Results: 5

1.

Figure 3. Bortezomib does not alter death receptor expression in TRAIL-resistant glioma cell lines. From: Bortezomib Sensitizes Malignant Human Glioma Cells to TRAIL, Mediated by Inhibition of the NF-?B signaling pathway.

(A). LNZ308 and U373 cells were seeded at 60% confluence, and allowed to attach overnight. Cells were treated with bortezomib as indicated. Cell extracts were prepared, and equal amounts of protein were separated by SDS-PAGE and subjected to Western blotting analysis with the indicated antibodies. (B). Five established glioma cells lines were treated with varying concentrations of bortezomib for 24 h. Cell extracts were prepared, and equal amounts of protein were separated by SDS-PAGE and subjected to Western blotting analysis with the indicated antibodies.

Esther P. Jane, et al. Mol Cancer Ther. ;10(1):198-208.
2.
Figure 2

Figure 2. TRAIL-resistant glioma cells can be sensitized by bortezomib. From: Bortezomib Sensitizes Malignant Human Glioma Cells to TRAIL, Mediated by Inhibition of the NF-?B signaling pathway.

LNZ308 and U373 cells were exposed to bortezomib (5 nM) with or without varying concentrations of TRAIL for 24 h. On the following day, the media was changed and complete media was added and cells were grown for additional 14 d in the absence of inhibitors. Control cells were treated with equivalent concentrations of vehicle (top). Colonies were fixed and processed as described in the Materials and Methods. In parallel, cells were treated with TRAIL (T, 5 ng/ml) or bortezomib (B, 5 nM) or the combination of both (TB) for 48 h. Cell extracts were prepared, and equal amounts of protein were separated by SDS-PAGE and subjected to Western blotting analysis with the indicated antibodies. β-actin served as loading control (bottom).

Esther P. Jane, et al. Mol Cancer Ther. ;10(1):198-208.
3.
Figure 5

Figure 5. p65/NF-κB Knockdown induces TRAIL cytotoxicity in TRAIL-resistant glioma cells. From: Bortezomib Sensitizes Malignant Human Glioma Cells to TRAIL, Mediated by Inhibition of the NF-?B signaling pathway.

(A). LNZ308 and U373 cells were transfected with non-target or specific shRNA against p65/NF-κB as described in Materials and Methods. Forty eight hours post-transfection, cell lysates were collected and protein was separated by SDS-PAGE. Western blot analysis and densitometric quantitation was performed as described in the Materials and Methods. β-actin served as loading control. p65/NF-κB/β-actin ratio shown below. This experiment was repeated two times, yielding comparable results (top). In parallel, the relationship between four different shRNA directed against p65/NF-κB and cell numbers was assessed semiquantitatively by spectrophotometric measurement of MTS bioreduction. Non-target shRNA served as control. Points represent the mean of three measurements ± S.D (bottom). ** P <0.005 values considered statistically significant. (B). LNZ308 and U373 cells were transfected with non-target shRNA or shRNA directed against p65/NF-κB (shRNA-1). After forty eight hours, media was aspirated and cells were treated with TRAIL at the indicated concentrations, and after 72 hours, MTS assays were performed as described in Materials and Methods. The relationship between p65/NF-κB and TRAIL concentration and cell numbers was assessed semiquantitatively by spectrophotometric measurement of MTS bioreduction. Points represent the mean of three measurements ± S.D.

Esther P. Jane, et al. Mol Cancer Ther. ;10(1):198-208.
4.

Figure 4. Bortezomib inhibits NF-κB DNA-binding activity in glioma cell lines. From: Bortezomib Sensitizes Malignant Human Glioma Cells to TRAIL, Mediated by Inhibition of the NF-?B signaling pathway.

(A). Nuclear extract was prepared from eight established malignant human glioma cell lines. NFκB DNA-binding was assessed by electrophoretic mobility shift assay (EMSA) as described in the Materials and Methods. (B). LN18, U87, LNZ308, and U373 cells were seeded at 60% confluence, allowed to attach overnight. Cells were treated with varying concentrations of bortezomib for 24 h. Nuclear extracts were prepared, and NF-κB DNA-binding activity was measured as described in the Materials and Methods. The values represent the mean ± standard deviation for two separate experiments performed in triplicate (*p < 0.05; **p < 0.005, bortezomib treated versus untreated control). (C). Glioma cells were treated with varying concentrations of bortezomib for 24 h. Cell extracts were prepared, and equal amounts of protein were separated by SDS-PAGE and subjected to Western blotting analysis and densitometric measurement was performed as described in the Materials and Methods. (D). LNZ308 and U373 were treated with TRAIL (5ng/ml) or bortezomib (5nM) or the combination of both for 24 h. NF-κB DNA-binding was assessed by electrophoretic mobility shift assay (EMSA) as described in the Materials and Methods.

Esther P. Jane, et al. Mol Cancer Ther. ;10(1):198-208.
5.

Figure 1. TRAIL inhibits cell proliferation and induces apoptosis in malignant human glioma cell lines. From: Bortezomib Sensitizes Malignant Human Glioma Cells to TRAIL, Mediated by Inhibition of the NF-?B signaling pathway.

(A). Logarithmically growing established human glioma cell lines were incubated with varying concentrations of TRAIL for 3 days. The relationship between TRAIL concentration and cell numbers was assessed semiquantitatively by spectrophotometric measurement of MTS bioreduction. Points represent the mean of three measurements ± S.D. Control cells (O) were treated with equivalent concentrations of vehicle (top). Eight established malignant human glioma cells were seeded on chamber slides and allowed to attach overnight. Cells were treated with or without TRAIL (5 ng/ml) for 24 h. Images were taken using phase contrast microscopy. Images were assembled using Adobe Photoshop CS2 software (Adobe Systems). TRAIL induces morphological changes such as cell shrinkage, rounding, and membrane blebbing in TRAIL-sensitive and moderately sensitive cells but not in the resistant cell lines (bottom). (B). LN18, U87, LNZ308, and U373 cells were seeded at 60% confluence, allowed to attach overnight, and treated with indicated concentrations of TRAIL. Cell extracts were prepared, and equal amounts of protein were separated by SDS-PAGE and subjected to Western blotting analysis with indicated antibodies (top). LN18, LN229, LNZ308, and U373 cells were exposed to TRAIL for 24 h. On the following day, the media was changed and complete media was added and cells were grown for additional 14 d in the absence of inhibitors. Control cells were treated with equivalent concentrations of vehicle. Colonies were fixed and processed as described in the Materials and Methods (bottom). (C). Eight established human glioma cells were seeded at 60% confluence, allowed to attach overnight. Cell extracts were prepared, and equal amounts of protein were separated by SDS-PAGE and subjected to Western blotting analysis with indicated antibodies. β-actin served as loading control. (D). LN18, U87, LNZ308, and U373 cells were treated with indicated concentrations of TRAIL for 2 days. Cell extracts were prepared, and equal amounts of protein were separated by SDS-PAGE and subjected to Western blotting analysis with indicated antibodies. β-actin served as loading control.

Esther P. Jane, et al. Mol Cancer Ther. ;10(1):198-208.

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