Figure 2. Lentivirus-mediated expression of R424H mutant subunits in cerebellar cultures decreases the density of Purkinje cells (PCs) but not of granule cells.

A–C, immunofluorescence images of cerebellar cultures infected with lentiviral vectors expressing green fluorescent protein (GFP) alone (Aa–c), WT subunits and GFP (Ba–c) or R424H mutant subunits and GFP (Ca–c; see Methods). Green fluorescent protein fluorescence was enhanced by immunostaining with guinea-pig anti-GFP antibody and AlexaFluor (AF) 488-conjugated goat anti-guinea-pig antibody. Purkinje cells were visualized by immunolabelling with rabbit anti-calbindin antibody (red signals in A–C). Ac′, Bc′ and Cc′, granule cells were selectively immunolabelled with mouse anti-NeuN mouse antibody (see Supplemental Fig. S3A and B). D, relative cell density of PCs plotted as a function of days in vitro (DIV). The density was normalized to the value of PCs expressing GFP alone at DIV 4. E, relative cell density of granule cells at DIV 14. The density was normalized to the mean cell density of granule cells in cultures lentivirally expressing GFP alone at DIV 14. F and G, R424H mutant-expressing PCs exhibiting chromatin condensation. F, representative fluorescence images of PCs at DIV 8. For nucleus detection, the PCs were stained with Hoechst 33342. Normal nuclei of PCs are indicated by arrows (GFP and WT), whereas a nucleus exhibiting chromatin condensation is marked with an arrowhead (R424H). G, summary of the percentages of PCs with chromatin condensation at DIV 8. The statistical analysis was conducted using Student’s unpaired t tests. In the following figures and tables, the statistical analysis was conducted between cells expressing the R424H mutant and those expressing GFP or between cells expressing R424H mutant and those expressing WT subunits. **P < 0.01 and ***P < 0.001.