Figure 6.

AZT-treated cells upregulate senescence-associated beta galactosidase (SA-B-Gal). Neurosphere-forming cells were treated with a single pulse of AZT (30 μM) 24 h after plating. Subsequent neurospheres were dissociated and the cells were processed for SA-B-Gal histochemistry. (A) Untreated control neurosphere cells show low levels of SA-B-Gal activity as compared to AZT-treated neurosphere cells (B). Graphical representation of the quantitative results (C) shows that SA-B-Gal labeling is approximately 15-fold higher in AZT-treated cells (2% of total control cells vs. 33% of total treated cells; unpaired t-test, p < 0.05). Similar results were obtained with MASC exposed to a single 48 h. pulse of either 0.3 or 30 μM AZT. In this case, untreated control astrocytes (D) show a higher baseline level of SA-B-Gal labeling than dissociated neurospheres, but the AZT-treated astrocytes (E) show a clear and substantial upregulation of this senescence marker. Graphical data for astroctyes (F) shows that 0.3 and 30 μM AZT for 48 h. (green bars) cause a respective 50 and 75% increase in SA-B-Gal+ cells (One-Way ANOVA, Dunnett's multiple comparison test of significance, p < 0.01). However, 24 h. exposure (gray bars) to these concentrations does not significantly alter SA-B-Gal expression. Asterisks indicate values significantly different from control; error bars represent standard deviation. N = 3 for all groups; error bars represent standard deviation.