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 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

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

MOESM1 of Risk factors for bone loss in patients with rheumatoid arthritis treated with biologic disease-modifying anti-rheumatic drugs

Additional file 1: Figure S1. These scatter plot show the reduction of BMD in lumber spine (A) and femoral neck (B)

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 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

RBPJ Is a Novel Target for Rhabdomyosarcoma Therapy

<div><p>The Notch pathway regulates a broad spectrum of cell fate decisions and differentiation processes during fetal and postnatal development. In addition, the Notch pathway plays an important role in controlling tumorigenesis. However, the role of <em>RBPJ</em>, a transcription factor in the Notch pathway, in the development of tumors is largely unknown. In this study, we focused on the role of <em>RBPJ</em> in the pathogenesis of rhabdomyosarcoma (RMS). Our data showed that Notch pathway genes were upregulated and activated in human RMS cell lines and patient samples. Inhibition of the Notch pathway by a γ-secretase inhibitor (GSI) decreased the <em>in vitro</em> proliferation of RMS cells. Knockdown of <em>RBPJ</em> expression by RNAi inhibited the anchorage-independent growth of RMS cells and the growth of xenografts <em>in vivo</em>. Additionally, overexpression of <em>RBPJ</em> promoted the anchorage-independent growth of RMS cells. Further, we revealed that <em>RBPJ</em> regulated the cell cycle in RMS xenograft tumors and decreased proliferation. Our findings suggest that <em>RBPJ</em> regulates the RMS growth, and that the inhibition of <em>RBPJ</em> may be an effective therapeutic approach for patients with RMS.</p> </div

Notch pathway molecules are overexpressed in rhabdomyosarcoma cells.

<p>Notch pathway genes (receptors <i>NOTCH1-4</i>, ligands <i>JAG1</i> and <i>DLL1</i>, target genes <i>HES1</i> and <i>HEY1</i>, and transcription factor <i>RBPJ</i>) were assessed by real-time PCR in a normal human skeletal muscle specimen and 2 human RMS biopsy specimens. The Ct values of all RMS samples were normalized to those of <i>ACTB</i>. The values of the human RMS specimens were compared with those of the human skeletal muscle sample, which is defined as a relative expression of 1.0. Columns, mean values of 3 independent experiments; bar, SD. *<i>p</i><0.05, **<i>p</i><0.01.</p

Knockdown of <i>RBPJ</i> suppresses anchorage-independent growth of rhabdomyosarcoma cells. A,

<p>The expression of <i>RBPJ</i> mRNA in RD cells was assessed by real-time PCR. The Ct values of all RMS cell lines were normalized to those of <i>ACTB</i>. The values of the RMS cell lines were compared with HSkMc cell, which is defined as a relative expression of 1.0. <b>B,</b> RBPJ protein levels in RD cells transfected with control and RBPJ siRNA were examined by western blotting analysis (top). <i>RBPJ</i> and <i>HES1</i> mRNA in RD cells transfected with control and <i>RBPJ</i> siRNA were assessed by real-time PCR analysis. Ct values of <i>RBPJ</i> and <i>HES1</i> were normalized to <i>ACTB</i>. The values of the cells transfected with <i>RBPJ</i> siRNA were compared to those the RD cells transfected with control siRNA, which is defined as a relative expression of 1.0 (bottom). <b>C,</b> Anchorage-independent growth of RD cells transfected with control and <i>RBPJ</i> siRNA were evaluated by colony formation assay. After 3 weeks, each of the colonies were counted and photographed. Scale bar is 200 µM. Columns, mean values of 3 independent experiments; bar, SD. *<i>p</i><0.05, **<i>p</i><0.01.</p