Figure 10. RNF10 nuclear trafficking regulates the expression level of specific target genes.
(A) Hippocampal neurons (DIV14) were treated with Bic in the presence or absence of MK801, immunolabeled for MAP2 (red), p21WAF1/cip1 (green) and stained with Dapi (blue); scale bar: 10 μm. (B) Histogram showing the quantification of p21WAF1/cip1 signal in the nucleus expressed as % of control (n=15, ***p<0.001 Bic vs control and Bic+MK801 vs Bic; one-way ANOVA, followed by Bonferroni post-hoc test). (C) WB analysis from cell lysates of hippocampal neurons treated with Bic in presence or absence of MK801. The histogram shows the quantification of p21WAF1/cip1 integrated density normalized on tubulin (n=5, **p<0.01 Bic vs control and Bic+MK801 vs Bic; one-way ANOVA, followed by Bonferroni post-hoc test). (D) WB analysis for p21WAF1/cip1 and tubulin performed on cell lysates from DIV14 hippocampal neurons treated for synaptic stimulation (Syn; see Materials and methods) in the presence or absence of MK801. The histogram shows the quantification of p21WAF1/cip1 levels normalized on tubulin (expressed as integrated density) (n=5, **p<0.01, Syn vs control; *p<0.05 Syn+MK801 vs Syn; one-way ANOVA, followed by Bonferroni post-hoc test). (E) WB analysis for RNF10, p21WAF1/cip1 and tubulin performed on cell lysates from hippocampal neurons virally infected with pLKO-shRNF10 or control vector and treated at DIV14 for Syn. The histogram shows the effect of RNF10 silencing on p21WAF1/cip1 expression levels normalized on tubulin (n=4, *p<0.05, Syn vs control and Syn+shRNF10 vs Syn; one-way ANOVA, followed by Bonferroni post-hoc test). (F) WB analysis for p21WAF1/cip1 and tubulin from hippocampal extracts following induction of cLTP in the presence or absence of viral infection with pLKO-shRNF10. The graph shows the modulation of p21WAF1/cip1 levels (following normalization on tubulin) (n=5, *p<0.05, cLTP vs control, **p<0.01, cLTP + pLKO-shRNF10 vs cLTP; one-way ANOVA, followed by Bonferroni post-hoc test). (G) WB analysis for Ophn1 and tubulin performed on cell lysates from hippocampal neurons virally infected with pLKO-shRNF10 or control vector and treated at DIV14 for Syn. The histogram shows the effect of RNF10 silencing on Ophn1 expression levels normalized on tubulin (n=3, *p<0.05, Syn+shRNF10 vs Syn; one-way ANOVA, followed by Bonferroni post-hoc test).