Figure 9.

Analysis of susceptibility of EL-4 cells with down-modulated GD2 to cell death induced by anti-GD2 antibodies. Cell viability (MTT assay, see Methods) of EL-4 cells treated with various concentrations of PDMP (2.5-100 μM) is shown in (A). Flow cytometry analysis of GD2 expression in the control cells vs. the cells with inhibited GD2 biosynthesis (with PDMP or siRNA GM2/GD2 synthase) is shown as the ratio of MFI of cells with reduced GD2 expression to MFI of control cells (B). The expression of GM2/GD2 synthase on mRNA level is shown in (C). The expression of GM2/GD2 synthase on a protein level in EL-4 cells transfected with siRNA that target GM2/GD2 synthase is shown in (D). Cytotoxic effects of anti-GD2 mAb 14G2a (5 μg/ml) on control EL-4 cells vs. cells with inhibited GD2 expression is shown in (E). In (C), RNA from control cells and cells transfected with GM2/GD2 synthase siRNA was isolated, and the expression of mRNA for GM2/GD2 synthase was determined by real time RT-PCR as described in Methods. In (D), expression of GM2/GD2 synthase was analyzed by Western blot as described in Methods. In (E), cytotoxicity was analyzed by PI assay as described in Methods.