Figure 4. scFv-EM48 suppresses cytotoxicity of mutant htt. (A and B) HEK293 cells were transfected with htt (1–208 aa) containing 23Q or 130Q and scFv-EM48 (+scFv). Cotransfection with a vector served as a control. SYTOX green staining of transfected HEK293 cells is shown in A, in which greater fluorescence is correlated with increased cell death. In B, the relative fluorescence values of SYTOX green–containing cells were obtained from six transfection experiments. Cell viability was also measured by a modified MTT assay (MTS) and expressed as absorbance at 490 nm (OD490 nm absorbance values; n = 9, P < 0.05). The decreased MTS correlates with reduced cell viability. (C) Immunofluorescent staining of cultured rat cortical neurons (7 days in vitro) that express transfected htt (htt-23Q or htt-130Q) or scFv-EM48 alone (top). Htt was labeled by rabbit EM48, and scFv-EM48-HA was labeled by antibody to the HA epitope. Note that mutant htt formed neuritic aggregates and caused neuritic fragmentation in transfected cells. Coexpression of scFv-EM48 reduced htt aggregation in neurites and degeneration of cultured neurons (bottom). Nuclei were stained with Hoechst blue dye in the merged images. (D) The percentage of cells showing disrupted neurites and fragmented nuclei. Data were obtained by counting 156–198 transfected cells in three transfection experiments. The data are presented as means ± standard error. *, P < 0.05; **, P, < 0.01. Bars: (A) 100 μm; (C) 8 μm.
Figure 4.

scFv-EM48 suppresses cytotoxicity of mutant htt. (A and B) HEK293 cells were transfected with htt (1–208 aa) containing 23Q or 130Q and scFv-EM48 (+scFv). Cotransfection with a vector served as a control. SYTOX green staining of transfected HEK293 cells is shown in A, in which greater fluorescence is correlated with increased cell death. In B, the relative fluorescence values of SYTOX green–containing cells were obtained from six transfection experiments. Cell viability was also measured by a modified MTT assay (MTS) and expressed as absorbance at 490 nm (OD490 nm absorbance values; n = 9, P < 0.05). The decreased MTS correlates with reduced cell viability. (C) Immunofluorescent staining of cultured rat cortical neurons (7 days in vitro) that express transfected htt (htt-23Q or htt-130Q) or scFv-EM48 alone (top). Htt was labeled by rabbit EM48, and scFv-EM48-HA was labeled by antibody to the HA epitope. Note that mutant htt formed neuritic aggregates and caused neuritic fragmentation in transfected cells. Coexpression of scFv-EM48 reduced htt aggregation in neurites and degeneration of cultured neurons (bottom). Nuclei were stained with Hoechst blue dye in the merged images. (D) The percentage of cells showing disrupted neurites and fragmented nuclei. Data were obtained by counting 156–198 transfected cells in three transfection experiments. The data are presented as means ± standard error. *, P < 0.05; **, P, < 0.01. Bars: (A) 100 μm; (C) 8 μm.

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