Article Figures & Data
Figures
- Fig. 1.
Notch and HES1 expression are correlated with metastatic phenotype in osteosarcoma cells and tumors of osteosarcoma patients. A, reverse transcription–PCR analysis of Notch pathway genes (ligand DLL1, receptors Notch1-Notch4, and downstream targets HES1, HES5, HERP2 and DTX1) in normal human osteoblast cells (hOSB), four human osteosarcoma cells, and two metastatic osteosarcoma tumor specimens (1 and 2). OS187 and COL are primary metastatic osteosarcoma cell lines. SAOS2 is a nonmetastatic osteosarcoma cell line, and LM7 is a metastatic subline of SAOS2. B, top, real-time PCR quantification of Notch1, Notch2, and HES1 in human osteoblastic and osteosarcoma cells. All Ct values of osteosarcoma cell lines are normalized to actin first. Comparison is made to the human normal osteoblast sample, which is defined as a relative expression of 1. Bottom, gel image shows the quantitative product of the PCR reaction.
- Fig. 2.
Effects of GSI on the cell proliferation and in vitro invasiveness of osteosarcoma cells. A, Notch signaling inhibition by GSI at the indicated concentrations was inferred by HES1 expression at mRNA and protein level. OS187 cells were cultured with GSI for 48 h. B, Matrigel transwell assay of OS187 cells (5 × 104 cells per well) treated by GSI. Histograms depict the mean number of transmigrated cells at 48 h from three wells for each treatment. Invasion of OS187 cells was decreased significantly by GSI at 0.1, 0.3, and 1 nmol/L concentration (**, P < 0.005). C, Matrigel transwell assay of SAOS2 (5 × 104 per well) and LM7 cells (5 × 104 per well) with GSI treatment. LM7 cells showed higher invasive ability than SAOS2 cells (**, P < 0.005), and in vitro invasiveness of LM7 cells was significantly reduced by GSI at 0.1, 0.3, and 1 nmol/L (**, P < 0.005). Top, histogram depicts quantified invasiveness as in B; bottom, gel depicts reverse transcription–PCR analysis of HES1 and actin expression in LM7 cells treated with GSI, suggesting the inhibition of Notch signaling. D, in vitro invasion assay of COL cells with GSI treatment. Data are depicted as in C. Invasiveness and HES1 expression of COL cells were not affected by GSI.
- Fig. 3.
Effects of manipulation of Notch signaling on osteosarcoma invasiveness in vitro. A-C, top, relative invasiveness in vitro of OS187 cells (1 × 104 per well; A), SAOS2 cells (1.5 × 105 per well; B), and LM7 cells (5 × 104 per well; C) transduced with ICN1, dnMAM, or HES1. Note: the input of cells for each cell line was adjusted to give roughly equal number of invading cells for each vector control sample. Histograms depict the quantified invasiveness. **, P < 0.005; *, P < 0.05. Bottom, gel depicts PCR analysis of HES1 and actin in the transduced cells.
- Fig. 4.
Effects of GSI (1 nmol/L) on in vitro invasiveness of OS187 cells transduced with empty vector, ICN1 (*, P < 0.05), and HES1 (*, P < 0.05). GSI (1 nmol/L) significantly suppressed invasion of OS187 cells transduced with empty vector. However, OS187 cells transduced with ICN1 (A) or HES1 (B) are resistant to GSI treatment. Data are displayed as in Fig. 2C.
- Fig. 5.
Effects of manipulation of Notch signaling on osteosarcoma invasiveness and metastasis in vivo. A and B, representative X-ray images (left) show the osteolytic lesion in the primary tumor site (tibia) of both vector and dnMAM group. Two representative images (right, 40×; left, 400×) show the H&E staining of orthotopic bone tumor (top two) and lung metastasis (bottom two) in vector (A) and dnMAM (B) groups. C, average tibial tumor volume of the OS187 orthotopic osteosarcoma xenograft model using cells transduced with dnMAM or empty vector as control. A representative experiment of three is shown. D, the average number of metastatic foci formed in lung tissues of vector and dnMAM groups is indicated. **, P < 0.005; n = 3 per group in each experiment. All tumor clusters with >10 cells were counted as micrometastases.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figures 1-5, Table 1 - PDF file - 51K
- Supplementary Figure Legends 1-5, Table Legend - PDF file - 27K
Volume 14, Issue 10
