Figure 3
Mechanism of DC-mediated AID induction. (A-B) Dependence of AID expression on DC–tumor cell contact. (A) Myeloma (U266, OPM2, CAG, ARK) or breast cancer (MCF-7) cells were cocultured with Mo-DCs as in Figure 1A, in the presence of transwell inserts to prevent cell-to-cell contact but permit interaction of soluble mediators. AID expression in purified tumor cells is analyzed by quantitative PCR. Data are represented as fold change compared with tumor cells cultured alone, as in Figure 1A. (B) U266 myeloma cells re-isolated from DC-tumor cocultures were cultured alone without DCs to analyze whether the expression of AID required continued DC–tumor contact. Tumor cells were harvested at indicated time points for analysis of AID expression. Data are expressed as fold change relative to tumor cells alone. (C) Effect of DC maturation status. MM cells (U266, OPM2, ARK, CAG) or breast cancer (MCF-7) cells were cultured with immature Mo-DCs, or those matured with LPS or cytokine cocktail. Induction of AID in purified tumor cells was analyzed by quantitative PCR as in Figure 1A. Data are expressed as fold change relative to tumor cells alone. (D-H) Role of RANK/RANKL interactions in DC-mediated AID induction. (D) Lysates from MM cells (U266) either cultured alone or after exposure to DCs as in Figure 1A was analyzed for the expression of RANKL protein by Western blot analysis with the use of an anti-RANKL Ab or β-actin as a loading control. (E) U266 cells were cocultured with or without DCs, and protein was isolated. Lysates were subjected to Western blot analysis with anti–NF-κB (total and phospho) Abs or β-actin as a loading control. (F) MM cells (U266, OPM2) were cocultured with Mo-DCs in the presence of OPG (0.5 μg/mL) to inhibit RANK/RANKL interactions or human IgG as a control. Data are represented as percentage change in induction of AID mRNA relative to control. (G) U266 cells were cocultured with Mo-DCs in the presence of OPG or human IgG as a control (0.5 μg/mL) to inhibit RANK/RANKL signaling, and protein was isolated. Lysates were subjected to Western blot analysis with anti–NF-κB (total and phospho) Abs or β-actin as a loading control. (H) MM cell line (U266) was cocultured with Mo-DCs in the presence of anti-RANKL Ab (1 μg/mL) to inhibit RANK/RANKL interactions or human IgG as a control. Data are represented as percentage change in induction of AID mRNA relative to control.

Mechanism of DC-mediated AID induction. (A-B) Dependence of AID expression on DC–tumor cell contact. (A) Myeloma (U266, OPM2, CAG, ARK) or breast cancer (MCF-7) cells were cocultured with Mo-DCs as in Figure 1A, in the presence of transwell inserts to prevent cell-to-cell contact but permit interaction of soluble mediators. AID expression in purified tumor cells is analyzed by quantitative PCR. Data are represented as fold change compared with tumor cells cultured alone, as in Figure 1A. (B) U266 myeloma cells re-isolated from DC-tumor cocultures were cultured alone without DCs to analyze whether the expression of AID required continued DC–tumor contact. Tumor cells were harvested at indicated time points for analysis of AID expression. Data are expressed as fold change relative to tumor cells alone. (C) Effect of DC maturation status. MM cells (U266, OPM2, ARK, CAG) or breast cancer (MCF-7) cells were cultured with immature Mo-DCs, or those matured with LPS or cytokine cocktail. Induction of AID in purified tumor cells was analyzed by quantitative PCR as in Figure 1A. Data are expressed as fold change relative to tumor cells alone. (D-H) Role of RANK/RANKL interactions in DC-mediated AID induction. (D) Lysates from MM cells (U266) either cultured alone or after exposure to DCs as in Figure 1A was analyzed for the expression of RANKL protein by Western blot analysis with the use of an anti-RANKL Ab or β-actin as a loading control. (E) U266 cells were cocultured with or without DCs, and protein was isolated. Lysates were subjected to Western blot analysis with anti–NF-κB (total and phospho) Abs or β-actin as a loading control. (F) MM cells (U266, OPM2) were cocultured with Mo-DCs in the presence of OPG (0.5 μg/mL) to inhibit RANK/RANKL interactions or human IgG as a control. Data are represented as percentage change in induction of AID mRNA relative to control. (G) U266 cells were cocultured with Mo-DCs in the presence of OPG or human IgG as a control (0.5 μg/mL) to inhibit RANK/RANKL signaling, and protein was isolated. Lysates were subjected to Western blot analysis with anti–NF-κB (total and phospho) Abs or β-actin as a loading control. (H) MM cell line (U266) was cocultured with Mo-DCs in the presence of anti-RANKL Ab (1 μg/mL) to inhibit RANK/RANKL interactions or human IgG as a control. Data are represented as percentage change in induction of AID mRNA relative to control.

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