Figure 4. ER‐located Mfn2 is necessary to maintain mitochondrial bioenergetics.

WT 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 three independent experiments). Data are presented as mean ± SEM.
• 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.
• C–F
  mt‐RFP and ER‐Mfn2 or control (CT) plasmid. After 48 h, the cells were fixed. (C) Representative images. Scale bar = 5 µm. (D) Mitochondrial morphology was analyzed (n = 113–422 cells analyzed in three independent experiments) and (E) aspect ratio and (F) circularity calculated (n = 360 mitochondria analyzed in three independent experiments). Data are presented as mean ± SEM.
• 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 three independent experiments). Data are presented as mean ± SEM.
• 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 four independent experiments). Data are presented as mean ± SEM.
• 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 three independent experiments). Data are presented as mean ± SEM.
• 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 three 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 were 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 in Fig 4A and Appendix Fig S4E, Fig 4D and Appendix Fig S4B, Figs 4K and 3I, 4L and 3J, 4M and 3K, 4N and 3L, 4G and Appendix Fig S4G, Fig 4H and Appendix Fig S4H, and Fig 4J and Appendix Fig S4J 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.