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. 2006 Sep 11;174(6):827–838. doi: 10.1083/jcb.200512066

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Copyright © 2006, The Rockefeller University Press

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Figure 2. — ApAF induced ERE-mediated gene expression and LTF by interacting with ApC/EBP. (A) Transcriptional activity of ApAF, ApC/EBP, or ApAF–ApC/EBP via the ERE sequence in A. kurodai sensory neurons. +, DNA constructs injected with ERE-luciferase reporter into sensory cells in the pleural ganglia. Either one (1×) or five pulses (5×) of 5-HT were applied to sensory cells. ERE-driven reporter gene expression was increased by ApAF (n = 4), ApC/EBP (n = 4), or ApAF–ApC/EBP (n = 4) overexpression in comparison to nonexpressing control cells (n = 4; ***, P < 0.001; one-way ANOVA and Newman-Keuls multiple comparison test for groups indicated by open bars). The reporter gene expression was further enhanced by 5-HT treatment in ApAF- (n = 4), ApC/EBP- (n = 5), and ApAF–ApC/EBP-introduced (n = 8) cells (*, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed unpaired t test). Each bar corresponds to normalized mean luciferase activity ± the SEM. Five pulses of 5-HT treatment significantly increased ERE-mediated gene expression (n = 4), whereas one pulse of 5-HT treatment did not (n = 5). (B) The effect and specificity of ApAF dsRNA. The expression level of ApAF mRNA was examined by in situ hybridization. The expression level of ApAF was significantly lower in ApAF dsRNA–injected neurons (n = 5) than in luciferase dsRNA–injected neurons (n = 9; *, P < 0.05; two-tailed unpaired t test), whereas the expression level of ApCREB1a was not affected by ApAF dsRNA injection (n = 6). The percentage of change in mRNA is calculated as the relative expression level of ApAF or ApCREB1a mRNA in ApAF dsRNA–injected neurons compared with luciferase dsRNA–injected neurons. The bar represents the mean ± the SEM. Bar, 25 μm. (C) The effect of ApAF–ApC/EBP heterodimer on LTF. +, DNA constructs injected into sensory cells. EPSPs were measured before and 24 h after one or five pulses of 5-HT were applied to the sensory-to-motor synapse. For shaded bars, injection of ApAF dsRNA completely blocked both the LTF induced by five pulses of 5-HT (n = 11) and the LTF induced by ApC/EBP overexpression combined with one pulse of 5-HT (n = 8), whereas the injection of luciferase dsRNA did not block either type of LTF (5×5-HT, n = 8; 1×5-HT, n = 4; **, P <0.01; one way ANOVA and Newman-Keuls multiple comparison test). For open bars, ApAF overexpression combined with one pulse of 5-HT did not produce LTF (n = 8), whereas ApC/EBP overexpression combined with 5-HT did (n = 13). However, ApAF overexpression enhanced the LTF induced by ApC/EBP and one pulse of 5-HT (n = 17; *, P < 0.05). The noninjected control cells produced normal LTF by five pulses of 5-HT (n = 10). Representative examples of recording traces are shown on the right. (D) STF was not affected by ApAF dsRNA microinjection. The mean percentage of changes in EPSP of ApAF dsRNA–injected cells (n = 4) was not significantly different from that of noninjected control cells (n = 4) or luciferase dsRNA–injected cells (n = 4; P > 0.05; one way ANOVA and Newman-Keuls multiple comparison test). Bars correspond to mean percentage changes ± the SEM in EPSP amplitudes.