Figure 4WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: A) mt-RFP and ER-GFP and ER-targeted Mfn2 (ER-Mfn2) or a control plasmid (CT). Mitochondria-ER colocalization was analyzed using Mander's coefficient (n= 15 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM. B Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity. WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: B) GFP and ERMITO-RLuc and ER-Mfn2 or control plasmid. After 24 h, the transfected cells were sorted and plated for another 24 h, after which RLuc was analyzed (n= 4 independent experiments). Data are presented as mean ± SEM. WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: mt-RFP and ER-Mfn2 or control (CT) plasmid. After 48 h, the cells were fixed. (C) Representative images. Scale bar= 5 μm. WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: mt-RFP and ER-Mfn2 or control (CT) plasmid. After 48 h, the cells were fixed. (D) Mitochondrial morphology was analysed (n=113-422 cells analyzed in 3 independent experiments) and (E) aspect ratio and (F) circularity calculated (n=360 mitochondria analyzed in 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity. WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: G) GFP and ER-Mfn2 or control (CT) plasmid. Mitochondrial Ca2+ was determined. Mitochondria were uncoupled with CCCP (10 μM) as indicated (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity. WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: H) LAR-GECO1 and ER-Mfn2 or control (CT) plasmid. ER Ca2+ levels were determined. ER Ca2+ release was induced with caffeine (20 mM) as indicated (n= 35 cells from 4 independent experiments). Data are presented as mean ± SEM Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity. WT and Mfn2 KO MEFs were co-transfected with plasmids encoding I) GFP and ER-Mfn2 or control (CT) plasmid. The effect of caffeine-induced (20 mM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: J-N) GFP and ER-Mfn2 or control (CT) plasmid. (J) MMP was determined. The values were normalized to surrounding untransfected cells. (n= 90 cells analyzed in 3 independent experiments). (K) After 24 h, the transfected cells were sorted and plated for another 24 h, after which cells were treated with 2-DG (10 mM) for 6 h, and ATP levels were measured (n= 3 independent experiments). (L) Oxygen consumption was measured and (M) RCR and (N) SRC was calculated (n= 4 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.