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1.
FIGURE 6

FIGURE 6. From: The Osterix Transcription Factor Down-Regulates Interleukin-1? Expression in Mouse Osteosarcoma Cells.

Osx expression correlates inversely with lytic phenotype. Nude mice received an intratibial injection of Dunn, Dunn-si-c, Dunn-si-2, DLM8, DLM8-si-c, or DLM8-si-1 cells. The mice were sacrificed 35 d after tumor cell injection. Representative radiographs of the tumor lesions are shown.

Ying Cao, et al. Mol Cancer Res. ;6(1):119-126.
2.
FIGURE 4

FIGURE 4. From: The Osterix Transcription Factor Down-Regulates Interleukin-1? Expression in Mouse Osteosarcoma Cells.

Mutating one Sp1-binding site on IL-1α promoter increased its activity. IL-1α promoter activity was increased after mutating the Sp1-binding site on the IL-1α promoter in Dunn and DLM8 cells. Wild-type, mutant, or irrelevant mutant promoter reporters were transfected into Dunn and DLM8 cells as indicated. After 24 h, luciferase activity was measured. Relative fold activity was calculated as the normalized luciferase value divided by the normalized luciferase value of wild-type promoter reporter.

Ying Cao, et al. Mol Cancer Res. ;6(1):119-126.
3.
FIGURE 2

FIGURE 2. From: The Osterix Transcription Factor Down-Regulates Interleukin-1? Expression in Mouse Osteosarcoma Cells.

Suppression of Osx by specific siRNA. A. Total RNA was extracted from Dunn and DLM8 cells. Osx expression was quantified by Northern blot. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. MC3T3 and K7M2 cells were used as positive and negative control cells, respectively. B. Total protein was extracted from Dunn and DLM8 parental cells, from Dunn-si-c and DLM8-si-c control vector – transfected cells, and from Dunn-si-2 and DLM8-si-1 Osx siRNA – transfected cells. Osx protein was quantified by Western blot.

Ying Cao, et al. Mol Cancer Res. ;6(1):119-126.
4.
FIGURE 3

FIGURE 3. From: The Osterix Transcription Factor Down-Regulates Interleukin-1? Expression in Mouse Osteosarcoma Cells.

IL-1α promoter activity is enhanced and IL-1α protein expression is increased following suppression of Osx. A. The IL-1α promoter reporter construct was transfected into different cells as indicated. Luciferase activity was measured 24 h later. Relative fold activity was calculated as the normalized luciferase value divided by the normalized luciferase value in Dunn or DLM8 cells. B. IL-1α protein in Dunn, Dunn-si-c, Dunn-si-2, DLM8, DLM8-si-c, and DLM8-si-1 cell lysate was quantified by ELISA. Relative fold was calculated as the normalized protein concentration divided by the normalized protein concentration in Dunn or DLM8 cells.

Ying Cao, et al. Mol Cancer Res. ;6(1):119-126.
5.
FIGURE 1

FIGURE 1. From: The Osterix Transcription Factor Down-Regulates Interleukin-1? Expression in Mouse Osteosarcoma Cells.

Effect of Osx on IL-1α protein production and IL-1α promoter activity. A. IL-1α protein levels in cell lysates were quantified by ELISA. The relative fold (pg/mg) was calculated as described in Materials and Methods. B. To determine the basal IL-1α promoter activity, K7M2 cells were transfected with the empty reporter (pGL3-basic), the IL-1α promoter reporter construct (pGL3-IL-1α), or the IL-1α promoter reporter vector in the reverse orientation [pGL3-IL-1α (reversed)]. Renilla luciferase construct was cotransfected for normalization. Luciferase activity was measured 24 h after transfection. Relative fold activity was calculated as the normalized pGL3-IL-1α or pGL3-IL-1α (reversed) activity divided by the normalized pGL3-basic value. C. K7M2 cells were cotransfected with IL-1α promoter reporter construct and either the expression plasmid for Osx (pTE-osx) or the control vector (pTE). Luciferase activity was measured 24 h after transfection. Relative fold activity was calculated as the normalized luciferase value divided by the normalized luciferase value in untreated K7M2 cells. D. IL-1α promoter reporter construct was transfected into K7M2, K7M2-neo, K7M2-osx-1, and K7M2-osx-2 cells. Luciferase activity was measured 24 h after transfection. Relative fold activity was calculated as described for C.

Ying Cao, et al. Mol Cancer Res. ;6(1):119-126.
6.
FIGURE 5

FIGURE 5. From: The Osterix Transcription Factor Down-Regulates Interleukin-1? Expression in Mouse Osteosarcoma Cells.

Osx inhibits IL-1α promoter activity through one Sp1-binding site on IL-1α promoter. Intracellular occupancy of Osx on the IL-1α promoter in Dunn and DLM8 cells. Top, primers used to amplify the putative regulatory and non-regulatory regions present in the IL-1α gene. P1, P2, P3, and P4, four pairs of primers used in the ChIP assay. Arrowheads, boundaries of the PCR products. Formaldehyde cross-linked chromatin samples (1 mg) from mouse osteosarcoma cell lines Dunn or DLM8 were subjected to immunoprecipitation with either affinity-purified Osx antibody or acetylated histone H3 antibody and subjected to PCR amplification with primer pair P1, P2, P3, and P4. Normal rabbit IgG (1 μg) was used as control for each ChIP. Input represents 0.2% of each soluble chromatin fraction used for immunoprecipitation.

Ying Cao, et al. Mol Cancer Res. ;6(1):119-126.

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