EMBO/SourceData huggingface dataset:
Figure 5A) Immunostaining of anterior horn of lumbar spinal cord for ChAT (in green) and p62 (in red) at 130 days. Scale bar, 10 µm.B) Quantification of the percentage of ChAT-positive motor neurons (MN) containing p62-positive inclusions at 130 days. PKO PKO/G93A MN have less SOD1 aggregates than G93A, while Non Tg and PKO MN have none. Data were collected from n= 4 (2 males and 2 females) mice for Non Tg and PKO and n= 6 (3 males and 3 females) for G93A and PKO/G93A (males and females). Number of images recorded for each genotype: n= 9 for Non Tg and PKO, n= 16 for G93A and n= 12 for PKO/G93A. Results are expressed as mean ± SEM; *p= 0.032, by unpaired one-tailed Student's t test.C) Representative Western blots of OPTN, p62 and VCP in spinal cord mitochondrial fractions at 130 days. Protein levels are normalized by Complex V. The quantifications show that mitochondria of PKO/G93A mice have less mitophagy adaptor proteins than G93A relative to Complex V.D) Quantification of OPTN relative to Complex V at 130 days. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); *p= 0.039 by paired Student's t test.E) Quantification of p62, relative to Complex V at 130 days. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); **p= 0.0029 by paired Student's t test.F) VCP was quantified using Complex V as loading reference at 130 days. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); *p= 0.044 by paired Student's t test.
Figure 6A) Kaplan-Meier survival analysis (Log-Rank test with Holm-Sidak's correction for multiple comparisons). Mean age of death (± SEM) for G93A was 169.3 ± 2.5 days and for PKO/G93A was 185.6 ± 2.2 days, indicating that Parkin knockout prolongs lifespan of SOD1-G93A mice; n=19 for Non Tg (9 males and 10 females), n= 20 for PKO (10 males and 10 females), n= 18 for G93A (8 males and 10 females) and n= 19 for PKO/G93A (9 males and 10 females); p= 0.0000285.B) Body weight changes over time, relative to body weight at 45 days that is set at 100% for each mouse. Parkin knockout delays body weight loss in SOD1-G93A mice. Results are expressed as mean ± SEM; n=19 for Non Tg (9 males and 10 females), n=20 for PKO (10 males and 10 females), n=18 for G93A (8 males and 10 females) and n=19 for PKO/G93A (9 males and 10 females); *p= 0.017 (at 150 days), *p= 0.004 (at 164 days), *p= 0.002 (at 171 days) and **p= 0.001 (at 178 days), by two-way ANOVA with repeated measures and with Holm-Sidak's correction for multiple comparisons.C) Clasping score changes over time. Parkin knockout does not affect clasping onset in SOD1-G93A mice. An intermediate score of 2.5 is indicated by the dotted line. Results are expressed as mean ± SEM; n=19 for Non Tg (9 males and 10 females), n=20 for PKO (10 males and 10 females), n=18 for G93A (8 males and 10 females) and n=19 for PKO/G93A (9 males and 10 females).D) Representative images of NMJs in the soleus muscle at 130 days. Muscles were immunostained for neurofilament H (NF-H, in red) and stained with α-bungarotoxin (α-BTX, in green). Scale bar, 20 µm.E) Quantification of the percentage of innervated NMJs at 130 days indicates that Parkin knockout decreases denervation in SOD1-G93A mice. Results are expressed as mean ± SEM and were collected from n= 6 mice per group (3 males and 3 females); n of images evaluated = 49 for Non Tg (with 483 NMJs counted), 46 for PKO (396 NMJs counted), 47 for G93A (413 NMJs assessed) and 38 for PKO/G93A (325 NMJs counted); ***p= 0.0001, by unpaired Mann-Whitney test.F) Representative images of lumbar spinal cord sections at 130 days immunostained with ChAT. Scale bar, 200 µm. G) Quantification of ChAT-positive MN in the spinal cord ventral horn (dotted areas) at 130 days shows that Parkin knockout decreases MN loss in the spinal cord of SOD1-G93A mice. Results are expressed as mean ± SEM; n = 10 mice per group in Non Tg, PKO and G93A (5 males and 5 females) and n= 9 for PKO/G93A (4 males and 5 females); *p=0.031, by unpaired Student's t test. H H) Representative images of lumbar spinal cords sections immunostained for GFAP at 130 days. Scale bar, 200 µm. I) Quantification of the average GFAP staining intensity at 130 days indicates that Parkin knockout decreases spinal cord astrogliosis in SOD1-G93Amice. Results are expressed as mean ± SEM; n = 10 mice per group (5 males and 5 females); *p= 0.033, by paired Student's t test.
Figure 7A) Citrate synthase activity in spinal cord homogenates at disease end stage. Results are expressed as mean ± SEM; n= 8 (4 males and 4 females) mice per group; *p= 0.024, by paired Student's t test. Activities are expressed as nmoles of substrate oxidized per minute per mg of protein.B) Cytochrome oxidase (COX) activity at end stage. Results are expressed as mean ± SEM; n = 8 (4 males and 4 females) mice per group; *p= 0.032, by paired Student's t test. Activities are expressed as nmoles of substrate oxidized per minute per mg of protein.C) qPCR (fold change) of PGC1α mRNA normalized by β-actin mRNA at 130 days of age. Results are expressed as mean ± SEM and as fold change of the Non Tg; n= 8 (4 males and 4 females). No statistically significant differences were found between G93A and PKO/G93A (p= 0.250 by paired Wilcoxon's test); *p= 0.035 paired Friedman's test with Dunn's correction (Non Tg vs. G93A). No other statistically significant differences were found. D) qPCR (fold change) of PGC1α mRNA normalized by β-actin mRNA at end stage. Results are expressed as mean ± SEM and as fold change of the Non Tg; n= 8 (4 males and 4 females). No statistically significant differences were found between G93A and PKO/G93A (p= 0.613 by paired Student's t test). **p= 0.0058 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A comparison) and ***p<0.0001 by paired one-way ANOVA with Tukey's correction (for PKO vs. PKO/G93A comparison). Parkin knockout improves PGC1α expression at 130 days (C) but the effect is lost at end stage (D). E E, F) Representative Western blots (E) and quantification (F) of PARIS at disease end stage. Protein levels are normalized by β-actin and results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females). No statistically significant differences were found between G93A and PKO/G93A by paired Wilcoxon's test (p= 0.99). PARIS levels are unchanged in SOD1-G93A mice relative to Non Tg at disease end stage.
Figure 8A, B) Western blots (A) and quantification (B) of Miro1 in spinal cord homogenates at disease end stage. β-actin is used as loading reference. Results are expressed as mean ± SEM and as percent of Non Tg; n = 8 (4 males and 4 females) mice per group; *p= 0.011 by paired Student's t test (for comparison G93A vs. PKO/G93A) and **p= 0.003 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A). Parkin knockout increases the levels of Miro1 in SOD1-G93A mice at disease end stage.C, D) Western blots (C) and quantification (D) of Mfn2 in spinal cord homogenates at disease end stage. Protein levels are normalized by β-actin. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females) mice per group; No statistically significant differences were found between G93A and PKO/G93A (p=0.078 by paired Wilcoxon's test); ***p= 0.0007 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A) and *p= 0.037 by paired Friedman's test with Dunn's correction (for PKO vs. PKO/G93A). Parkin knockout increases the levels of Mfn2 in SOD1-G93A mice at disease end stage.E) Western blots of spinal cord mitochondria at disease end stage, probed for lysine 48 (left panel) and lysine 63 (right panel) ubiquitin chains. Citrate synthase is used as loading control. Asterisks indicate ubiquitinated proteins with different abundance in SOD1-G93A samples.F, G) Representative Western blot (F) and quantification (G) of LC3 II in spinal cord mitochondria at end stage. Protein levels are normalized by Complex V. LC3 II is increased in both G93A and PKO/G93A mitochondria and Parkin knockout does not affect LC3 II levels. Results are expressed as mean ± SEM and as percent of Non Tg; n = 8 (4 males and 4 females) mice per group; No statistically significant differences were found between G93A and PKO/G93A (p=0.504 by paired Student's t test). *p= 0.035 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A) and **p= 0.0014 by paired one-way ANOVA with Tukey's correction (for PKO vs. PKO/G93A)H) Representative Western blot of March5 and Mul1 in spinal cord mitochondria at end stage.I) Quantification of March5 at disease end stage. Results are expressed as mean ± SEM and percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between G93A and PKO/G93A by paired Wilcoxon's test (p= 0.742). Protein levels are normalized by Complex V. March5 levels are unaffected by Parkin knockout.J) Quantification of Mul1 at disease end stage. Results are expressed as mean ± SEM and percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between G93A and PKO/G93A by paired Wilcoxon's test (p= 0.94). Protein levels are normalized by Complex V. Mul1 levels are unaffected by Parkin knockout.
Figure 1B. Beta-hydroxybutyrate (ketone body) concentrations, plasma.C. Creatine kinase (CK) concentrations, plasma.D. Myoglobin concentrations, plasma. Dashed line: upper limit of control range. The individual patients are shown separately, and controls as mean with standard deviation (n=10). Arrow: mAD endpoint in PEO patients; lines under graphs: duration of mAD.E-G. PEO patient (P1) muscle before diet:E. Ultrathin section of abnormal muscle fiber (arrowhead) with subsarcolemmal accumulations of mitochondria. Scale bar 50 µmF. Electron micrograph of subsarcolemmal accumulation of abnormal mitochondria (same cell as in E).G. Enlargement of the area marked in F; mitochondria with paracrystalline "parking lot" inclusions (arrow) and concentric cristae (asterisk). Scale bar 1µmH-J. PEO patient (P1) muscle after mAD (H-J):H. Ultrathin section, lytic muscle fiber with centrally located organellar debris (arrow). Scale bar 50 µmI. Electron micrograph of a degrading muscle fiber with an invaded macrophage (arrow) (same cell as in H).J. Enlargement of the fiber in I: paracrystalline inclusions within mitochondria (asterisk). Scale bar 1µmK. Quantification of necrotic or apoptotic cells in PEO muscle from ultrathin sections. n.a., not available.
Figure 2A-B. Cleaved caspase-3 positive muscle fibers (*).A. PEO patient (P3) on normal diet (inset: caspase staining in healthy control individual)B. PEO patient (P3) after modified Atkins diet. Arrowhead: caspase negative necrotic fiber.C. Western blot analysis of autophagy receptor p62 and pax7 marking muscle satellite cell activation; left, blot; right, signal quantification in each patient.D - E. Immunohistochemistry analysis of p62 and mitochondrial mass marker, consecutive sections. Asterisks indicate p62-positive fibers and corresponding affected ragged-red fibers.F - G. p62 and mitochondrial mass after mAD; consecutive sections. Arrowheads indicate fibers that show p62 positivity but have normal mitochondrial mass.H. p62 and LC3 positive ragged-red fibers (*) in PEO patient (P2), normal diet.I. Muscle fibers showing granular LC3-positivity, PEO patient (P1), after mAD. Immunohistochemistry on frozen sections; bars: 50 µm, except for H, 100 µm.
Figure 3A-C. Muscle strength and performance of PEO patients (P1-P4) on normal diet (ND) and after modified Atkins diet (mAD) (one and six months and 2.5 years after initiation of diet).D-G. Spiroergometry results from PEO patients and control subjects on normal diet (ND), as well as one month and six months after mAD initiation. PEO patients had higher serumlactate levels, and attenuated respiratory response to exercise. These effects were further enhanced after mAD and remained after 6 months of the study. mAD increased patients' serumammonium (NH4+) levels. Data presented as mean and SD.
Figure 5A. PEO patients, relative amino acid levels on normal diet (PEO), immediately after finishing mAD (PEO_mAD), and one month after mAD.B. Control subjects, relative amino acid levels on normal diet (Control) and immediately after finishing mAD (Control_mAD).C. Relative values of plasma metabolites in creatine pathway, compared to controls on ND. Right: illustration of major metabolites and pathway of creatine synthesis.D. Relative values of plasma purine and pyrimidine degradation pathway intermediates, compared to controls on ND. Right: illustration of major metabolites and synthesis of pyrimidine and purines.
Figure 1 (B) Box plots show the MIG-6 (ERRFI1) gene expression in different breast tumor subtypes in the Servant dataset of 343 primary breast cancer carcinomas, analyzed using Illumina HumanWG-6_v3 Arrays. The gene expression levels are determined using the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). In the box plot, error bars are the 95% confidence interval, the bottom and top of the box are the 25th and 75th percentiles, the line inside the box is the 50th percentile (median), and any outliers are shown as open circles. **p < 0.01, by Student's t-test.  (C) Immunoblotting analysis for MIG-6 protein expression in various human breast cancer cell lines.  (D) Representative images of histological analyses of MIG-6 in TNBC patients in low-grade (stage I) and high-grade (stage III) tumors. Scale bar, 200 µm. (E) Quantification analysis of MIG-6 expression in 85 cases of resected TNBCs. In the box plot, error bars are the 95% confidence interval, the bottom and top of the box are the 25th and 75th percentiles, the line inside the box is the 50th percentile (median). The p-value 0.011 is used for the comparison of all three groups, determined by the Kruskal-Wallis H test. (F) Kaplan-Meier plot analysis of the disease-specific survival of 85 TNBC breast cancer patients with low or high expression of MIG-6.  (G) Kaplan-Meier plot analysis of the metastasis-specific survival of 85 TNBC patients with low or high expression of MIG-6. MIG-6 protein expression in the 85 TNBC specimens was determined by H-score and widely distributed. MIG-6 expression greater than or equal to the median is classified as "high," while expression less than the median is classified as "low."
Figure 2 (A) Cell proliferation and immunoblotting assays in BT549 cells with Luciferase or MIG-6 knockdown.  (B) Cell proliferation and immunoblotting assays in MDA-MB-231 cells with Luciferase or MIG-6 knockdown.  (C) Representative images and quantification results for a colony formation assay in BT549 cells with GFP or MIG-6 knockdown.  (D Immunoblotting analysis for EGFR signaling in BT549 cells with GFP or MIG-6 knockdown.  E) Immunoblotting analysis for EGFR signaling in MDA-MB-231 cells with GFP or MIG-6 knockdown.  (F) Cell growth assay in BT549 cells with GFP or MIG-6 knockdown in the absence and presence of gefitinib for 72 hours.
Figure 3 (A) Immunoblotting analysis for MIG-6 expression in BT549 cells under normoxia and hypoxia.  (B) Extracellular acidification rate (ECAR) in BT549 cells with GFP or MIG-6 knockdown. The ECAR flux profile of GFP and MIG-6 knockdown BT549 cells upon stimulation of glucose, oligomycin, and 2-DG is shown on the left. Quantitative results for the basal glycolysis and maximal glycolysis capacities in GFP and MIG-6 knockdown BT549 cells are indicated by orange and green boxes, graphed on the right. Results are presented as mean ± SEM (n=15; biological replicates).  (C) A heatmap diagram shows the expression profile of genes involved in glycolysis, the TCA cycle, the pentose phosphate pathway, pyruvate oxidation, gluconeogenesis, and glycogen metabolism obtained from two sets of GFP and MIG-6 knockdown BT549 cells.  Histograms show the percentage of gene dysregulation in glycolysis, TCA cycle, upon MIG-6 knockdown in BT549 cells. For each gene, the change was calculated based on the equation: (shGFP-shMIG6)/shMIG-6. Results are presented as mean ± SEM (n=3; biological replicates).  F) Histograms show the percentage of pyruvate oxidation pathways upon MIG-6 knockdown in BT549 cells. For each gene, the change was calculated based on the equation: (shGFP-shMIG6)/shMIG-6. Results are presented as mean ± SEM (n=3; biological replicates).  (H) Lactate production assay in BT549 cells with GFP or MIG-6 knockdown (n=3). The medium was collected to measure lactate concentration using lactate test strips and an Accutrend Lactate analyzer. The rate of lactate production was determined (lactate production rate = lactate concentration/cells/time) and normalized to the rate detected in the control group. The quantified results are presented as mean ± SEM (n=3; biological replicates).
Figure 4 (A) Box plots show the GLUT1 (SLC2A1) gene expression in different breast tumor subtypes in the Servant dataset of 343 primary breast cancer carcinomas, analyzed using Illumina HumanWG-6_v3 Arrays. The gene expression levels are determined using the R2 platform. In the box plot, error bars are the 95% confidence interval, the bottom and top of the box are the 25th and 75th percentiles, the line inside the box is the 50th percentile (median), and any outliers are shown as open circles. **p < 0.01, by Student's t-test.. (B) Real-Time PCR analysis for GLUT1 and MIG-6 mRNA expression in GFP and MIG-6 knockdown BT549 cells (n=3). (C) Immunoblotting analysis for GLUT1 protein expression in BT549 cells with GFP or MIG-6 knockdown.  (D) Confocal image analysis results for membrane-bound GLUT1 in GFP and MIG-6 knockdown BT549 cells are shown in the top panel. The arrow indicates membrane-localized GLUT1. Scale bar, 25 μm. Quantitative analysis results of the percentage of GFP and MIG-6 knockdown BT549 cells expressing membrane-localized GLUT1 are shown in the bottom panel (n=3).  (E) Representative flow cytometry images (upper) and quantitative results (lower) of measuring glucose uptake in GFP and MIG-6 knockdown BT549 cells (n=3). Cells were grown in the presence of the fluorescent analog 2-NBDG for various time periods, and glucose uptake was quantified using flow cytometric analysis. The red box indicates the gating for 2-NBDG+ cells and the number indicates the percentage of 2-NBDG+ cells.  (F) Immunoblotting analysis for GLUT1 protein expression in GFP and MIG-6 knockdown BT549 cells with or without MG132 treatment.
Figure 5 (B) Immunoblotting analysis for HIF1α protein expression in BT549 cells with GFP or MIG-6 knockdown. (C) Immunoblotting analysis for HIF1α protein expression in MDA-MB-231 cells with GFP or MIG-6 knockdown.  (D) Real-Time PCR analysis for HIF1α mRNA expression in BT549 cells with GFP or MIG-6 knockdown. The quantified results are presented as mean ± SD (n=4). ***p < 0.001, by Student's t-test.  (E) Immunoblotting analysis for HIF1α protein expression in GFP and MIG-6 knockdown BT549 cells in the absence or presence of the proteasome inhibitor MG132.  (F) GFP and MIG-6 knockdown BT549 cells transfected with the indicated plasmids were treated with MG132, subjected to immunoprecipitation (IP) with Flag-tag or HIF1α antibody, followed by immunoblotting analysis. MG132 was used to rescue HIF1α protein degradation mediated by MIG-6 knockdown, leading to similar HIF1α protein levels in control and MIG-6 knockdown BT549 cells, which were used as the input to examine the role of MIG-6 in the interaction between HIF1α and HAUSP.  (G) Luciferase and MIG-6 knockdown 293 cells were transfected with the indicated plasmids and subjected to MG132 treatment. Afterward, cells were harvested for IP with HA antibody, followed by immunoblotting analysis to determine the level of K48-linked ubiquitination of HIF1α. MG132 was used to preserve the degradative K48-linked ubiquitination signals.  (H) Immunoblotting analysis for MIG-6 and HIF1α expression in Luciferase and MIG-6 knockdown 293 without MG132 treatment.  (I) Immunoblotting analysis for GLUT1 protein expression upon MIG-6 overexpression in GFP and HIF1α knockdown BT549 cells.  (J) Immunoblotting analysis for GLUT1 protein expression upon MIG-6 overexpression in GFP and HAUSP knockdown BT549 cells.  (K) Immunoblotting analysis for HIF1α protein expression upon HAUSP overexpression in GFP and MIG-6 knockdown BT549 cells.
Figure 6 (A and B) Lactate production assay in BT549 (A) and MDA-MB-231 (B) cells with Luciferase or GLUT1 knockdown.  (C) Cell proliferation assay in BT549 cells with Luciferase or GLUT1 knockdown.  (D) Cell proliferation assay in MDA-MB-231 cells with GFP or GLUT1 knockdown.  (E and F) Immunoblotting analysis for GLUT1 protein expression in control and MIG-6 knockdown BT549 (E) and MDA-MB-231 (F) cells with or without GLUT1 overexpression.  (G Lactate production assay in control and MIG-6 knockdown BT549 cells with or without GLUT1 overexpression.  H) Lactate production assay in control and MIG-6 knockdown MDA-MB-231 cells with or without GLUT1 overexpression.  (I and J) Cell proliferation assay in BT549 (I) and MDA-MB-231 (J) cells with control knockdown, MIG-6 knockdown, or MIG-6 knockdown plus GLUT1 overexpression.
Figure 7 (A and B) In vivo primary tumor growth derived from BT549 cells with Luciferase or MIG-6 knockdown (six mice per group). Cells were injected into the mammary fat pads of nude mice, and tumor sizes were measured weekly by caliper. Kaplan-Meier plot analysis is used to determine the incidence of Luciferase or MIG-6 knockdown BT549-xenograft tumors (A). Volumes of Luciferase or MIG-6 knockdown BT549 tumors at week 10 are presented as mean ± SEM (B).  (C In vivo primary tumor growth derived from MDA-MB-231 cells with Luciferase or MIG-6 knockdown (nine mice per group). Volumes of Luciferase or MIG-6 knockdown MDA-MB-231 tumors were measured weekly by caliper are presented as mean ± SEM  D) In vivo primary tumor derived from MDA-MB-231 cells with Luciferase or MIG-6 knockdown (nine mice per group). Tumor weights of MDA-MB-231-derived xenografts were measured at the endpoint (day 33) and are presented as mean ± SEM (D).  (E) Immunoblotting analysis for MIG-6 expression in BT549 cells with MIG-6 inducible knockdown (iMIG-6-shRNA) and the non-targeting shRNA control (iNT-shRNA) upon doxycycline treatment.  (F) In vivo primary tumor growth derived from BT549 cells containing iNT-shRNA and iMIG-6-shRNA (n=7 per group). The arrow indicates the time point when the diet was given to induce MIG-6 knockdown in vivo. Volumes of iNT-shRNA and iMIG-6-shRNA BT549 tumors are presented as mean ± SEM.  (G) Representative images for tumor growth and quantitative results for tumor weight at the experimental endpoint (seven mice per group). Results are presented as mean ± SEM.
Figure 8(A) Representative images of histological analysis for GLUT1 protein expression in BT549-derived xenograft tumors. Scale bar, 100 μm. (B) Representative images of histological analysis for GLUT1 protein expression in xenograft tumors derived from BT549 cells containing iNT-shRNA and iMIG-6-shRNA. Scale bar, 100 μm. (C) Representative images of histological analyses for GLUT1 and MIG-6 expressions in TNBC patients in low-grade (stage I) and high-grade (stage III) tumors Scale bar, 200 µm.  (D) Scatterplot analysis for the correlation between GLUT1 and MIG-6 expression levels (n=85). The r value was calculated by Spearman's rank correlation coefficient analysis.  (E) Representative images of the immunofluorescence multiplex assay for colocalization of MIG-6 and HIF1α protein expression in TNBC tumor specimens. The triangle indicates nuclear colocalization, and the star indicates cytoplasmic colocalization.