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Figure 2


Fig. 2. LETM1 downregulation causes mitochondrial swelling. (A) Total-cell lysates from HeLa cells transfected once or twice with siRNA for either GFP (mock) or LETM1 were subjected to immunoblotting with antibodies to LETM1 and GAPDH as a loading control. (B) HeLa cells that stably expressed mitochondrion-targeted Su9-DsRed were transfected with siRNA targeting either GFP (a, control) or LETM1 (b). Then, the expression plasmid, which coexpressed LETM1 and nucleus-targeted GFP, was introduced into the cells transfected with the LETM1-targeting siRNA (c). Live images were obtained by confocal microscopy. Typical images were superimposed. Scale bar, 10 µm. (C) Cells transfected with siRNA (as described for B) were analyzed by immunofluorescence microscopy. Data represent the mean ± s.e. of three independent experiments; 100-200 individual cells were counted. (D) HeLa cells expressing Su9-DsRed were transfected with siRNAs targeting DRP1 (a), DRP1 and LETM1 (b), OPA1 (e), OPA1 and LETM1 (f), or LETM1 (panel d). After siRNA transfection, the plasmid expressing Drp1(K38A)-EGFP was transfected (c,d). Live images were collected by confocal microscopy. Arrowheads indicate large dot structures on the filaments, asterisks indicate cells expressing Drp1-EGFP. Scale bar, 10 µm. Data represent the mean ± s.e. of three independent experiments; 100-200 individual cells were counted. (E) HeLa cells transfected with siRNA targeting GFP (a-d) or LETM1 (e-h) were fixed, permeabilized and immunostained using antibodies against the inner membrane protein mitofilin and the matrix protein mtHsp70. Scale bar, 10 µm. (F) HeLa cells transfected with siRNA targeting either GFP (a, control) or LETM1 (b,c) were fixed and thin sections were visualized by electron microscopy. Arrowheads indicate swollen mitochondria. Scale bar, 950 nm. (G) Cells stably expressing Su9-DsRed were transfected with plasmid expressing LETM1-EGFP and analyzed by confocal microscopy. Inset, sigh-magnification image. Arrowhead indicates the cell overexpressing LETM1-EGFP. Scale bar, 10 µm.