Fig. 3.

Nuclear viability in stressed astrocytes. Astrocytes were treated with the indicated concentrations of MG132 24h (1^st hit) and 48h (2^nd hit) after plating. On the third day, astrocytes were stained with the nuclear marker Hoechst and analyzed by a blinded observer using ImageJ software. A–C) Frequency distributions of the number of nuclei corresponding to the indicated nuclear area. The vehicle-treated group is included on all three figures for comparison. D) Average nuclear size of all cellular profiles. E) Average size of all nuclei except those smaller than 53 μm². F) Representative high-power image of Hoechst-stained nuclear profiles. G–I) Frequency distribution of the number of nuclei as a function of Hoechst nuclear staining intensity. The vehicle-treated group is included on all three figures for comparison. J) Average nuclear staining intensity of all cellular profiles. K) Average nuclear staining intensity of all profiles except cells with nuclei smaller than 53 μm². L–O) Nuclear staining intensity as a function of nuclear size. P) Astrocytes were divided into two groups: those with small, bright nuclei (50–150 μm²; staining intensity greater than 20 arbitrary units for the mean gray value measurements) or large nuclei (greater than 150 μm²; all staining intensities). Raw data for this graph are shown in Supplementary Figure 2. Q) Number of TUNEL⁺ cells relative to the total number of Hoechst⁺ nuclei. R) Data presented in panel Q were expressed as a function of the 0 μM 2^nd MG132 hit groups. S) Representative images of TUNEL staining. * p ≤ 0.05, ** p ≤ 0.01 vs 0 μM 2^nd MG132 hit; + p ≤ 0.05, ++ p ≤ 0.01, +++ p ≤ 0.001 vs 0 μM 1^st MG132 hit; ^ p ≤ 0.05, ^^ p ≤ 0.01 vs small, bright nuclei; two- or three-way ANOVA followed by Bonferroni post hoc correction