Mitochondrial tethering assay and mitochondrial contact in swelled cells. (A) Scheme of the in vitro mitochondrial tethering assay. HEK293T cells were transfected with siRNA against MFN1/2 (siMFN1/2). After 2 d, cells were co-transfected with an equal amount of NME3-HA and mito-YFP (or mito-DsRed). Isolated mitochondria labeled with YFP and DsRed were mixed for in vitro tethering. (B) Mitochondria large intracellular vesicles (LICVs) distribution in exogenous NME3 expressing cells. Hela cells expressing indicated Tom20-GFP or NME3-GFP together with Tom20-mCherry were incubated in 5% DMEM for 10 min. Cells before and after the hypotonic swelling were imaged and shown. Scale bar, 10 μm. (C) The representative mitochondrial LICVs were magnified and shown in C, scale bar of 2 μm. (D and E) The contact angel and length between closely positioned mitochondrial LICVs were quantified by ImageJ and shown in D and E, respectively. Data are expressed as mean ± SD and analyzed with one-way ANOVA. *P < 0.05; ***P < 0.001; ns, not significant.