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. 2011 Jan;21(1):12–20. doi: 10.1101/gr.108696.110

Figure 3.

Figure 3.

OPA1-exon4b N terminus colocalizes with mtDNA and rescues exon 4b silencing. The human exon 4b peptide sequence (top) stands above the corresponding wild-type and mutagenized exon 4b nucleotide sequences. The sequence targeted by exon 4b siRNA is boxed on the wild-type sequence, and the mutagenized sequence used in expression vectors to prevent cross-reaction with the siRNA 4b is underlined on the bottom sequence. (A) Merged Cherry and anti-DNA fluorescences from HeLa cells transfected by the plasmids expressing NT-OPA1-exon4-cherry (top) and NT-OPA1-exon4b-cherry (bottom) show the preferential colocalization of the mitochondrial nucleoid with the NT-OPA1-exon4b peptide. (Bottom) Quantification of the colocalization in both conditions (n = 12 cells, t-test; [*] P-value inferior to 0.01). (B, left) Quantification of mtDNA copy number from wild-type cells, cells transfected by the exon 4b siRNA (si4b), or cells transfected with exon 4b siRNA plus a plasmid expressing NT-OPA1-exon4 (si4b + NT-4) or NT-OPA1-exon4b (si4b + NT-4b) illustrates the full recovery of mtDNA abundance in cells transfected by exon 4b siRNA and expressing the NT-OPA1-exon4b peptide and partial recovery in those expressing the NT-OPA1-exon4 peptide. (n = 3, one-way ANOVA; P-value inferior to 0.01 [*] and 0.001 [**]). (Right) Merged fluorescence pictures using anti-DNA and Mitotracker, showing the mtDNA distribution in control and exon 4b–silenced HeLa cells. (Bottom) Merged fluorescences of antiDNA and Cherry protein in cells cotransfected by the exon 4b siRNA and the NT-OPA1-exon4 (left) and NT-OPA1-exon4b encoding plasmids, showing the rescue of mtDNA distribution in cells expressing NT-OPA1-exon4b.