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. 2011 Nov 16;31(46):16619–16636. doi: 10.1523/JNEUROSCI.1639-11.2011

Figure 5.

Figure 5.

Dnmts can mediate DNA damage-induced apoptosis of differentiated neurons. A, B, Dnmt1 and Dnmt3a protein levels do not change significantly in DIV5 NSC34 cells exposed to 10 μm CPT for 4–24 h. Representative Western blots are shown along with membranes reprobed for actin to show protein loading. Immunoreactivity optical densities for DNMT1 and Dnmt3a were normalized to actin immunoreactivity. Values in graphs are mean ± SEM of optical density units. C, D, Dnmt1 and Dnmt3a protein levels increase in DIV20 NSC34 cells exposed to 10 μm CPT for 4–24 h. Values in graphs are mean ± SEM of optical density units. Asterisks denote a significant difference from control for Dnmt1 (p < 0.01) and Dnmt3a (p < 0.05). Representative Western blots are shown along with membranes reprobed for actin to show protein loading. E, Dnmt inhibitors protect against CPT-induced apoptosis. DIV20 NSC34 cells were treated with either 20 μm RG108 or 0.5 mm procainamide for 2.5 h in optimem medium. The medium with inhibitors was removed, and the cells were treated with 10 μm CPT for 24 h. The cells were fixed and stained with Hoechst 33258. The percentages of apoptotic cells were determined by counting total cell nuclei (n = 1000 cells per condition) and the proportion of apoptotic nuclei. Values in graphs are mean ± SEM. Double asterisks denote a significant difference from control for CPT (p < 0.001). There is a significant difference from CPT-treated cells for RG108 (++p < 0.01) and procainamide (+p < 0.05). F, RNAi for Dnmt3a protects against CPT-induced apoptosis. DIV14 NSC34 cells were transfected with siRNA construct targeting Dnmt3a or BACE1 (as an independent target), and 24 h later, the cells were treated with 10 μm CPT for 24 h. Cells were prepared as in E. Values in graphs are mean ± SEM. ++Significant difference from control for CPT (p < 0.001); +Significant difference from CPT-treated cells for siRNAi cells (p < 0.01). BACE1 knockdown (control siRNA + CPT) did not protect. G, Western blot analysis of phospho-p53Ser15 in NSC34 cells treated with RG108 (20 μm) and Dnmt3a-siRNA and challenged with 10 μm CPT for 24 h. RG108 dramatically blocked the CPT-induced activation of p53. Dnmt3a-siRNA attenuated the activation of p53 in CPT-treated cells.