcellulose medium to allow formation of colonies in the presence of 20 ng/ml SCF, 20 ng/ml TPO, 20 ng/ml IL-3, and 3 u/ml EPO. Absolute numbers of LPP and HPP-CFCs (middle panel) are shown as the mean ± S.D. for triplicate cultures. Absolute numbers of each colony type are shown in the right panel. (C) Flow cytometric analysis of CD34^-LSK HSCs at day14 of culture. Representative flow cytometric profiles of LSK cells in cultures of CD34^-LSK HSCs from <i>Tie2-Cre</i> (Control) and <i>Tie2-Cre;R26Stop^FLBmi1</i> (Bmi1) mice are depicted. The proportion of Lin^- and LSK cells in total cells are indicated. (D) Quantitative RT-PCR analysis of the expression of <i>p19^Arf,</i> and <i>Bmi1</i> in <i>Tie2-Cre</i> (Control) and <i>Tie2-Cre;R26Stop^FLBmi1</i> (Bmi1) LSK cells. LSK cells were purified by cell sorting from CD34^-LSK cultures in (C) at day 14 of culture. Each value was normalized to <i>Hprt1</i> expression and the expression level of each gene in control cells was arbitrarily set to 1. Data are shown as the mean ± S.D. for triplicate analyses. * <i>p</i><0.05, **<i>p</i><0.01.</p

Generation of mice overexpressing <i>Bmi1</i> in hematopoietic cells.

<p>(A) Strategy for making a knock-in allele for <i>Bmi1</i> downstream of the <i>Rosa26</i> promoter. A <i>loxP</i>-flanked <i>neo^r</i>-stop cassette followed by Flag-tagged <i>Bmi1</i>, an <i>frt</i>-flanked <i>IRES</i>-<i>eGFP</i> cassette, and a bovine polyadenylation sequence was knocked-in the <i>Rosa26</i> locus. (B) Quantitative RT-PCR analysis of <i>Bmi1</i> in BM LSK cells from <i>Tie2-Cre</i> and <i>Tie2-Cre;R26Stop^FLBmi1</i> mice. mRNA levels were normalized to <i>Hprt1</i> expression. Expression levels relative to that in <i>Tie2-Cre</i> LSK cells are shown as the mean ± S.D. (n = 3). (C) Western blotting analysis of Bmi1 in c-Kit⁺ BM cells from <i>Tie2-Cre</i> and <i>Tie2-Cre</i>;<i>R26Stop^FLBmi1</i> mice. α-tubulin was used as the loading control. (D) Hematopoietic analysis of 10-week-old <i>Tie2-Cre</i> and <i>Tie2-Cre;R26Stop^FLBmi1</i> mice. Absolute numbers of BM cells, CD34^-LSK cells, and LSK cells in bilateral femurs and tibiae are presented as the mean ± S.D. (upper panels, <i>Tie2-Cre</i>; n = 7, <i>Tie2-Cre;R26Stop^FLBmi1</i>; n = 8). PB analysis of 10-week-old <i>Tie2-Cre</i> and <i>Tie2-Cre;R26Stop^FLBmi1</i> mice. White blood cell (WBC) counts and lineage contribution of myeloid, B, and T cells are shown as the mean ± S.D. (lower panels, <i>Tie2-Cre</i>; n = 7, <i>Tie2-Cre;R26Stop^FLBmi1</i>; n = 8). ** <i>p</i><0.01.</p

Overexpression of <i>Bmi1</i> protects HSCs during serial transplantation.

<p>(A) Serial transplantation of BM cells. BM cells (5×10⁵) from <i>Tie2-Cre</i> (denoted as “C”) and <i>Tie2-Cre;R26Stop^FLBmi1</i> (denoted as “B”) mice (CD45.2) along with 5×10⁵ competitor BM cells (CD45.2) were transplanted into CD45.1 recipient mice lethally irradiated at a dose of 9.5 Gy. For serial transplantation, BM cells were collected from all recipient mice at 12–20 weeks after transplantation and pooled together. Then, 5×10⁶ BM cells were transplanted into lethally irradiated recipient mice without competitor cells. Third and fourth transplantation were similarly performed using 5×10⁶ pooled BM cells. Percent chimerism of donor cells in the recipient PB and BM LSK cells was determined at 16 weeks post-transplantation. Results are shown as the mean ± S.D. (n = 6, 3^rd transplantation; n = 4). (B) Serial transplantation of cultured CD34^-LSK cells. CD34^-LSK cells were cultured in the SF-O3 serum-free medium supplemented with 50 ng/ml of SCF and TPO for 10 days. Then, the cells in culture corresponding to the 20 initial CD34^-LSK cells were injected into a recipient mouse along with 2×10⁵ competitor BM cells (CD45.2) as described in (A) (n = 6, 4^th transplantation; n = 5). * <i>P</i><0.05, ** <i>P</i><0.01.</p

DNA damage response of <i>Tie2-Cre;R26Stop^FLBmi1</i> HSCs.

<p>(A) DNA damage response of CD34^-LSK cells from <i>Tie2-Cre</i> (Control) and <i>Tie2-Cre;R26Stop^FLBmi1</i> (Bmi1) mice <i>in vitro</i>. Purified CD34^-LSK cells were irradiated (IR) at a dose of 2 Gy. At 2 and 4 hours after irradiation, cells were stained with anti-γH2AX. Numbers of γH2AX foci expressed per cell are depicted. (B) DNA damage response of CD34^-LSK cells from <i>Tie2-Cre</i> (Control) and <i>Tie2-Cre;R26Stop^FLBmi1</i> (Bmi1) mice <i>in vivo</i>. LSK cells were purified from the recipients of tertiary transplantation and stained with anti-γH2AX. Numbers of γH2AX foci expressed per cell are depicted as the mean ± S.D. (n = 3).</p