GFP-Mfn1, but not E209A, is capable of rescuing morphology. (A) Representative images of U-2 OS cells transfected with control siRNA and pAcGFP-C1 empty vector; anti-Mfn1 siRNA and pAcGFP-C1 empty vector; anti-Mfn1 siRNA and siRNA-resistant GFP-Mfn1; or anti-Mfn1 siRNA and siRNA-resistant GFP-Mfn1-E209A. Mitochondria were visualized by immunofluorescence using an anti-TOM20 antibody and native GFP fluorescence of transfected constructs. (B) Graph representing the mean mitochondrial area from cells treated and imaged as in A; ns and ****, P < 0.0001 as calculated by Kruskal–Wallis test. (C) Western blot showing relative protein levels of endogenous and exogenous Mfn1 after control or anti-Mfn1 siRNA treatment. (D) Graph showing the relative fold change of the number of puncta per mitochondrial area in cells transfected with either GFP-Mfn1 or GFP-Mfn1-E209A. *, P = 0.0377 by two-tailed t test; normality determined by Shapiro–Wilk test; n = 5 cells. Scale bar: 5 µm.