EMBO/soda-nel huggingface dataset:
Enhancing protective microglia activities with a dual function TREM2 antibody to the stalk region (2020)
Figure 2

Figure 2. 4D9 antibody stimulates TREM2-dependent SYK signaling and blocks ADAM17-mediated TREM2 shedding

(A) Flow cytometry dose response curve for cell binding of 4D9 mAb (EC50= 0.29nM), 4D9 Fab (EC50=0.17nM), and isotype to HEK cells stably overexpressing mouse TREM2. Data represent the mean ± SEM (n=2).

(B) In vitro ADAM17 sheddase activity is blocked by 4D9-effectorless mAb and 4D9 Fab fragment but not an isotype control. Fluorescence polarization of FAM conjugated TREM2 stalk peptide was detected in the presence or absence of ADAM17 and 4D9 mAb, 4D9 Fab and isotype control. Data represent the mean ± SEM (n=6). One-way ANOVA, Tukey's post hoc test; p (4D9 Fab vs 4D9 mAb) = 0.8855; p (4D9 Fab vs uncleaved) < 0.0001; p (4D9 mAb vs uncleaved) < 0.0001; n.s., not significant.

(C) ELISA-mediated quantification of sTREM2 in conditioned media from HEK293 cells stably overexpressing mouse TREM2 treated with a dose titration of 4D9 mAb (EC50=2.3nM), 4D9 Fab, or isotype for 18 hours. Data represent the mean ± SEM (n=3).

(D) AlphaLISA-mediated quantification of pSYK levels in HEK293 Flp-In cells stably overexpressing mouse TREM2 and mouse DAP12 treated with a dose titration of 4D9 mAb, 4D9 Fab or isotype for 5 minutes. Data represent the mean ± SEM (n=3).

(E) AlphaLISA-mediated quantification of pSYK levels in HEK293 Flp-In cells stably overexpressing mouse TREM2 and mouse DAP12 or empty vector treated with 1mg/ml POPC/POPS liposomes and 20µg/ml 4D9 mAb, 4D9 Fab or isotype for 5 minutes. pSYK levels were also determined for cells treated with liposomes only. Data represent the mean ± SEM (n=3). Two-way ANOVA, Tukey's post hoc test (cell line effect: F1,16=365.7, p=<0.0001; treatment effect: F3,16=39.35, p<0.0001; cell line x treatment effect: F3,16=38.75, p<0.0001); p (no Ab vs isotype) = 0.6218; p (no Ab vs 4D9 mAb) < 0.0001; p (no Ab vs 4D9 Fab) = 0.7301; p (isotype vs 4D9 mAb) < 0.0001; p (isotype vs 4D9 Fab) > 0.9999; p (4D9 mAb vs 4D9 Fab) < 0.0001; n.s., not significant.

(F) Schematic representation of the proposed mechanism of action of antibody 4D9. Binding of 4D9 to TREM2 leads to receptor clustering on the cell surface thereby driving downstream pSYK signaling. At the same time, cell surface levels are enhanced by inhibition of ectodomain shedding, potentially because of the inability of proteases to cleave dimeric substrates.
Enhancing protective microglia activities with a dual function TREM2 antibody to the stalk region (2020)
Figure 3

Figure 3. 4D9 antibody modulates TREM2 expression in macrophages and stimulates survival

(A) Flow cytometry detection of cell surface binding of 4D9 (blue) and isotype (green) to wild type and Trem2-/- mouse bone marrow derived macrophages. Representative histograms are shown as MFI (mean fluorescent intensity). Graphs represent the median signal intensity ± SEM (n=3). Student's t-test (two-tailed); Trem2+/+: p = 0.0005; Trem2-/-: p = 0.4435; n.s., not significant.

(B) Immunoblot analysis of membrane fractions of bone marrow derived macrophages (BMDM) upon treatment with 4D9 antibody reveals increased levels of membrane-bound TREM2 relative to an isotype antibody. BMDM from TREM2 knockout mice were included to show the specificity of the anti-TREM2 antibody. Calnexin served as a loading control. Levels of membrane-bound TREM2 were quantified by MSD ELISA. TREM2 antibody clone 5F4 was used for detection. Data represent the mean ± SEM (n=5-6). Unpaired t-test (two-tailed) with Welch's correction; p<0.0001. No gender-specific differences could be observed.

(C) Immunoblot analysis of sTREM2 in conditioned media from BMDM upon treatment with 4D9 and isotype antibodies. BMDM from TREM2 knockout mice were included to show the specificity of the anti-TREM2 antibody. Soluble APP served as a loading control. Note that heavy and light chains of the antibodies used for treatment are also detected and annotated as follows: Ab hc= antibody heavy chain; Ab lc= antibody light chain. Levels of sTREM2 were quantified by MSD ELISA. TREM2 antibody clone 5F4 was used for detection. Data represent the mean ± SEM (n=6). Unpaired t-test (two-tailed) with Welch's correction; p<0.0001. No gender-specific differences could be observed.

(D) Wildtype, Trem2+/-, and Trem2-/- BMDMs were plated in low concentration of M-CSF on 4D9 or isotype coated wells for 5 days and cellular ATP levels were measured by luminescence detection to indicate cell viability. 4D9 and isotype data for each genotype represent the mean ± SEM (n=2 and n=1, respectively).
Enhancing protective microglia activities with a dual function TREM2 antibody to the stalk region (2020)
Figure 4

Figure 4. 4D9 antibody promotes myelin and Aβ(1-42) phagocytosis in primary microglia

(A) Flow cytometry detection of cell surface 4D9 (red) binding and isotype control (blue) on primary wild type and Trem2-/- mouse microglia. Data are shown as MFI (mean fluorescent intensity).

(B) Uptake assay for fluorescently labelled myelin, Aβ(1-42) and inactivated E.coli particles. The top row shows that myelin and Aβ (pseudocoloured in white) accumulate within the plasma membrane of primary microglia (labeled with dylight-649 isolectin and pseudocoloured in red). Overnight pre-treatment with antibody 4D9 significantly increased the percentage of substrate positive cells (middle row). Pre-treatment with the isotope control did not affect the uptake rate (bottom row). No changes in the uptake of E.coli particles were observed. Substrate uptake in the absence of antibody is shown in the top row. Hoechst 33342 was used to counter stain the nuclei (in cyan) and to assess cell density. Scale bar= 20µm.

(C, D, E) Quantification of the change in uptake of myelin (C), Aβ(1-42) (D), and E.coli (E) upon antibody treatment relative to the uptake of the respective substrate in the absence of antibody. The number of substrate positive cells relative to the total number of cells was quantified in each condition. Data represent the mean ± SEM (n=3). Two-way ANOVA (myelin), Sidak's multiple comparisons test (time effect: F1,4=0.01123, p=0.9207; treatment effect: F1,4=27.98, p=0.0061; time x treatment effect: F1,4=0.5948, p=0.4836); p (1h) = 0.1362; p (2h) = 0.0185; unpaired t-test (two-tailed) with Welch's correction (Aβ(1-42) and E.coli); p (Aβ(1-42)) = 0.0151; p (E.coli) = 0.5751; n.s., not significant.
Enhancing protective microglia activities with a dual function TREM2 antibody to the stalk region (2020)
Figure 5

Figure 5. Pharmacokinetics and In vivo target engagement with 4D9 antibody

(A) 4D9 demonstrates standard IgG pharmacokinetics in vivo. Peripheral clearance rates of 4D9 on a human IgG effectorless backbone compared to an isotype control were determined in wild type mice by hIgG ELISA detection of plasma antibody concentrations 1 and 24 hours, 4 and 7 days after 10 mg/kg intravenous injection of antibody.

(B) Brain antibody concentration was measured 24hrs post intravenous dosing of isotype hIgG at 100mg/kg and 4D9-hIgG dosed intravenously at 100, 50 and 10mg/kg. Detection of hIgG levels by ELISA demonstrated dose dependent brain concentrations of 4D9 in the single digit nM range. Animals were perfused to minimize IgG contribution from the plasma. Data represent the mean ± SEM (n=5).

(C) Schematic diagram depicting the target engagement assay setup for bound and total sTREM2 detection. For the bound assay, a secondary anti-human IgG detects 4D9 antibody bound to soluble TREM2 in CSF and plasma. For the total assay, a saturating amount of 4D9 antibody is added to eliminate unbound sTREM2. The ratio of 4D9-bound sTREM2 to total sTREM2 is calculated to determine the level of target engagement achieved by the concentration of antibody present.

(D) Target engagement timecourse demonstrated near 100% 4D9-bound sTREM2 in CSF of wildtype mice at 24 hrs post dose. Over a 10 day timecourse with timepoints at day 1, 2, 4, 7 and study termination at day 10, the bound/total sTREM2 reduces gradually to reach ~50% by day 10. Animals were dosed intravenously with 50mg/kg of isotype and 4D9 antibodies. Data represent the mean ± SEM (n=5).

(E) Target engagement dose response demonstrated saturated bound sTREM2 at 50 and 10mg/kg with >50% bound sTREM2 at 1mg/kg of antibody. 4D9-bound sTREM2 was undetectable at 0.1mg/kg and lower. 4D9 was IV-dosed at 50, 10, 1, 0.1, 0.01, 0.001, 0.0001 mg/kg, and isotype dosed at 50mg/kg. CSF bound: total sTREM2 in wildtype mice was measured at 24hrs. Data represent the mean ± SEM (n=5).

(F) 4D9 antibody demonstrates dose dependent increase of total brain TREM2 levels. Quantification of total TREM2 in brain lysates from wildtype mice dosed with 4D9 or isotype control was performed by a MSD-platform based ELISA. Data represent the mean ± SEM (n=5) and are shown as pg TREM2 per ug of total protein. One-way ANOVA, Dunnett's post hoc test, p (isotype vs 4D9 (100mg/kg)) < 0.0001; p (isotype vs 4D9 (50mg/kg)) = 0.0007.

Data information: only male mice were used.
Enhancing protective microglia activities with a dual function TREM2 antibody to the stalk region (2020)
Figure 6

Figure 6. 4D9 antibody modulates TREM2 functions within the brain

(A) Schematic outlining study design and timeline of intraperitoneal injections of either isotype control of 4D9 antibody in 6 months old APP-NL-G-F and age-matched WT mice.

(B) Immunohistochemical costainings of TREM2 (magenta) and IBA1 (green) microglia in the cortex of isotype control and 4D9 injected WT and APP-NL-G-F mice. Side panel shows images from each staining at a larger magnification indicated by the dotted white boxes. Scale bar = 10µm; scale bar (inset) = 2.5µm.

(C) Quantification of cortical TREM2 stainings shown in B. Two-way ANOVA, Tukey's multiple comparison test (Genotype effect: F1,23=70.63, p=<0.0001; Treatment effect: F1,23=14.33, p=0.0010; Genotype x Treatment effect: F1,23=14.51, p=0.0009); p (WT isotype vs WT 4D9) > 0.9999; p (WT isotype vs APP isotype) = 0.02; p (WT 4D9 vs APP 4D9) < 0.0001; p (APP isotype vs APP 4D9) = 0.0001; n.s., not significant.

(D) Quantification of cortical IBA1 staining shown in B. Two-way ANOVA, Tukey's multiple comparison test (Genotype effect: F1,21=215.4, p=<0.0001; Treatment effect: F1,21=0.5994, p=0.4475; Genotype x Treatment effect: F1,21=0.2992, p=0.5902); p (WT isotype vs APP isotype) < 0.0001; p (WT 4D9 vs APP 4D9) < 0.0001; p (APP isotype vs APP 4D9) = 0.9986; n.s., not significant.

(E) Confocal images of P2RY12 (red), IBA1 (green), and Aβ (gray) costainings from cortex. Top panel: Dotted white boxes indicate the area in P2RY12 and IBA1 costainings that are magnified as inset. Bottom panel: Insets show P2RY12, IBA1 and Aβ costainings at a larger magnification. Of note, we did not observe a complete co-localization of P2RY12 and IBA1 suggesting different microglial populations. Scale bar= 10µm; scale bar (insets) = 5µm.

(F) Quantification of P2RY12 stained cells in the cortex shown in E. Two-way ANOVA, Tukey's multiple comparison test (Genotype effect: F1,23=71.99, p=<0.0001; Treatment effect: F1,23=9.029, p=0.0063; Genotype x Treatment effect: F1,23=5.93, p=0.0231); p (WT isotype vs APP isotype) = 0.0018; p (WT 4D9 vs APP 4D9) < 0.0001; p (APP isotype vs APP 4D9) = 0.0051.

Data information: Each data point for all quantifications represents an average of n=3 replicates per mouse. All data represent the mean ± SEM (n=6-7). No gender-specific differences could be observed.
Enhancing protective microglia activities with a dual function TREM2 antibody to the stalk region (2020)
Figure 7

Figure 7. 4D9 antibody reduces plaque burden

(A, B) Overview of the cortex immunohistochemically stained for Aβ plaques in 6 months old APP-NL-G-F mice treated with isotype control (A) and 4D9 antibody (B). Scale bar= 100µm.

(C) Higher magnification images of cortical Aβ plaques in APP isotype and 4D9 injected mice. Scale bar= 10µm.

(D) Quantification of cortical plaque area from stainings shown in C. Mann-Whitney U test, p=0.0082.

(E) Quantification of total plaque coverage of Aβ stainings in the cortex shown in C. Mann-Whitney U test, p=0.014.

(F) Immunoblot analysis of soluble (DEA fraction, top) and insoluble Aβ levels (FA fraction, bottom) in brains of APP-NL-G-F mice treated with either isotype control or 4D9 antibody. While a reduction in levels of soluble Aβ is evident from the DEA fractions no change in levels of insoluble Aβ could be detected in the FA fractions. Note that the higher background in the DEA immunoblot is due to a much longer exposure time, which was needed to visualize soluble Aβ.

(G) MSD ELISA quantification of soluble Aβ42 in DEA extracts as shown in (F). Mann-Whitney U test, p=0.026.

(H) MSD ELISA quantification of insoluble Aβ42 in FA extracts as shown in (F). Unpaired t-test (two-tailed) with Welch's correction; p=0.0877.

Data information: Each data point for both quantifications represents an average of n=3 replicates per mouse. All data represent the mean ± SEM (n=6-7). No gender-specific differences could be observed.
High-throughput phenotypic screen for genetic modifiers in patient-derived OPA1 mutant fibroblasts identifies PGS1 as a functional suppressor of mitochondrial fragmentation (2021)
Figure 1

Figure 1: Inhibition of mitochondrial division prevents mitochondrial fragmentation caused by OPA1 deficiency in DOA+ patient-derived fibroblasts

(A) Schematic of supervised machine learning (ML) mitochondrial morphology imaging and quantification pipeline. Fibroblasts plated in 384 well plates are stained for mitochondria (anti-TOM40, green), nuclei (DAPI, blue), and cell body (CellMask, blue). Supervised ML training performed on cells with fragmented (OPA1 or YME1L siRNA), normal (non-targeting NT siRNA), and hypertubular (DNM1L siRNA) mitochondria. Automatic single-cell trinary classification of control (CTL-1, 2, 3) and OPA1S545R patient fibroblasts by supervised ML.

(B) Representative confocal images of control (CTL-1, 2, 3) and DOA+ patient fibroblasts carrying indicated monoallelic mutations imaged as described in A. Scale bar=20μm. Passage number between P12-15.

(C) Mitochondrial morphology quantification of B. Data represent mean ± SD of two independent experiments, (195-2496 cells per cell line), One-way ANOVA; ** p < 0.01, ****p < 0.0001, ns; not significant

(D) Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with OPA1, DNM1L, or non-targeting (NT) siRNAs for 72 hours and imaged as described in A. Scale bar=20μm. Passage number between P12-14.

(E) Mitochondrial morphology quantification of D. Data represent mean ± SD of three independent experiments, (3219-5857 cells per cell line), One-way ANOVA; ****p < 0.0001, ns; not significant.

(F) Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with 50μM cycloheximide (CHX) where indicated for 6 hours. Imaging as described in A. Scale bar=20μm. Passage number between P14-P15.

(G) Mitochondrial morphology quantification of F. Data represent mean ± SD of two independent experiments, (879-4154 cells per cell line), One-way ANOVA; ****p < 0.0001, ns; not significant.
High-throughput phenotypic screen for genetic modifiers in patient-derived OPA1 mutant fibroblasts identifies PGS1 as a functional suppressor of mitochondrial fragmentation (2021)
Figure 3

Figure 3: PGS1 depletion rescues mitochondrial fragmentation in OPA1-deficient human and mouse fibroblasts

(A) Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with OPA1, DNM1L, PGS1, and non-targeting (NT) siRNAs or indicated combinations for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=20μm. Passages number between P10-15.

(B) Mitochondrial morphology quantification of (A) using control fibroblasts with fragmented (OPA1 siRNA), normal (non-targeting NT siRNA), and hypertubulated (DNM1L siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (905-3695 cells per cell line), (% fragmented); ; ****p < 0.0001, ns; not significant..

(C) Representative confocal images of wild type (WT) and Opa1Crispr MEFs treated with NT or Pgs1 siRNA for 72 hours. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm.

(D) Mitochondrial morphology quantification of (C) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) ground truth training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (6613-8758 cells per cell line), (% fragmented) ; ****p < 0.0001, ns; not significant.

(E) Representative confocal images of WT, Opa1Crispr MEFs complemented with pLenti-Opa1, Opa1CrisprPgs1Crispr MEFs and Pgs1Crispr MEFs complemented with pLenti-Pgs1 by lentiviral delivery. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm.

(F) Supervised ML mitochondrial morphology quantification of (E) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (691-3990 cells per cell line), (% fragmented) ; ****p < 0.0001, ns; not significant..

(G, H) (G) Equal amounts of protein extracted from MEFs were separated by SDS-PAGE, immunoblotted with anti-OPA1 antibody, and quantified (H) by densitometry relative to Stain-Free. Data represent mean ± SD of three independent experiments, One-way ANOVA; *p < 0.05, ***p < 0.001, ****p < 0.0001, ns; not significant..
High-throughput phenotypic screen for genetic modifiers in patient-derived OPA1 mutant fibroblasts identifies PGS1 as a functional suppressor of mitochondrial fragmentation (2021)
Figure 5

Figure 5: Interfering with the cardiolipin synthesis pathway can prevent mitochondrial fragmentation in Opa1 deficient fibroblasts

(A) Schematic of cardiolipin (CL) biosynthesis pathway in mitochondria. Phosphatidic acid (PA) is transported to the inner membrane by PRELID1 where it is converted to CDP-diacylglycerol (CDP-DAG) and glycerol 3-phosphate (G3P) by TAMM41. Phosphatidylglycerol phosphate (PGP) is dephosphorylated to phosphatidylglycerol (PG) by PTPMT1. PG is either degraded to DAG or reacts with CDP-DAG to form CL in a reaction catalyzed by cardiolipin synthase (CLS1). Tafazzin (TAZ) catalyzes the remodeling of monolysocardiolipin (MLCL) to mature CL. CL is transported to the outer membrane and converted to PA by mitoPLD. PA is converted to DAG by Lipin1. PA can be supplied to the inner membrane from DAG conversion by Acylglycerol Kinase (AGK).

(B) Representative confocal micrographs of MEFs WT and Opa1Crispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm.

(C) Supervised ML mitochondrial morphology quantification of (B) using WT MEFs with fragmented (Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubular (Dnm1l siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (726-4236 cells per cell line), (% fragmented); *** p < 0.001, ****p < 0.0001, ns; not significant.

(D) quantitative RT-PCR (qRT-PCR) measurement of Prelid1, Tamm41, Pgs1, Ptpmt1, and Cls1 expression in Opa1Crispr and WT MEFs. Fold change is indicated relative to WT control. Data represent mean ± SD of three independent experiments, One-way ANOVA.

(E) Whole cell phospholipidome of WT and Opa1Crispr MEFs treated with NT (non-targeting), Tamm41 or Pgs1 siRNAs. Data represent mean ± SD of five independent experiments; *p < 0.05, *** p < 0.001, ****p < 0.0001, ns; not significant.

(F) Representative confocal micrographs of MEFs WT, Pgs1Crispr and Dnm1lCrispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm.

(G) Supervised ML mitochondrial morphology quantification of (G) using WT MEFs with fragmented (Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubulated (Dnm1l siRNA) mitochondria. Data represent mean ± SD of >3 independent experiments, (3096-7238 cells per cell line), One-way ANOVA (% fragmented) ; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.
High-throughput phenotypic screen for genetic modifiers in patient-derived OPA1 mutant fibroblasts identifies PGS1 as a functional suppressor of mitochondrial fragmentation (2021)
Figure 6

Figure 6: Pgs1 depletion does not rescue apoptotic sensitivity nor cristae structure in Opa1-deficient MEFs.

(A, B) (Top) MEFs of the indicated genotypes were subjected to 4μM Actinomycin D and 10μM ABT-737 in the presence or absence of the pan-caspase inhibitor qVD. Dead cells (PI+ nuclei, orange) and total cells (NucBlue, blue) were imaged every hour for 25 hours. PI+ nuclei number divided by the total nuclei number was then quantified over time. (Bottom) Representative confocal images of (Top). Scale bar=100μm. Data represent mean ± SD of three independent experiments, (1380-2157 cells per cell line), One-way ANOVA; ****p < 0.0001, ns; not significant.

(C) Representative transmission electron micrographs of MEFs of the indicated genotypes showing loss of lamellar cristae in Opa1Crispr and Opa1CrisprPgs1Crispr MEFs. Scale bar=200nm.

(D) Quantification of (C) of mitochondrial ultrastructure; outer membrane/inner membrane ration (IMM/OMM) and cristae number per mitochondrion. Violin plot of >50 mitochondria per cell line, One-way ANOVA; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.
High-throughput phenotypic screen for genetic modifiers in patient-derived OPA1 mutant fibroblasts identifies PGS1 as a functional suppressor of mitochondrial fragmentation (2021)
Figure 7

Figure 7: Pgs1 depletion enhances respiration in wild type and Opa1-deficient MEFs.

(A-C) (A) Mitochondrial respiration measured in adherent MEFs of the indicated genotypes using Seahorse FluxAnalyzer. Oxygen consumption rate (OCR) normalized to protein concentration. Following basal respiration, cells were treated sequentially with 1μM Oligomycin (Omy), 2μM CCCP, Antimycin A 1μM + 1μM Rotenone. Bar graphs of (A) representing basal (B) and maximum (C) respiration. Data represent mean ± SEM of 7-12 independent OCR measurements, One-way ANOVA; *p < 0.05, ** p < 0.01, ***p < 0.001, ns; not significant.

(D) Mitochondrial membrane potential measured by fluorescence microscopy in WT, Opa1Crispr, Opa1Crispr + pLenti-Opa1, Opa1CrisprPgs1Crispr, Opa1CrisprPgs1Crispr + pLenti-Pgs1, Pgs1Crispr and Pgs1Crispr MEFs + pLenti-Pgs1. Membrane potential is represented as the ratio between TMRE/mitoYFP. WT MEFs treated with 20μM CCCP serve as a negative control for TMRE. Data represent mean ± SD of three independent experiments, number of analyzed cells indicated in inset, One-way ANOVA; **p < 0.01, ***p < 0.001, ****p < 0.0001, ns; not significant.

(E) mtDNA content in MEFs from (F) was quantified by amplification of MTTL1, 16S and MT-ND1 genes relative to the GAPDH nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One-way ANOVA; ****p < 0.0001, ns; not significant.

(F) mtDNA content in WT and mutant MEFs treated with indicated siRNAs for 72 hours was quantified by amplification of MTTL1, 16S and ND1 genes relative to the GAPDH nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One-way ANOVA; **p < 0.01, ****p < 0.0001, ns; not significant.

(G, H) (G) Equal amounts of protein extracted from WT and mutant MEFs were separated by SDS-PAGE (horizontal line denotes separate membranes), immunoblotted with indicated antibodies and quantified by densitometry (H). Data represent mean ± SD of three independent experiments, One-way ANOVA; **p < 0.01, ***p < 0.001, ****p < 0.0001, ns; not significant.

Table 1: Clinical features of DOA+ patients

Description of clinical features of Dominant Optic Atrophy plus (DOA+) patients from which primary, non-senescent fibroblasts were derived.

Extended View