Figure EV2. Mitochondrial parameters in ATPIF1‐overexpressing HEK293T cells.

• A–D
  Representative rendering (A) and quantification of morphological parameters (count, B; average volume, C; total volume, D) in HEK293T cells transiently transfected with pcDNA3 or with a plasmid for the overexpression of ATPIF1. The results are representative of three independent experiments. Scale bar = 10 μm.
• E, F
  Representative images (E) and quantification (F) of TMRM staining in HEK293T cells transiently transfected with pcDNA3 or with a plasmid for the overexpression of ATPIF1. The results are representative of five independent experiments. *P = 0.0001 (unpaired Student's t‐test). Scale bar = 10 μm.
• G, H
  Representative traces (G) and quantification (H) of mitochondrial ATP‐dependent light emission in HEK293T cells transiently transfected with pcDNA3 or with a plasmid for the overexpression of ATPIF1. 75 μM N,N‐dicyclohexylcarbodiimide (DCCD) was employed to completely inhibit mitochondrial ATP synthesis. The results are representative of five independent experiments.
• I, J
  Representative traces (I) and quantification (J) of mtAlphi/ECFP ratio in HEK293T cells transiently co‐transfected with pcDNA3 or with a plasmid for the overexpression of ATPIF1. 30 mM Na acetate or 30 mM NH₄Cl was employed to detect the minimum and maximum ratio, respectively. The results are representative of four independent experiments.
• K, L
  Representative immunoblotting for the detection of the ATP5A1 subunit after blue‐native PAGE (K) and quantification (L) of F₁F_O ATP synthase dimers (D) and monomers (M) in HEK293T cells transiently transfected with pcDNA3 or with a plasmid for the overexpression of ATPIF1. The results are representative of three independent experiments. *P = 0.0095 (unpaired Student's t‐test).

Data information: All results are expressed as mean ± SEM.