Mitochondrial genome maintenance and replication require OPA1-exon4b. (A) Western blot using OPA1 and ACTB1 antibodies of protein extracts (top), and evaluation by Q-PCR (middle) and Southern blot (bottom) of mtDNA copy number from control HeLa cells (Cont) or cells transfected for 72 h with siRNAs targeting all OPA1 mRNA (OPA1), or those including exon 4 (Ex4), 4b (Ex4b), or 5b (Ex5b), show a specific mtDNA loss in cells silenced for OPA1-exon4b transcripts. Note that because OPA1 isoforms including the exon 4b domain account for <25% of all OPA1 isoforms, their silencing can barely be detected by Western blot. (n = 6, one-way ANOVA; [**] P-value inferior to 0.001). (B) Quantification of the relative abundances of OPA1 transcripts including exon 4b (top) and of mtDNA in HeLa cells transfected by the exon 4b siRNA during a 24-h period time course reveals a delay between OPA1-exon4b silencing and the mtDNA loss, without evidencing mtDNA degradation or deletions on the Southern blot (bottom). (n = 3, one-way ANOVA; P-value inferior to 0.01 [*] and 0.001 [**]). (C) Southern–Western blot using anti-BrdU antibodies on DNA extracts (top) and quantification of the replication rate (bottom) in control HeLa cells (Cont) or cells transfected for 48 h with the siRNAs targeting all OPA1 mRNA (OPA1) or those including exon 4 (Ex4), 4b (Ex4b), or 5b (Ex5b) shows a significant 60% decrease of BrdU incorporation in mtDNA, in cells silenced for OPA1-exon4b (n = 3, one-way ANOVA; [*] P-value inferior to 0.01). (D) Southern–Western blot of DNA extracts (top) and quantification of the replication rate (bottom) from HeLa cells transfected with the exon 4b siRNA for the indicated times illustrates a progressive inhibition of mtDNA replication starting readily after OPA1-exon4b silencing (n = 3, one-way ANOVA; [*] P-value inferior to 0.01).
