EMBO/soda-nel huggingface dataset:
Mitochondrial F1FO ATP synthase determines the local proton motive force at cristae rims (2021)
Figure 8

Figure 8 -The activity of ATP synthase determines lateral, radial and transmembrane pH gradients.

A) Mitochondrial pH at CV SU e is compared for HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions.

B) Mitochondrial pH at CV SU γ measured in HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions.

C) pH in the matrix bulk (MPP60 as mitochondrial targeting sequence) measured in HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions.

D) pH at respiratory complex IV at SU CoxVIIIa in the ICS measured in HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions.

E) pH differences between SU e and SU γ of ATP synthase SU e is part of FO, SU g of F1. Triple stars in columns indicate that the difference between the compared pH values constituting the ∆pH was significant; p≤0.001.

F) Radial pH gradient between matrix bulk and subunit γ of ATP synthase.

G) pH difference between SU e of ATP synthase and matrix bulk.

H) Transmembrane pH difference between local pH at CIV (CoxVIIIa) and matrix pH.

I) Lateral pH gradient between CIV SU CoxVIIIa and CV SU e. Be aware that the pH under glycolytic conditions at SU e was lower than the pH at CIV in the ICS, resulting in a "negative" ∆pH. (A-I) One-way ANOVA comparison of sets of pH values at the indicated localizations. Box (75%) and whisker plots, median (horizontal line in box), mean (box in box). p≤0.001: ***, p≤0.01: **, p≤0.05: *; n.s.: non-significant (N=3). Statistics: one-way ANOVA with post hoc Schéffe test for normal distribution.

J) pH measurements in mitochondria in live cells expressing sEcGFP as the pH sensor at SU γ. The pH was color coded according to respective emission ratio. The pH at SU γ in IF1-H49K-expressing cells is compared with WT cells. (Scale bar: 1 µm).

K) Schematic drawing showing the resolved pH values and resulting gradients under respiratory and glycolytic conditions. The color gradient was generated following the ratiometric pH code used throughout the study.

Data information: (A-D) One-way ANOVA comparison of sets of pH values at the indicated localizations. Box (75%) and whiskers plots (outlier included, coefficient 1.5). The error bars denote SD, the boxes represent the 25th to 75th percentiles. The vertical lines in the boxes represent the median values, whereas the square symbols in the boxes denote the respective mean values. The minimum and maximum values are denoted by x. error bars = SD. p≤0.001: ***, p≤0.01: **, p≤0.05: *; n.s.: non-significant (N=3, technical replicates from same cell clones, data from individual cells are shown). Statistics: one-way ANOVA with post hoc Schéffe test for normal distribution. (E-I) One-way ANOVA comparison of measured pH values; shown is ∆pH as the calculated difference between the mean pH values from data shown in A-D. p≤0.001: ***, p≤0.01: **, p≤0.05: *; n.s.: non-significant.
Mitochondrial F1FO ATP synthase determines the local proton motive force at cristae rims (2021)
Figure 9

Figure 9 - IF1 is beneficial for ATP production even under OXPHOS conditions.

A) Mitochondrial ATP production rates in HeLa cells the presence and absence of IF1. Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were determined with an automatic flux analyzer (Seahorse/Agilent). After monitoring basal respiration, oligomycin (1 µM), and rotenone plus antimycin A (Rot; 0.5 µM; AA, 0.5 µM) were added sequentially to determine glycolytic and mitochondrial ATP production rates. ~12 technical replicates per measurement, N=4 independent assays.

B) Cytosolic ATP production rates. Each data point represents the mean ATP production rate of one well (~12 technical replicates per measurement, N=4 independent assays.).

C) Determination of mitochondrial ATP levels with the fluorogenic dye ATP-red™ (5 µM, prestaining for 15 min). To normalize on mitochondrial mass, mitochondria were stained with MitoTracker™ Green (MTG, 100 nM for 30 min). Two technical replicates from same clones.

Data information: (A-C) Statistics: One-way ANOVA with post hoc Scheffé test. The error bars denote SD, the boxes represent the 25th to 75th percentiles. Whiskers: outliners include, coefficient 1.5. The vertical lines in the boxes represent the median values, whereas the square symbols in the boxes denote the respective mean values. The minimum and maximum values are denoted by x.***, p≤0.001; **, p≤0.01; n.s.: non-significant.
NDP52 acts as a redox sensor in PINK1/Parkin-mediated mitophagy (2022)
Figure 1

Figure 1. NDP52 forms DLC in response to oxidative stress or mitochondrial damage

A, HeLa cells were treated with H2O2 (5 mM, 1 min) or PR-619 (10 µM, 10 min) and analysed by immunoblotting for endogenous p62, NDP52 and OPTN in either reducing (2.5% β-mE) or non-reducing conditions.

B, NDP52 and OPTN are reducible by the thioredoxin antioxidant system. HeLa cells were pre-treated with TrxR inhibitors, curcumin (50 μM) or auranofin (5 μM) for 30 min, then treated with H2O2 (500 μM) at different time points as indicated. Immunoblotting was performed to detect NDP52, OPTN and PRX-3 (dimer formation reflecting oxidation of the protein) in non-reduced conditions and PRX-SO3 (marker of oxidative stress (Carroll et al., 2018)) and actin in reduced conditions.

C, D, Immunoblotting for NDP52 in either reducing or non-reducing conditions of whole cell samples (C) and mitochondrial fractions (D) from HeLa cells treated with the combination of 4 µM antimycin A1 and 10 µM oligomycin (AO) in the presence or absence of 400 nM bafilomycin A1 (Baf) for 2 h. NDP52 DLC in mitochondrial fraction was quantified (D). Data are mean ± s.e.m (D). P values were calculated by one-way ANOVA followed by Sidak test on four independent experiments (D). ***, p<0.001; ns (non-significant).

Data information: #, non-specific band or a post-translational modification.
NDP52 acts as a redox sensor in PINK1/Parkin-mediated mitophagy (2022)
Figure 2

Figure 2. Cysteine residues 18, 153, 163 and 321 of NDP52 are required to form DLC

A-D, Single (A), double (B), triple (C), and quadruple (D) cysteine mutant NDP52 plasmids were generated and transfected into HeLa PentaKO cells. Cells were treated with 20 µM PR-619 for 10 min and analysed by immunoblotting for NDP52 in either reducing or non-reducing conditions. The constructs highlighted in red were subjected to the next round of Cys-Ala screen or further analyses.

E, Schematic diagram of the NDP52 domain organisation showing the location of the redox sensitive cysteines.

F, Multiple protein sequence alignment of NDP52 sequences in different species. Increasing conservation is shown by light-dark red; cysteine residues 18, 153, 163 and 321 are indicated by green boxes, and these are conserved in primates.
NDP52 acts as a redox sensor in PINK1/Parkin-mediated mitophagy (2022)
Figure 3

Figure 3. Modelling of the NDP52 tetramer and the properties of NDP52 C18,153,163,321S

A, Calculated conformation of the antiparallel NDP52 tetramer and residue interactions after 100 ns of MD simulation. The schematic summarises the predicted orientation of Cys residues within CC domains of the NDP52 tetramer.

B, Recombinant proteins of NDP52 WT and C18,153,163,321S mutant (Mut) were exposed to the indicated concentrations of H2O2 for 5 min and subjected to SDS-PAGE and CBB gel staining.

C, Analytical size-exclusion chromatography (SEC) of the NDP52 WT and Mut proteins.

D, Recombinant NDP52 WT and Mut proteins (5 ng) were subjected to NATIVE-PAGE analysis. S.E, short exposure; L.E. long exposure.
NDP52 acts as a redox sensor in PINK1/Parkin-mediated mitophagy (2022)
Figure 4

Figure 4. Mitochondrial ROS-dependent oxidation of NDP52 is required for the efficient activation of mitophagy

A, B, Fluorescence microscopy images (A) and quantification (B) of mitophagy of HeLa wild-type (WT), PentaKO + empty vector, PentaKO + NDP52 WT or NDP52 C18,153,163,321S (Mut) cells, stably expressing YFP-Parkin and mt-mKeima were pre-treated with or without MitoQ for 22 h and treated with 4 µM / 10 µM AO for the indicated times.

C, HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM / 10 µM AO for 2 h in the absence or presence of 10 μM MG132 (MG) and 400 nM Baf, followed by a mitochondrial fractionation and immunoblotting for NDP52 in non-reducing conditions. The mitochondrial protein UQCRC2 was used as a loading control.

D, E, HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima were treated with 1 mM DFP for 24 h (D), or pre-treated with 500 nM MitoQ for 22 h and treated with 4 µM / 10 µM AO for 2 h (D, E) in the presence or absence of 400 nM Baf (E). Cells were then analysed by MitoSOX staining (D) and immunoblotting for NDP52 in reducing conditions (E).

Data information: #, non-specific band or a post-translational modification. Data are mean ± s.e.m. (B, D) or displayed as cell popular violin plots (B). P values were calculated by one-way ANOVA followed by Sidak test on three independent experiments (B, D). *, p<0.05; **, p<0.01; ***, p<0.001; §, p<0.05, §§§, p<0.001 (relative to MitoQ-untreated condition); ns (non-significant). Scale bars: 20 μm (A, D).
NDP52 acts as a redox sensor in PINK1/Parkin-mediated mitophagy (2022)
Figure 5

Figure 5. NDP52 oxidation is mediated by ROS produced by mitochondrial CI and CIII

A, Fluorescence microscopy images and quantification of mitophagy in HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM rotenone (R), 4 µM A, 10 µM O, or the combination of RO or AO for 3h.

B, Fluorescence microscopy images and quantification of MitoSOX staining in HeLa PentaKO + NDP52 WT cells in the same conditions as (A).

C-E, HeLa PentaKO + NDP52 WT cells (D) or cells stably expressing YFP-Parkin and mt-mKeima (C, E) were pre-treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h, followed by mitophagy measurement (C), MitoSOX staining (D) and immunoblotting for NDP52 in non-reducing conditions (E).

F, G, HeLa PentaKO + NDP52 WT stably expressing YFP-Parkin and mt-mKeima were pre-treated with or without 10 mM DTT for 30 min and treated with AO for 2 h followed by mitophagy measurements (F) and immunoblotting for NDP52 in non-reducing conditions (G).

Data information: #, non-specific band or a post-translational modification. Data are mean ± s.e.m. (A, B, C, D, F) or displayed as cell popular violin plots (A, B, C, F). P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated (A, B, C, D, F). *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm (A, B, C, D, F).
NDP52 acts as a redox sensor in PINK1/Parkin-mediated mitophagy (2022)
Figure 6

Figure 6. Oxidation of NDP52 facilitates the recruitment of autophagy proteins to damaged mitochondria

A-D, HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf, followed by immunofluorescence analyses (A), immunoblotting for LC3 and NDP52 in mitochondrial fraction (B) or whole cell lysate (C) in reducing conditions, and co-immunoprecipitation assay to analyse the interaction of NDP52 with FIP200, ULK1 and ATG13 (D). The number of foci of the indicated proteins colocalised with NDP52, or foci of NDP52 colocalised with Parkin, was quantified.

E, MEFs stably expressing YFP-Parkin, mt-mKeima and empty, human NDP52 (hNDP52) WT or hNDP52 Mut were pre-treated with or without 500 nM MitoQ for 21 h and treated with AO for 3 h followed by mitophagy measurement.

F, Schematic representation of the mechanism of the damage-induced mitophagy mediated by NDP52 DLC.

Data information: Data are mean ± s.e.m. (A, B, C, D, E) or displayed as cell popular violin plots (E). P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments (A, B, C, D, E). *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 (relative to MitoQ-untreated condition); ns (non-significant). Scale bars: 20 μm (A, E).

Expanded View Figure Legends
Spironolactone is an Antagonist of NRG1-ERBB4 Signaling and Schizophrenia-Relevant Endophenotypes in Mice (2017)
Figure 1

Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.

(A) The ERBB4-PIK3R1 split TEV assay monitors NRG1-ERBB4 signaling in PC12 cells. The Nrg1 ligand (green) is stably expressed in the signal-sending cell population A. The signal-receiving cell population B (or split TEV assay cells) is transfected with plasmids encoding the assay components ERBB4 (red) fused to NTEV-tevS-GV (ERBB4-NTEV-tevS-GV), the adapter molecule PIK3R1 (purple, the regulatory subunit alpha of the PI3K) fused to CTEV (PIK3R1-CTEV), and a UAS-driven firefly luciferase reporter (Fluc). Upon Nrg1 binding to the extracellular domain of ERBB4 (1), ERBB4-NTEV-tevS-GV dimerizes and cross-phosphorylates itself (2). PIK3R1-CTEV binds to the phosphorylated ERBB4 receptor leading to the functional reconstitution of TEV protease activity and the concomitant release of the artificial co-transcriptional activator Gal4-VP16 (GV) through proteolytic cleavage at a TEV protease cleavage site (tevS) (3). In turn, released GV translocates to the nucleus and binds to UAS sequences (open box) to activate the transcription of a firefly reporter gene (4). (B) Dose-response assay using increasing numbers of Nrg1 type I β1a-expressing PC12 cells. For each 96-well, 40,000 split TEV assay cells were co-plated with increasing numbers of Nrg1-expressing cells, and incubated for 24h. Half-maximal activation is reached at 5,000 Nrg1-expressing cells. The Z’ factor is 0.5 indicating a large separation band for this assay. (C) Adding 10ng/ml EGFld resulted in a two-fold activation. Per 96-well, 40,000 split TEV assay cells were co-plated with empty PC12 cells (no Nrg1 expression), 10,000 Nrg1-expressing cells, and 10,000 Nrg1-expressing cells plus 10ng/ml EGFld. (D) Lapatinib antagonizes ERBB4-PIK3R1 signaling in a dose-dependent manner. Per 96-well, 40,000 split TEV assay cells were incubated with increasing amounts of lapatinib, followed by co-plating 10,000 Nrg1 type I β1a expressing cells. The inset depicts the IC 50 value in µM. After compound/stimulus addition, each assay was incubated for 24h. RLU, relative luciferase units; Fluc, firefly luciferase activity (black line); Rluc, Renilla luciferase activity (grey line); n=6; data are shown as mean, error bars represent SEM.
Spironolactone is an Antagonist of NRG1-ERBB4 Signaling and Schizophrenia-Relevant Endophenotypes in Mice (2017)
Figure 2

Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen.

(A) Flow chart of the compound screen. PC12 cells (population A) were transfected in-solution with the split TEV assay plasmids ERBB4-NTEV-tevS-GV and PIK3R1- CTEV and incubated for 2 h before seeded onto 96-well plates. Population A cells were allowed to express the plasmids for 24 h. Compounds were added in a concentration of 10 µM, followed by seeding the Nrg1-expressing PC12 cells (population B) half an hour later. After 24 h of compound incubation, cells were lysed and subjected to a dual luciferase assay. The screening data was analyzed using the cellHTS2 package in R Bioconductor. (B) Graphic visualisation of the primary screen data of the NIH-NCC library set 1. All counts (320 compounds and 64 controls) from the Nrg1-ERBB4-PIK3R1 split TEV compound screen were plotted against the Z-score using the Mondrian programme, with pathway activators displaying high values and inhibitors low values. For the secondary analysis, we selected all candidates that were at least three standard deviations away from the mean. EGFld positive and Lapatinib/CI-1033 negative controls are shown in red. (C and D) Spironolactone antagonizes Nrg1-ERBB4-PIK3R1 signaling. In dose-response assays using ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids transfected into PC12 cells, spironolactone was administered at increasing concentrations before seeding Nrg1 type I (C) or Nrg1 type III (D) expressing PC12 cells. (E) Spironolactone inhibits ERBB4 receptor dimerization. Dose-dependent dimerization of the ERBB4 receptor was analyzed using a split TEV assay encompassing ERBB4-NTEV-tevS-GV and ERBB4-CTEV plasmids transfected into PC12 cells. 10 ng/ml EGFld was applied as Nrg1 stimulus. Fluc, firefly luciferase activity (black lines); Rluc, Renilla luciferase activity (grey lines, indicating toxicity levels); n=6; data are shown as mean, error bars represent SEM. The insets depict IC 50 values in µM.
Spironolactone is an Antagonist of NRG1-ERBB4 Signaling and Schizophrenia-Relevant Endophenotypes in Mice (2017)
Figure 3

Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.

(A) Spironolactone, molecular structure shown left, inhibits ERBB4/PIK3R1 split TEV assay activity (black line), with an IC 50 value of 2.76 µM. (B-D) The spironolactone metabolites (B) canrenone and (C) 7α-thiomethyl-spironolactone as well as the second-generation drug (D) eplerenone do not attenuate the ERBB4/PIK3R1 assay activity (black lines). Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6. (E) Schematic representation of the most critical ERBB dimers tested in spironolactone dose-response assays using the split TEV protein-protein interaction detection technique. Note that not all possible combinations are depicted. (F) Heat map showing the IC 50 values obtained from individual ERBB dimerization split TEV assays. All single dose-response assays can be found in Fig EV4; the combination ERBB4-ERBB4 is shown in Fig 2E. Fluc, firefly luciferase activity (black lines); Rluc, Renilla luciferase activity (grey lines, indicating toxicity levels); n=6; data are shown as mean, error bars represent SEM. The insets depict IC 50 values in µM.
Spironolactone is an Antagonist of NRG1-ERBB4 Signaling and Schizophrenia-Relevant Endophenotypes in Mice (2017)
Figure 4

Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.

(A) Spironolactone reduces ERBB4 levels. T-47D cells were stimulated with 10 ng/ml EGFld, 10 µM lapatinib and 10 µM spironolactone for 5 min as indicated. Cell lysates were probed for ERBB4 phosphorylation levels at Tyr1056 and Tyr1284. (B) Quantification of band intensities for phospho-ERBB4 levels (n=4 per condition) shown in (A) using ImageJ. Phosphorylation levels are normalized to protein levels of ERBB4. Data shown as mean, error bars represent SD; t-test, with *, p=0.0356 for p-ERBB4 (Y1056), and **, p=0.0079 for p-ERBB4 (Y1284). (C) Experimental design for western blot analysis shown in (D) and (E). Nrg1-tg and wt animals were treated daily with spironolactone (50 mg/kg, s.c.) or vehicle (n=2 per genotype and per treatment) for 21 days. (D) Spironolactone reduces phospho-Erbb4 levels in Nrg1-tg mice. Mice were treated with spironolactone for 21 days and sacrificed for western blot analysis. Lysates were probed with indicated antibodies. (E) Quantification of band intensities for phospho-Erbb4 and phospho-Limk1 levels shown in (D) using ImageJ. Phosphorylation levels are normalized to protein levels of Erbb4 and Limk1. Data shown as mean, error bars represent SD; t-test, with *, p=0.0330 for p-Erbb4, and p=0.0201 for p-Limk1. (F) Canrenone (applied as 10 µM) showed no effects on frequencies and amplitudes of sIPSCs in pyramidal neurons of the prefrontal cortex (PFC). Representative traces of sIPSCs (upper) and a histogram of mean sIPSC (lower) are shown for both before and after addition of canrenone. (G) Spironolactone (applied as 10 µM) significantly increases frequencies (n=12; p=0.0454) and amplitudes (n=12, p=0.0478) of sIPSCs in pyramidal neurons of PFC. Representative traces of sIPSCs (upper) and a histogram of mean sIPSC (lower) are shown for both before and after addition of spironolactone. (H) Spironolactone increases amplitudes of evoked IPSCs in pyramidal neurons of PFC (n=15, p=0.0286). Spironolactone (5 µM) was applied in the presence of canrenone (10 µM). (I) Lapatinib increases amplitudes of evoked IPSCs in pyramidal neurons of PFC (n=15, p=0.0124). Lapatinib was applied in 5 µM. For (H, I), sample recording (upper) and a histogram of averaged eIPSC (lower) is shown for both before and after drug application. For (F-I), the numbers displayed inside the histogram bars indicate the number of recorded slices / number of animals. Data are shown as mean, error bars represent SEM (n=4); paired t-test, with *, p≤0.05; Spiro, spironolactone; Canre, canrenone; Lap, lapatinib.
Spironolactone is an Antagonist of NRG1-ERBB4 Signaling and Schizophrenia-Relevant Endophenotypes in Mice (2017)
Figure 5

Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.

(A) Experimental design. Nrg1-tg and wt animals were treated daily with spironolactone (50 mg/kg, s.c.) or vehicle (n=12 per genotype and per treatment) for 3 weeks, followed by behavioral phenotyping using the tests as indicated. Spironolactone or vehicle treatment was continued throughout the phenotyping phase. (B) Nrg1-tg mice travelled longer distances in the open field arena (effect of genotype F (1,44) =10.53; p=0.0022; 2-way ANOVA). Bonferroni post-hoc analysis revealed a significant genotype-dependent difference between vehicle-treated groups (p=0.0044) but not in spironolactone-treated groups (p=0.3783). Genotype differences were abolished upon spironolactone treatment. (C) When vehicle treated animal were analyzed in 1 min intervals, transgenic mice showed an increased activity throughout the entire test (effect of genotype F (1,22) =9.27; p=0.0060; 2-way ANOVA), most prominent in intervals 2, 7, and 8 (p=0.0427, p=0.0385 and p=0.000036, respectively, Bonferroni post-hoc test). (D) There was no significant difference between the genotypes when treated with spironolactone (effect of genotype F (1,22) =2.19; p=0.1535; 2-way ANOVA). However, the interaction of genotype and treatment was significant (F (9,198) =1.94; p=0.0481; 2-way ANOVA). (E) Nrg1-tg mice treated with spironolactone spent more time in the light compartment during the light-dark test (interaction gene × treatment F (1,41) =4.90; p=0.0324; 2-way ANOVA, and p=0.0219, Bonferroni post-test). (F) In the Y-maze test, transgenic mice performed less alterations (effect of genotype F (1,44) =11.50; p=0.0015; 2-way ANOVA). The Bonferroni test confirmed this phenotype in vehicle-treated groups (p=0.0011), but not in spironolactone-treated animals (p=0.5950). Spironolactone treatment had a significant effect on the number of alterations (F (1,44) =4.12; p=0.0484; 2-way ANOVA). (G) Spironolactone treatment significantly enhanced PPI in Nrg1-tg mice (effect of treatment F (1,21) =5.07; p=0.0325; 2-way repeated measures ANOVA). Data are shown as mean, error bars represent SEM. *, p<0.05, **, p<0.01 and ***, p<0.001, Bonferroni test following 2-way ANOVA; Spiro, spironolactone; Veh, vehicle. n=12 per genotype and treatment with an exception of (E) (Nrg1-tg vehicle, n=11; Nrg1-tg Spiro, n=10; wt vehicle, n=12; wt Spiro, n=12) and (G) (Nrg1-tg vehicle, n=11; Nrg1-tg Spiro, n=12; wt vehicle, n=12; wt Spiro, n=12).