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. 2023 Mar 28;6(6):e202301914. doi: 10.26508/lsa.202301914

Figure 2. Mitochondrial fragmentation in SLC25A46 knock-out cells and hyperfusion in pathogenic variants.

Figure 2.

(A) Mitochondrial morphology of control fibroblasts, the knock-out cell line, cells with the reintroduced SLC25A46 protein (WT, p.T142I, p.R257Q, and p.E335D), and the Leigh syndrome patient cell line (p.T142I) was analyzed by immunofluorescence. Representative images of fibroblasts decorated with anti-PRDX3 (mitochondria) in white and DAPI (nuclei) in blue. A zoomed image of the indicated area is shown on the right. Scale bars: 10 μm. (B) Quantification of cells according to their mitochondrial network morphology (randomized, n > 60 cells per condition, N = 3; data are shown as the mean + SEM). Statistical analysis was done using an unpaired t test. Blue stars indicate the t test analysis fraction of elongated cells from the total amount of cells compared with the control cell line. Red stars indicate t test analysis fraction of fragmented cells from total cells compared with knock-out. (C) Live-cell imaging analysis of fibroblasts expressing a mitochondrial-targeted photoactivatable GFP (OCT-PAGFP) probe. Representative images of the OCT-PAGFP probe diffusion over a 3-min period after activation with the 405-nm laser. Scale bars: 10 μm. (D) Quantification of the GFP signal area 3 min after stimulation for each cell type. Statistical analysis was done by a Wilcoxon signed-rank test. Data represent n > 10. (E) Representative TEM images of the indicated fibroblast cells showing impaired mitochondrial cristae morphology in KO cells and in cells with pathogenic variants. Scale bars: 800 nm.