EMBO/soda-nel huggingface dataset:
TLR3 downregulates expression of schizophrenia gene Disc1 via MYD88 to control neuronal morphology (2016)
Figure 2

Figure 2. TLR3 acts through MYD88 to regulate neuronal morphology.

(A, B) Quantitation of dendrite morphology of Trif mutant neurons (A) and Myd88–/– neurons (B) after poly(I:C) stimulation. Trif mutant or Myd88–/– neurons were transfected with a GFP construct at 4 DIV and treated with poly(I:C) one day later. Neuron morphology was analyzed at 6 DIV according to the GFP signal. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by unpaired t-test. (C) MYD88 but not TRIF expression reduces dendrite outgrowth. HA-tagged MYD88 and TRIF were co-transfected with a GFP construct into cultured wild-type (WT) neurons at 4 DIV, and cell morphology was analyzed at 6 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by one-way ANOVA with Bonferroni’s multiple comparison test. (D) MYD88 knockdown, but not TRIF knockdown, robustly increases dendritic arbors and loses the response to poly(I:C). Three DIV neurons were transfected with control shRNA (shCtrl), MYD88 shRNA (shMYD88) or TRIF shRNA (shTRIF) and poly(I:C) was applied into the culture at 5 DIV for 24 h before harvest. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni’s multiple comparison test. Scale bar, 20 μm. *** P < 0.0001.
TLR3 downregulates expression of schizophrenia gene Disc1 via MYD88 to control neuronal morphology (2016)
Figure 3

Figure 3. TLR3 interacts with MYD88.

(A) Coimmunoprecipitation of TLR3 and MYD88. HEK293T cells were transfected with the indicated constructs. Twenty-four h later, immunoprecipitation (IP) was performed using HA antibody. The precipitates were immunoblotted (IB) with Myc and HA antibodies as indicated. EC, TLR3 ectodomain. (B) Poly(I:C) treatment increases the interaction between TLR3 and MYD88. One day after transfection, cells were treated with saline or poly(I:C) for 30 min before harvesting. (C) Upper, schematic of MYD88 constructs. DD, death domain; ID, intermediate domain, TIR, TIR domain. Lower, coimmunoprecipitation of TLR3 and the N-terminal region of MYD88. For (A)-(C), representatives of three independent experiments are shown. (D) Full-length and N-terminal MYD88 inhibit dendrite outgrowth. Myd88–/– neurons were transfected with control vector (Ctrl), HA-tagged MYD88 (MYD88), N-terminal MYD88 (MYD88-N), or C-terminal MYD88 (MYD88-C) with a GFP construct. Poly(I:C) was applied into the culture at 5 DIV and neuronal morphology was analyzed at 6 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni’s multiple comparison test. Scale bar, 20 μm. (E) Expression patterns of full-length and truncated fragments of MYD88 in Myd88–/– neurons. Representative images are shown.
TLR3 downregulates expression of schizophrenia gene Disc1 via MYD88 to control neuronal morphology (2016)
Figure 4

Figure 4. TLR3 activation does not use secreted factors to regulate neuronal morphology.

(A) qPCR analysis of proinflammatory cytokines Il-6, Tnfα, Il-1β and antiviral cytokine Ifnβ in WT cultured cortical and hippocampal neurons. Data were analyzed by unpaired t-test. Il-6 and Tnfα N=8; Ifnβ and Il-1β, N=6. (B, C) Poly(I:C) treatment inhibits dendrite outgrowth of Il-6–/– neurons (B) and Tnfα–/– neurons (C). Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by unpaired t-test. (D) Left, schematic of experiment using conditioned medium. Upper right, conditioned medium applied to WT neurons. Lower right, conditioned medium applied to Tlr3–/– neurons. Dendrite morphology was analyzed one day after adding conditioned medium. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by unpaired t-test. * P < 0.05, *** P < 0.0001.
TLR3 downregulates expression of schizophrenia gene Disc1 via MYD88 to control neuronal morphology (2016)
Figure 6

Figure 6. TLR3 activation downregulates expression of neuropsychiatric disorder-related genes.

(A) Expression of 12 neuropsychiatric disorder-related genes in saline- and poly(I:C)-treated WT neurons. (B) Expression of 9 neuropsychiatric disorder-related genes in poly(I:C)-treated Tlr3–/– neurons. Relative expression levels compared with vehicle control are shown. (C, D) Expression of Aust2, Disc1, Fmr1, Pten and Ube3a in poly(I:C)-treated Myd88–/– neurons (C) and poly(I:C)-treated P5 WT mouse brains (D). Similar to (B), relative levels compared with vehicle control are shown. In (B-D), dashed lines indicate the level of saline control. Data were analyzed by unpaired t-test. N=6 in A, B, C and N=5 in D. (E) DIV 5 WT neurons were treated with poly(I:C) for 24 h. The total neuronal lysates were subjected to immunoblotting with the indicated antibodies. VCP and β-actin were used as loading controls. The protein levels were normalized with β-actin. Data were analyzed by unpaired t-test. Numbers of experimental repeats (N) are shown in each column. * P < 0.05, ** P < 0.001, *** P < 0.0001.
TLR3 downregulates expression of schizophrenia gene Disc1 via MYD88 to control neuronal morphology (2016)
Figure 7

Figure 7. DISC1 overexpression rescues the effects of TLR3 activation on dendritic arborization in vitro and in vivo.

(A) Overexpression of WT DISC1, but not the DISC1 L604F mutant, in cultured neurons suppresses dendrite withdrawal induced by TLR3 activation. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni’s multiple comparison test. Scale bar, 20 μm. * P < 0.05, ** P < 0.001, *** P < 0.0001. (B-C) Overexpression of DISC1 in cortical neurons of mouse brain is resistant to poly(I:C)-triggered reduction of dendritic arborization. Vector control (Ctrl) (B) or Myc-DISC1 (C) was co-expressed with GFP in cortical layer 2/3 neurons in mouse brain. After poly(I:C) injection at P4 and P5, the neuronal morphology was analyzed at P7 by tracing the GFP signals. The data of dendrite length and tip number were analyzed by unpaired t-test. The Sholl data were analyzed by two-way ANOVA with Bonferroni’s multiple comparison test. The numbers of analyzed neurons collected from 3-4 mice of each group are indicated. Mean values SEM are shown. Scale bar, 30 μm. * P < 0.05, *** P < 0.0001.
TLR3 downregulates expression of schizophrenia gene Disc1 via MYD88 to control neuronal morphology (2016)
Figure 8

Figure 8. TLR3 activation alters spine morphology in vitro and in vivo.

(A) Cortical and hippocampal mixed neuronal cultures were transfected with a GFP construct at 12 DIV and treated with poly(I:C) (pIC) at 17 DIV for 24 h. Spine morphology was examined at 18 DIV. Three secondary dendrites of each neuron were selected to analyze the spine density, the width of the spine head and the spine length. Mean values SEM of representatives of three independent experiments are shown. For the saline group, 15 neurons/45 dendrites were examined; for the poly(I:C) group, 16 neurons/48 dendrites were examined. Data were analyzed by unpaired t-test. Scale bar, 20 μm in right upper panel and 5 µm in lower left panel. *** P < 0.0001. (B) Schematic of the experimental procedure for in vivo spine morphology analysis in C and D. Representative images of somatosensory cortical layer 5 neurons and a high magnification image of a secondary dendrite are shown. Scale bar, 100 μm in the brain section (black) and 10μm in the dendrite segment (white). (C, D) Spine morphology of P21 Thy1-Yfp (C) and Tlr3–/–; Thy1-Yfp mice (D) after poly(I:C) stimulation at P4 and P5. One secondary dendrite of an apical dendrite of each somatosensory layer 5 cortical neuron was selected to examine the spine morphology. Three mice were analyzed for each group. Saline-treated Thy1-Yfp, 44 neurons; poly(I:C)-treated Thy1-Yfp, 43 neurons; saline-treated Tlr3–/–;Thy1-Yfp, 46 neurons; poly(I:C)-treated Tlr3–/–;Thy1-Yfp, 50 neurons. Mean values SEM are shown. Data were analyzed by unpaired t-test. Scale bar, 5 μm. *** P < 0.0001.
FTLD-TDP assemblies seed neoaggregates with subtype-specific features via a prion-like cascade (2021)
Figure 1

Figure 1: FTLD patient-derived TDP-43 aggregates seed neoaggregates in cells with subtype-specific potency

A) Seeding efficiency was compared between control non-neurodegenerative and FTLD-TDP patient samples. Cells were transfected using Lipofectamine after mitotic arrest with AraC and were fixed 3 to 6 days post-transfection with SarkoSpin fractions (Laferrière et al, 2019). Surface masks generated with Imaris show DAPI (blue), nuclear TDP-43 (NuclTDP-43, cyan) and neoaggregates (NeoAgg, orange). All images are presented at the same scale, 20 μm as indicated in AraC panel Day 6.

B) Higher magnification imaging of cells seeded for 6 days revealed presence of large cytoplasmic TDP-43-HA aggregates in cells seeded with FTLD-TDP-A and multiple cytoplasmic TDP-43-HA aggregates in cells seeded with FTLD-TDP-C. Surface representation generated with Imaris showing DAPI, TDP-43-HA (green), phosphorylated TDP-43 (pTDP-43, magenta) and neoaggregates (NeoAgg, orange). All images are presented at the same scale, 5 μm as indicated in FTLD-TDP-C top panel.

C) Quantification of the number of neoaggregates over time (n=3 independent experiments, with multiple biological replicates: non-neurodegenerative control (n=5), FTLD-TDP-A (n=7) and FTLD-TDP-C (n=6) ). Whiskers in the box plot represent median and minimum/maximum values. 2-way ANOVA repeated measure, with post-hoc Fisher's LSD, F(3,15)=1.3, p=0.3110, NS difference for Day 3 and 4. Day 5: Non-neurodegenerative control vs FTLD-TDP-A p=0.0344, non-neurodegenerative control control vs FTLD-TDP-C p=0.4606. Day 6: non-neurodegenerative control vs FTLD-TDP-A p=0.1028, non-neurodegenerative control vs FTLD-TDP-C p=0.2919.

D) Linear regression model of neoaggregate formation over time. Best-fit values: non-neurodegenerative control control (y=0.06902x-0.1281, R2=0.3655, p=0.0048), FTLD-TDP-A (y=0.1810x-0.4836, R2=0.3532, p=0.0009), FTLD-TDP-C (y=0.1902x-0.5896, R2=0.2581, p=0.0112). Null-hypothesis slope=0.06902: non-neurodegenerative control vs FTLD-TDP-A p=0.0278, non-neurodegenerative control vs FTLD-TDP-C p=0.0918.

E) Comparison of pTDP-43 score to neoaggregate formation per patient showed positive correlation for FTLD-TDP-A and no correlation for FTLD-TDP-C. Trendline is shown per subtype. Best-fit value of linear regression model: FTLD-TDP-A (y=1.286x+1.123, R2=0.6099, p=0.0667), FTLD-TDP-C (y=-0.2655x+2.805, R2=0.03133, p=0.7758).
FTLD-TDP assemblies seed neoaggregates with subtype-specific features via a prion-like cascade (2021)
Figure 2

Figure 2: TDP-43 seeding depends on specific aggregate conformation(s)

A) Strategy to explore the prion-like properties of TDP-43 seeds.

B) Bar graph showing the seeding effect at day 5 and day 6 post-seeding in untreated, temperature denaturation or autoclaving denaturation conditions. While seeding in untreated condition showed a significant increase in neoaggregates (One-way ANOVA, with post-hoc Fisher's LSD test, Day 5: F=(5,12)=6.713, p=0.0033, non-neurodegenerative control vs FTLD-TDP-A p=0.0016, Day 6: F(5,12)=4.271, p=0.0183, non-neurodegenerative control vs FTLD-TDP-A p=0.0397), this effect was abolished in denaturing conditions (N=3 biological replicates for non-neurodegenerative control, FTLD-TDP-A and FTLD-TDP-C, bar graph showing mean ± SEM).

C) TOP: Aggregation kinetics of full length (fl) recombinant human TDP-43 (rec-hTDP-43) measured as an increase in absorbance. The addition of TEV protease induces the cleavage of the MBP tag and subsequent aggregation of rec-hTDP-43. Rec-hTDP-43 aggregates used for seeding were collected 18 hours after the addition of TEV protease. BOTTOM LEFT: rec-hTDP-43 aggregates stained by Thioflavin T (ThT). BOTTOM RIGHT: Transmission Electron Microscopy (TEM) image of the rec-hTDP-43 aggregates. All images are presented at the same scale of 1 μm.

D) Rec-hTDP-43 aggregates were transfected on mitotically arrested cells and fixed after 5 or 6 days post-transfection. Surface masks generated with Imaris depict DAPI (blue), NuclTDP-43 (cyan) and pTDP-43 (magenta) for non-transfected AraC control and 1 μg rec-hTDP-43. All images are presented at the same scale, 20 μm as indicated in AraC panel.

E) Quantification of neoaggregates (left y-axis) and pTDP-43 (right y-axis) at day 5 and day 6 post-transfection for increasing concentrations of rec-hTDP-43. 2-way ANOVA repeated measure, with post-hoc Fisher's LSD, F(7,80)=5.886, p<0.0001, Day 5: AraC vs 50 ng p=0.0497, AraC vs 500 ng p=0.0002, AraC vs 5 µg p=0.0161. 50 ng vs 100 ng p=0.0208, 50 ng vs 500 ng p=0.0169, 50 ng vs 2.5 µg p=0.0272, 100 ng vs 1 µg p<0.0001, 100 ng vs 5 µg p=0.006, 250 ng vs 1 μg p=0.0005, 250 ng vs 5 µg p=0.034, 500 ng vs 1 µg p<0.0001, 500 ng vs 5 µg p=0.0047, 1 µg vs 2.5 µg p<0.0001, 2.5 µg vs 5 µg p=0.0081, Day 6: AraC vs 250 ng p=0.0385, AraC vs 1 µg p=0.0006, 50 ng vs 1 µg p=0.0016, 100 ng vs 1 µg p=0.0101, 100 ng vs 5 µg p=0.0173, 250 ng vs 5 µg p=0.0006, 500 ng vs 1 µg p=0.0099, 500 ng vs 5 µg p=0.0176, 1 µg vs 2.5 µg p=0.0144, 1 µg vs 5 µg p<0.0001, 2.5 µg vs 5 µg p=0.0121. To reduce complexity, only comparisons with AraC are marked in the graph. N=6 biological replicates for each concentration; bar graph showing mean ± SEM.
FTLD-TDP assemblies seed neoaggregates with subtype-specific features via a prion-like cascade (2021)
Figure 4

Figure 4: Superresolution analysis shows distinct features of type A and C cellular neoaggregates

A-C) dSTORM images of cells seeded for 6 days with non-neurodegenerative control (non-ND control), FTLD-TDP-A and FTLD-TDP-C seeds extracted from post-mortem brain tissue. Staining shows localization of TDP-43 (HA tag, cyan) along with LaminB1 (yellow, A), nucleopore complexes (nucleopores, yellow, B) and nucleolus (yellow, C). Cells seeded with FTLD-TDP seeds show coaggregation of nuclear membrane proteins upon neoaggregate formation. All images are presented at the same scale, 2 μm as indicated in the non-neurodegenerative control top panels.

D) dSTORM images of cells containing aggregates after 6 days of seeding with SarkoSpin fractions of postmortem brain from patients with FTLD-TDP-A or FTLD-TDP-C. Red arrows point to cytoplasmic aggregates. Cells were labelled with TDP-43 (HA-tag, cyan) and phosphorylated TDP-43 pS403/404 (pTDP-43, magenta). All images are presented at the same scale, 2 μm as indicated in the FTLD-TDP-A top panel.

E) Violin plots showing the size of analyzed neoaggregates using TDP-43-HA area. Non-parametric unpaired t- test FTLD-TDP-A vs FTLD-TDP-C, p<0.0001. (Continuous line represent median, dashed lines represent quartiles , n = 3 independent experiments, 6 biological replicates for each condition)

F) Bar graph showing the number of neoaggregates per cell. Non-parametric unpaired t test FTLD-TDP-A vs FTLD-TDP-C, p=0.0003. Error bars represent mean ± SEM, n=3 independent experiments, 6 biological replicates for each condition.

G) Heatmap representation of the density of TDP-43-HA and pTDP-43 particles for four representative neoaggregates of FTLD-TDP-A and FTLD-TDP-C seeded cells. All images are presented at the same scale, 1 μm as indicated in the FTLD-TDP-C top right panel.

H) Violin plots showing the density of particles TDP-43 per µm2. Non-parametric unpaired t test FTLD-TDP-A vs FTLD-TDP-C, p=0.0083. (Continuous line represent median, dashed lines represent quartiles , n=3 independent experiments, 6 biological replicates for each condition)
The biochemical basis of mitochondrial dysfunction in Zellweger Spectrum Disorder (2021)
Figure 1

Figure 1. Peroxin gene expression is maintained in the absence of functional peroxisomes.

(A) The indicated yeast strains (wild type, pex19Δ, pex3Δ, msp1Δ, pex19Δmsp1Δ, and pex3Δmsp1Δ) expressing RFP-SKL (peroxisomal marker) and mitochondria-targeted green ﬂuorescent protein (mGFP) were grown to mid-log phase and imaged by ﬂuorescence microscopy. Representative images are shown, scale bar, 2 μm. DIC, differential interference contrast. Enhanced: the red signal intensity was set to 'best-fit' in ZENPro microscopy software analysis.

(B) The indicated yeast strains (wild type, msp1Δ, pex19Δ, pex19Δmsp1Δ, pex3Δ, and pex3Δmsp1Δ) were grown to mid-log phase, back-diluted to OD600=1, and serial dilutions were spotted onto agar plates containing synthetic media supplemented with glucose (S-D), glycerol (S-Gly), glycerol and oleate (S-Gly-Ole), or oleate (S-Ole).

(C) Wild type versus pex19Δ RNA levels. Outliers are indicated. Peroxin genes are indicated in red. The blue shading emphasizes that the RNA levels of the indicated genes are lower in pex19Δ compared to wild type.

(D) Cumulative distributions of fold changes (log2) in RNA between wild type and pex19Δ yeast (Kolmogorov-Smirnov test). Peroxin genes are indicated in red.

(E) Translational efficiency (TE) of wild type versus pex19Δ. Outliers are indicated. Peroxin genes are indicated in red. The red shading emphasizes that the translation efficiency of the indicated genes is higher in pex19Δ compared to wild type.

(F) pex19Δ versus pex19Δmsp1Δ RNA levels. Outliers are indicated. Peroxin genes are indicated in red. The red shading emphasizes that the RNA levels of the indicated genes are higher in pex19Δmsp1Δ compared to pex19Δ.

(G) Cumulative distributions of fold changes (log2) in RNA between pex19Δ and pex19Δmsp1Δ yeast (Kolmogorov-Smirnov test). Peroxin genes are indicated in red.

(H) Translational efficiency (TE) of pex19Δ versus pex19Δmsp1Δ. Outliers are indicated. Peroxin genes are indicated in red. The blue shading emphasizes that the translation efficiency of the indicated genes is lower in pex19Δmsp1Δ compared to pex19Δ.
The biochemical basis of mitochondrial dysfunction in Zellweger Spectrum Disorder (2021)
Figure 2

Figure 2. Peroxisomal proteins accumulate on mitochondria in absence of peroxisomes in yeast.

(A) Experimental flow of sample generation for quantitative mass spectrometry.

(B) Volcano plot showing the most enriched/decreased proteins in the mitochondrial proteome of pex19Δ versus pex19Δmsp1Δ strains as detected by quantitative mass spectrometry (n=7, biological replicates; n=2, technical replicates). Student's t-tests were performed using scipy.stats.ttest_ind() (Mckinney, 2010). Effect sizes were calculated for Cohen's d (Sawilowsky, 2009; Cohen, 2013). A protein's relevance was determined using a p-value threshold of < 0.05 with an absolute effect size threshold of 1 or greater (large effect). Gray dots represent all measured proteins and red dots highlight peroxisomal-associated proteins.

(C) Heatmap of peroxin proteins from pex19Δ and pex19Δmsp1Δ stains as detected by quantitative mass spectrometry (n=5). Protein classes are indicated in the row labels. Values were z-score normalized by protein.

(D) The indicated yeast strains expressing PEX13-RFP and mitochondria-targeted green ﬂuorescent protein (mGFP) were grown to mid-log phase and visualized by ﬂuorescence microscopy. Representative images are shown, scale bar, 2 μm. DIC, differential interference contrast.

(E) The indicated yeast strains expressing PEX11-RFP and mitochondria-targeted green ﬂuorescent protein (mGFP) were grown to mid-log phase and visualized by ﬂuorescence microscopy. Representative images with enhanced contrast and brightness are shown, scale bar, 2 μm. DIC, differential interference contrast.

(F) Total yeast cell lysate, post mitochondrial supernatant, and HistodenzTM-purified mitochondria from the indicated strain (wild type, msp1Δ, pex19Δ, pex19Δmsp1Δ, pex3Δ, and pex3Δmsp1Δ) expressing Pex13-V5 (under its endogenous promotor) were separated by SDS-PAGE and immunoblotted for Pex13 (α-V5), cytochrome c (α-cyt c), and HSP70 (α-hsp70).

(G) Total yeast cell lysate, post mitochondrial supernatant, and HistodenzTM-purified mitochondria from the indicated strain (wild type, msp1Δ, pex19Δ, pex19Δmsp1Δ, pex3Δ, and pex3Δmsp1Δ) expressing Pex11-V5 (under its endogenous promotor) were separated by SDS-PAGE and immunoblotted for Pex11 (α-V5) and porin (α-porin), cytochrome c (α-cyt c), and HSP70 (α-hsp70).
The biochemical basis of mitochondrial dysfunction in Zellweger Spectrum Disorder (2021)
Figure 3

Figure 3. Peroxisomal proteins are incorporated into the mitochondrial membrane in absence of peroxisomes in yeast.

(A) Volcano plot (log10) of the most enriched/decreased proteins in the mitochondrial proteome of pex3Δ versus pex3Δmsp1Δ strains as detected by quantitative mass spectrometry (n=3, biological replicates, n=1, technical replicate). Student's t-tests were performed using scipy.stats.ttest_ind() (Mckinney, 2010). Effect sizes were calculated for Cohen's d (Sawilowsky, 2009; Cohen, 2013). A protein's relevance to the model was determined using a p-value threshold of < 0.05 with an absolute effect size threshold of 1 or greater (large effect). Gray dots represent all measured proteins. Red dots highlight peroxisomal-associated proteins.

(B) Heatmap (log2) of peroxin protein levels from pex3Δ and pex3Δmsp1Δ as detected by quantitative mass spectrometry (n=3). Protein classes are indicated in the row labels. Values were z-score normalized by protein.

(C) Mitochondria from the indicated yeast strains (wild type, pex19Δ, pex19Δmsp1Δ), expressing Pex13-V5 (under its endogenous promotor) were either left untreated (lane 1,2,3) or exposed to Na2CO3 and separated into the membrane pellet (4,5,6) or supernatant fraction (7,8,9). After separation by SDS-PAGE the fractions were immunoblotted for Pex13-V5 (α-V5) and porin (α-porin).
The biochemical basis of mitochondrial dysfunction in Zellweger Spectrum Disorder (2021)
Figure 5

Figure 5. Peroxins target to mitochondria in cells derived from ZSD patients and overexpression of ATAD1 rescues their mitochondrial morphology.

(A) Fluorescence microscopy of human fibroblast cell lines derived from ZSD patients-cWT, cWT+ATAD1, PEX3-/-, and PEX3-/-+ATAD1-expressing PEX13-GFP and stained with Mitotracker Deep Red FM. Representative images are shown, scale bar, 10 μm.

(B) Electron microscopy of human fibroblast cell lines cWT, cWT+ATAD1, PEX3-/-, and PEX3-/-+ATAD1. Representative images are shown, scale bar 500 nm.

(C) Quantification of electron microscopy imaging. Mitochondrial length and width were analyzed using the Fiji length analysis tool. Per cell line >50 mitochondria were graphed. Statistical significance was calculated using a non-paired t-test, p-values are indicated as: ns (not significant), ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. The horizontal lines represent the mean length and width in each cell line, respectively.

(D) Quantification of electron microscopy imaging. Three independent experiments were quantified. Mitochondria were counted (cWT n = 232, PEX3-/- n = 266, and PEX3-/-+ATAD1 n = 257) and binned into 3 categories: mitochondria with many cristae and low electron density (Type 1), mitochondria with fewer cristae (Type 2), and mitochondria with high electron density and/or almost no cristae (Type 3).