with pcDNA3 or with a plasmid for the overexpression of ATPIF1. The results are representative of three independent experiments. Scale bar = 10 lm.
EMBO reports
F1FO ATP synthase disassemble for MPT
Massimo Bonora et al
Expanded View Figures Figure EV1. Validation of F1FO ATP synthase dimerization status. High-resolution image of F1FO ATP synthase dimers and monomers isolated from rat heart mitochondria (HM) by the use of n-dodecyl b-D-maltoside (DM) or digitonin (DIG), upon separation by blue-native PAGE and immunostaining for ATP5A. B Schematic representation of PLA assay setting in physiological conditions (left) or in the course of MPT (right). C Immunoreactivity of the ATP5H-specific antibody used in the PLA assay as compared to an antibody specific for the mitochondrial marker TOMM20. D Maximum intensity projection of z-stack from a representative HEK293T cell stained for the (immuno)fluorescence microscopy-based detection of DNA (DAPI, blue), mitochondria (TOMM20, red), and F1FO ATP synthase dimers (PLA, green). Quantification of the PLA signal colocalizing with TOMM20 is reported (whiskers: max and min; box: 10th and 90th percentile; line: median). E Assessment of transfection efficiency in HEK293T cells transfected with a control siRNA (siCTR) or a siRNA targeting ATP5H (siATP5H) for 96 h. GAPDH levels were monitored to ensure equal lane loading. F, G Representative images (F) and quantification (G) of PLA assays for F1FO ATP synthase dimers in HEK293T cells transfected with siCTR or siATP5H for 96 h. The results are representative of three independent experiments. *P = 0.0001 (unpaired Student’s t-test). H, I Representative images (H) and quantification (I) of PLA assays for F1FO ATP synthase dimers in q0 cells and their wt counterpart HPA11. The results are representative of three independent experiments. ****P = 0.0001 (unpaired Student’s t-test). A
Data information: All results are expressed as mean SEM. Scale bar = 10 lm.
EV1
EMBO reports
ª 2017 The Authors
▸
Massimo Bonora et al
EMBO reports
F1FO ATP synthase disassemble for MPT
A
B F1FO ATP synthase
Signal
(ATP5A)
DM
DIG
-
+
-
+
1.2 MDa (Dimer)
No signal
MPT
0.6 MDa (Monomer) F1FO ATP synthase monomers
F1FO ATP synthase dimer
C
D
TOMM20
siATP5H
GAPDH ATP5H
10000 5000
C
TR
0
PLA TOMM20
4000000 3000000
****
2000000 1000000 0
ρ0
ρ
C on t ce rol lls
PLA TOMM20
15000
I 0
PLA intensity (A.U.)
H
PLA TOMM20
*
20000
si
PLA TOMM20
CTRL
0.0
G siCTR
PLA intensity (A.U.)
si AT P5 H
F si C TR
E
MERGE
0.5
si AT P5 H
ATP5H
PLA DAPI MERGE TOMM20
Mander's Green
1.0
Figure EV1.
ª 2017 The Authors
EMBO reports
EV2
EMBO reports
F1FO ATP synthase disassemble for MPT
Massimo Bonora et al
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 lm. 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 lm. 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 lM 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 F1FO 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.
EV3
EMBO reports
ª 2017 The Authors
▸
Massimo Bonora et al
A
EMBO reports
F1FO ATP synthase disassemble for MPT
pcDNA3
ATPIF1
C
D
Average mitochondrial volume (μm3)
60
40
20
5
250
4
200
3 2 1
150 100 50
0
0
pcDNA3 ATPIF1 E
0
G
F
*
pcDNA3
Δψm (AFU)
6
4
2
H
100
Luminescence (%)
8
TMRM
ATPIF1
pcDNA3 ATPIF1
pcDNA3 ATPIF1
Luciferin
DCCD
100 80
ATP (%)
Mitochondria (n)
80
Total mitochondrial volume (μm3)
B
50
60 40 20
0
0
120 sec 0
pcDNA3 ATPIF1
pcDNA3 ATPIF1 J
K
0.5
10 sec
AT PI F1 pc DN A3
NH4 Cl Na acetate
1.0
0.0
L
0.8
480/425 normalized ratio
480/425 normalized ratio
1.5
0.6
D
0.4
0.2
M
0.0
4
* Dimers/Monomers
I
3
2
1
0
pcDNA3 ATPIF1
pcDNA3 ATPIF1
Figure EV2.
ª 2017 The Authors
EMBO reports
EV4
EMBO reports
F1FO ATP synthase disassemble for MPT
B
D
M
Figure EV3. Dimerization status during ATP5I overexpression.
1.5
Dimers/Monomers
AT P5 G pc 1 DN A3
A
Massimo Bonora et al
A, B Representative immunoblotting for the detection of the ATP5A1 subunit after blue-native PAGE (A) and quantification (B) of F1FO ATP synthase dimers (D) and monomers (M) in HEK293T cells transiently transfected with pcDNA3 or with a plasmid for the overexpression of ATP5G1. The results are representative of three independent experiments and expressed as mean SEM.
1.0
0.5
0.0
pcDNA3 ATP5G1
EV5
EMBO reports
ª 2017 The Authors
Massimo Bonora et al
EMBO reports
F1FO ATP synthase disassemble for MPT
A ATP5G1 + pcDNA3
50
6L
AT
P5
P5
I G2
I
A3 40
6L
I G2
I AT
P5
P5 AT
A3
0 ATP5G1+
N
120 sec
D
6L
AT
P5
P5
60
20
I G2
I
A3 N D
N 80
0
pc
100
pc
2
DCCD Luciferin
ATP (%)
Luminescence (%)
**
AT
P5
D
6L
I G2
I
H 100
4
0 ATP5G1+
20
AT
D pc
*
AT
ATP5G1+ ATP5IG26L
Δψm (AFU)
ATP5G1 + ATP5I
P5
A3
6L
P5 AT
8 6
40
ATP5G1+
G
ATP5G1 + pcDNA3 TMRM
60
0
F
E
80
Total mitochondrial volume (μm3)
0.5
AT
Average mitochondrial volume (μm3)
1.0
0.0 ATP5G1+
I G2
I P5 AT
pc
D
0 ATP5G1+
A3
50
N
Mitochondria (n)
100
D
1.5
pc
C
150
N
B
ATP5G1 + ATP5IG26L
ATP5G1 + ATP5I
Figure EV4. Mitochondrial parameters in ATP5G1- and ATP5I-co-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 a construct for the overexpression of ATP5G1 plus pcDNA3 or a plasmid for the overexpression of ATP5I or ATP5IG26L. The results are representative of three independent experiments. Scale bar = 10 lm. E, F Representative images (E) and quantification (F) of TMRM staining in HEK293T cells transiently transfected with a construct for the overexpression of ATP5G1 plus pcDNA3 or a plasmid for the overexpression of ATP5I or ATP5IG26L. The results are representative of three independent experiments. *P = 0.0060, **P = 0.0345 (Kruskal–Wallis test with Dunn’s correction for multiple comparison). Scale bar = 10 lm. G, H Representative traces (G) and quantification (H) of mitochondrial ATP-dependent light emission in HEK293T cells transiently transfected with a construct for the overexpression of ATP5G1 plus pcDNA3 or a plasmid for the overexpression of ATP5I or ATP5IG26L. 75 lM DCCD was employed to completely inhibit mitochondrial ATP synthesis at the end of the assay. The results are representative of three independent experiments. Data information: All results are expressed as mean SEM.
ª 2017 The Authors
EMBO reports
EV6
