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. 2007 Apr 25;27(17):4562–4571. doi: 10.1523/JNEUROSCI.5110-06.2007

Figure 5.

Figure 5.

Functional inhibition of p21Waf1/Cip1 reverses the neuroprotective effect of corticosterone. A, Cortical neurons were transiently transfected with rat p21 antisense and sense oligonucleotides 24 h before pretreatment with 50 μm corticosterone (Cort.). Immunocytochemistry was performed with antibodies against p21 (green) or Map-2 (red). Transfection of either antisense or sense oligodeoxynucleotide did not influence the viability of control cultures. Nuclear morphology was assessed by Hoechst staining, and cell counts were performed as described in Materials and Methods. B, Immunocytochemical analysis of p21Waf1/Cip1 expression shows a marked loss of p21Waf1/Cip1 in AF64A- and Cort. (50 μm)-treated neurons after transfection with p21 antisense oligonucleotide with subsequent failure of the neuroprotective effect of corticosterone. Transfection with antisense oligodeoxynucleotide revealed neurons with pronounced (filled arrow), moderate (arrowhead), or weak (open arrow) effects on endogenous p21 levels after antisense transfection. Scale bar, 30 μm. C, Primary cortical neurons derived from p21Waf1/Cip1+/+ or p21Waf1/Cip1−/− mice were treated with 10 μm Cort. and 20 h later with AF64A. LDH release into the supernatant was measured after 72 h. #p < 0.001 versus corresponding control; *p < 0.001 versus AF64A plus Cort. in p21Waf1/Cip1−/− cultures; +p < 0.001 versus AF64A in p21Waf1/Cip1+/+ cultures. D, Naive cells were incubated with propidium iodide (PI; 48 h after AF64A), and digital images were taken with inverse fluorescence or phase contrast. Viable neurons were counted and shown as a percentage of all neurons as described in Materials and Methods. #p < 0.001 versus corresponding control; *p < 0.001 versus AF64A plus Cort. in p21Waf1/Cip1−/− cultures; +p < 0.001 versus AF64A in p21Waf1/Cip1+/+ cultures. E, Representative pictures in phase contrast and red fluorescent PI were merged and shown from cortical neurons derived from p21Waf1/Cip1+/+ and p21Waf1/Cip1−/− mice. Neurons were treated with Cort. (10 μm) and 20 h later with AF64A. PI staining was performed 48 h later. Scale bar, 30 μm.