EMBO/soda-nel huggingface dataset:
Antigen delivery by filamentous bacteriophage fd displaying an anti-DEC-205 single-chain variable fragment confers adjuvanticity by triggering a TLR9-mediated immune response. (2015)
Figure 5

fdsc-αDEC localizes with TLR9 in DCs

A–D BMDCs from C57BL/6 mice were stably transduced with YFP-tagged TLR9. Cells grown on coverslips were then incubated with TRITC-conjugated wild-type (A, B) or fdsc-αDEC (C, D) bacteriophages for 6 h. Cells were then washed, fixed and analyzed by confocal microscopy. Nuclei were counterstained with Hoechst. (B, D) are enlargements of a single cell. Representative images are shown. Numbers at bottom of right panels indicate the percentage of TLR9 bacteriophage co-localization (mean values ± SD, N = 2, n = 100). Scale bars, 10 mm.
Mitochondria and quality control defects in a mouse model of Gaucher disease--links to Parkinson's disease. (2013)
Figure 1

Loss of gba Impairs Both Autophagic and Proteasomal Degradation Machinery

(A) Autophagic markers from mixed midbrain gba+/+, gba+/−, and gba−/− cultures were analyzed via western blotting using antibodies as indicated.

(B) Autophagic flux was analyzed in gba+/+ and gba−/− midbrain neurons. Neurons were treated with 100 nM bafilomycin A1 and 1 μM rapamycin and flux assayed by comparing LC3I and LC3II levels via western blotting. β-actin was used as a loading control.

(C) Densitometry analyzes of autophagy of (B) expressed as a ratio of LC3II/LC3I. Error bars, ± SEM. *p < 0.05.

(D) Accumulation of p62/SQSTM1 was analyzed via immunoblotting. β-actin was used as a loading control.

(E) Midbrain neurons were treated with either 10 μM MG132 or vehicle control DMSO. Proteins were extracted and analyzed via western blotting and stained with anti-ubiquitin antibodies.

(F) Ubiquitin linkage of isolated midbrain analyzed using western blotting with antibodies as indicated. β-actin was used as a loading control.

(G) Proteasomal activity (chymotrypsin activity) was measured using the Proteasome Glo assay where activity is proportional to the released luciferase. RFU, relative luciferase units. Data represent the mean ± SEM (n = 3, each experiment contained triplicates for each genotype). *p < 0.05.
Mitochondria and quality control defects in a mouse model of Gaucher disease--links to Parkinson's disease. (2013)
Figure 2

Accumulation of α-Synuclein as a Consequence of Impaired Quality Control

(A) Levels of soluble and insoluble α-synuclein from midbrain of P1 gba+/+ and gba−/− mice. TX-100 soluble and insoluble (SDS/Urea) factions were isolated and analyzed via western blotting with α-synuclein antibodies. β-actin was used as a loading control.

(B) Sagittal brain stem sections from gba+/+, gba+/−, and gba−/− mice were stained with α-synuclein antibodies. Scale bar, 1,000 μm.
Mitochondria and quality control defects in a mouse model of Gaucher disease--links to Parkinson's disease. (2013)
Figure 3

Mitochondrial Physiology Is Affected in gba−/− Neurons and Astrocytes

(A) Neurons and astrocytes from gba+/+, gba+/−, and gba−/− mice were stained with TMRM. The mean florescence intensity in mitochondria was analyzed via confocal microscopy (n = 3, >32 cells analyzed/experiment).

(B) Neurons from gba+/+ and gba−/− bathed in TMRM containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 1 min, 1 μM oligomycin was added followed by 1 μM FCCP (n = 5, three cells analyzed/experiment).

(C) Neurons from gba+/+ and gba−/− incubated with 5 mM methyl pyruvate 5 min prior to imaging. Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 90 s, 1 μM oligomycin was added, followed by 1 μM FCCP (n = 3, three cells analyzed/experiment).

(D) As in (C), except cells were bathed in 10 mM methyl succinate, (n = 3, four cells/experiment). All data in this figure represent the mean ± SEM, *p < 0.05.
Mitochondria and quality control defects in a mouse model of Gaucher disease--links to Parkinson's disease. (2013)
Figure 4

gba−/− Mitochondria Have a Reduced Respiratory Chain Activity

(A) Oxygen consumption rates of gba+/+, gba+/−, and gba−/− mixed midbrain cultures. Basal oxygen consumption was measured over 3 min. The maximal (uncoupled) rate was measured via the addition of FCCP and the nonmitochondrial oxygen consumption analyzed via the addition of antimycin A (n = 3, three runs/experiment).

(B) Complex I–NADH: Ubiquinone reductase activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).

(C) Complex II–III: Succinate dehydeogenase, cytochome c reductase activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).

(D) Complex IV activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).

(E) Neurons were treated with 3 nM decylTPP and MitoQ10 for 48 hr and stained with TMRM and fluorescence intensity analyzed via confocal microscopy (n = 3, >16 cells/experiment).

(F) gba+/+and gba−/− cells treated with 3 nM decylTPP and MitoQ10 for 48 hr were subjected to immunoblotting using the indicted antibodies. All data in this figure represent the mean ± SEM, *p < 0.05.
Mitochondria and quality control defects in a mouse model of Gaucher disease--links to Parkinson's disease. (2013)
Figure 5

Mitochondrial Morphology Is Affected in gba−/− Cells

(A) Neurons and astrocytes were immunostained for cytochrome c and stained with Hoechst. Far left panels were additionally immunostained for GFAP and MAP2 to mark neurons and astrocytes. “A” in nucleus denotes astrocytes. Scale bar, 20 μm.

(B) Mitochondrial morphology was blind counted from cells in (A) with neurons scored as MAP2 positive and astrocytes as GFAP positive. A “normal” morphology is classified as a mixture of fused and fragmented mitochondria that comprise a wild-type network. (n = 3; 100 cells counted/experiment).

(C) Levels of morphology proteins from gba+/+, gba+/−, and gba−/− isolated mitochondria were analyzed via immunoblotting with the indicated antibodies.

(D) Midbrain gba+/+, gba+/−, and gba−/− astrocytes expressing GFP-DRP1K38A were immunostained for cytochrome c and mitochondrial morphologies analyzed; concurrently cells were stained with TMRM and fluorescence intensity analyzed via confocal microscopy. Scale bar, 20 μm (n = 3, >15 cells/experiment). All data in this figure represent the mean ± SEM.
Mitochondria and quality control defects in a mouse model of Gaucher disease--links to Parkinson's disease. (2013)
Figure 6

Impaired Mitophagy in gba−/− Neurons and Astrocytes

(A) Midbrain astrocytes were transfected with GFP-LC3, treated with 100 nM bafilomycin A1, and stained with MitoTracker Red and imaged using confocal microscopy.

(B) Midbrain gba+/+ and gba−/− neurons and astrocytes were transfected with YFP-Parkin. Half the transfected cells were treated for 1 hr with 10 μM FCCP and all bathed in TMRM containing recording solution and imaged using confocal microscopy. Scale bar, 20 μm.

(C) TMRM fluorescence intensity of cells from (A) was analyzed via confocal microscopy. Data represent the mean ± SEM, (n = 3, >4 cells analyzed per experiment).

(D) Mitochondria within midbrain neurons were uncoupled by the addition of 10 μM FCCP for 1 and 6 hr. PINK1 expression analyzed via immunoblotting on isolated mitochondria from gba+/+ and gba−/− neurons. Mitochondrial CI subunit NDUFS4 was used as a loading control.
Cerebrospinal fluid tau, neurogranin and neurofilament light in Alzheimer’s disease (2016)
Figure 1

Figure 1. Associations between CSF T-tau, Ng and NFL (panels A-C) and between these biomarkers and CSF A42 (panels D-F). Black squares: CN (n=109), red circles: MCI (n=187), blue triangles: AD (n=93). Associations are shown for Spearman correlations. Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).
Cerebrospinal fluid tau, neurogranin and neurofilament light in Alzheimer’s disease (2016)
Figure 2

Figure 2. CSF T-tau (panel A), Ng (panel B) and NFL (panel C) in different combinations of clinical diagnosis and A pathology (CN Aβ-, n=69; CN Aβ+, n=40; MCI Aβ-, n=50; MCI Aβ+, n=137; AD Aβ-, n=8; AD Aβ+, n=85). See Table 5 for comparisons between groups. Biomarker levels are standardized to z-scores and shown in boxplots (indicating median and interquartile ranges; whiskers are defined as quartiles 1 and 3 +/- 1.5 * interquartile range, respectively). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).
Cerebrospinal fluid tau, neurogranin and neurofilament light in Alzheimer’s disease (2016)
Figure 3

Figure 3. Baseline associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the main effects of the biomarkers, capturing the effects at study baseline. Effects were significant (*) for MMSE: T-tau (p=.034) and NFL (p<.0001) in A-negative; for ADAS-cog: NFL in A-negative (p=.0013) and A-positive (p=.024); for hippocampal volume: NFL in A-negative (p<.0001); for lateral ventricles: T-tau (p=.0019), Ng (p=.00092) and NFL (p=.0024) in A-negative and T-tau (p=.00083) and Ng (p<.0001) in A-positive; and for FDG-PET: NFL in A-negative (p=.046) and A-positive (p=.0041) people. Biomarkers and outcomes were standardized to z-scores. Note that the y-axes for ADAS-cog, ventricle size and WMH are flipped, so that the lower ranges constantly reflect “worse” outcomes. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).
Cerebrospinal fluid tau, neurogranin and neurofilament light in Alzheimer’s disease (2016)
Figure 4

Figure 4. Longitudinal associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the effect of time plus the biomarker by time interactions, capturing the longitudinal effects of the biomarkers. For each model, the “average” effect of time is also shown for comparison. Effects were significant (*), meaning that biomarker levels affected the slopes of the outcome, for MMSE: T-tau (p=.021) and NFL (p<.0001) in A-negative and for T-tau (p<.0001), Ng (p=.0031) and NFL (p<.0001) in A-positive; for ADAS-cog: NFL in A-negative (p<.0001) and for T-tau (p<.0001), Ng (p=.0015) and NFL (p=.0027) in A-positive; for hippocampal volume: NFL in A-negative (p=.0048) and for T-tau (p=.0015) and Ng (p=.0027) in A-positive; for lateral ventricles: Ng (p=.0051) and NFL (p<.0001) in A-negative and NFL (p<.0001) in A-positive; and for FDG-PET: T-tau (p=.00038) and Ng (p=.0019) in A-positive people. Biomarkers and outcomes were standardized to z-scores. Note that the y-axes for ADAS-cog, ventricle size and WMH are flipped, so that the lower ranges constantly reflect “worse” outcomes. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).
Post-metaphase correction of aberrant kinetochore-microtubule attachments in mammalian eggs (2019)
Figure 1

Figure 1. 3D in vivo tracking of chromosome movements from anaphase I to metaphase II arrest.

Time-lapse imaging of an oocyte expressing CENP-C-EGFP (centromeres, green) and H2B-mCherry (chromatin, red). Upper row shows maximum intensity z projection images from representative timepoints of four stages from anaphase I onset to CSF-dependent MII arrest. The 3D positions of centromeres are shown as black dots in a side view (along the spindle equator, middle row) and top view (perpendicular to the equator plane, bottom row). Tracks of individual centromeres are color-coded according to the speed, as indicated by the colour bar. Time is shown after anaphase I onset (min:sec). Scale bars, 5 μm.
Post-metaphase correction of aberrant kinetochore-microtubule attachments in mammalian eggs (2019)
Figure 2

Figure 2. Chromosomes undergo rapid movements concurrent with inter-centromere stretching in prometaphase II.

Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. On the schemes above the charts in (A) and (C), chromosomes are red with green centromeres.

(A) Changes in the distance to the centre for all chromosomes in a representative oocyte undergoing transition from anaphase I to metaphase II from Fig 1. The black arrow on the scheme above the chart indicates chromosome-centre distance.

(B) Changes in the speed of all chromosomes observed for the oocyte from Fig 1.

(C) Changes in inter-centromere distance (indicated on a scheme by the black arrow), observed for the oocyte from Fig 1.

(D) Kinetochore-MT attachments at the prometaphase II stage were visualized using an anti-tubulin antibody (white), an anti-centromeric ACA antibody (green) and DAPI to label chromatin (red). The image represents maximum intensity projection through all z-planes containing MTs. Representative chromosomes displaying syntelic, amphitelic or merotelic/lateral attachments are enclosed in white frames and their enlarged single z-plane images are shown to the right. PB: polar body. Scale bar, 10μm.

(E) Chromosomes with syntelic (red), amphitelic (green) and merotelic/lateral (grey) attachments were plotted according to their inter-centromere distance (on the horizontal axis) and distance to spindle equator (vertical axis). Data shown for 156 chromosomes taken from 8 prometaphase II oocytes.
Post-metaphase correction of aberrant kinetochore-microtubule attachments in mammalian eggs (2019)
Figure 3

Figure 3. 3D in vivo tracking of chromosome movements from metaphase II to anaphase II.

Time-lapse imaging of a representative MII oocyte with a normal segregation pattern (i.e. all 20 chromosomes show synchronous equational chromatid separation at anaphase onset, leaving no chromatids behind at the midzone), expressing CENP-C- EGFP (centromeres, green) and H2B-mCherry (chromatin, red). Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II. Number next to the circles enclosing segregating chromatids denotes amount of chromatids segregated to each of the poles. The 3D positions of centromeres are shown as black dots in a side view (along the spindle equator, middle row) and top view (perpendicular to the equator plane, bottom row). Tracks of individual centromeres are color-coded according to their speed, as indicated by the colour bar. Time is shown relative to anaphase II onset (min:sec). Scale bars, 5 μm.
Post-metaphase correction of aberrant kinetochore-microtubule attachments in mammalian eggs (2019)
Figure 4

Figure 4. Chromosomes maintain stable positions and constant inter-centromere distances until anaphase II onset.

Chromosome parameters obtained for a representative oocyte with normal segregation pattern are shown in A-D on the vertical axis, and time in min on the horizontal axis relative to the anaphase II onset. Thin grey lines in A-D represent individual chromosomes; thick black line in C-D shows the mean value for each timepoint. On the schemes near the charts chromosomes are red with green centromeres; spindle equator in A and spindle axis in B-C are indicated by dashed lines.

(A) Changes in distance to the equator plane for all chromosomes in the representative oocyte with normal segregation pattern, shown in Fig 3. The analysed distance is indicated by a black arrowed line on the scheme above the chart.

(B) Changes in distance to the spindle axis (shown on the scheme by a black arrowed line) for all chromosomes in oocyte from Fig 3.

(C) Changes in chromosome orientation for all chromosomes in oocyte from Fig 3. The orientation is characterized by the angle (highlighted black) between the spindle axis and the line connecting the sister centromeres (black solid line on the scheme). (D) Changes in inter-centromere distance, shown on the scheme by black arrowed line, for all chromosomes in oocyte from Fig 3.

(E) Kinetochore-MT attachments in oocytes released from the CSF-dependent metaphase arrest were visualized using an anti-tubulin antibody (white), an anti-centromeric ACA antibody (green) and DAPI to label chromatin (red). The image represents maxiumum intensity projection through all z-planes containing MTs. Single z-planes of representative chromosomes displaying amphitelic or merotelic/lateral attachments are shown to the right. Scale bar, 10μm.

(F) Chromosomes with amphitelic (green) and merotelic/lateral (grey) attachments were plotted according to their inter-centromere distance (on the horizontal axis) and distance to spindle equator (vertical axis). The inter-centromere distance in fixed oocytes is 1.2±0.2 μm for amphitelic and 1.0±0.2 μm for merotelic attachments (Mean±SD). Data shown for 95 chromosomes taken from 5 oocytes released from the CSF-dependent metaphase II arrest.
Post-metaphase correction of aberrant kinetochore-microtubule attachments in mammalian eggs (2019)
Figure 5

Figure 5. 3D in vivo tracking of chromosome movements in oocytes displaying lagging chromatids at anaphase II.

A time-lapse imaging of a representative MII oocyte with visible lagging chromatid at midzone during anaphase II expressing CENP-C- EGFP (centromeres, green) and H2B-mCherry (chromatin, red). Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II. A lagging chromatid is indicated by a blue arrowhead. Number next to the circles enclosing the segregating chromatids denotes number of chromatids at each pole. The bottom row depicts 3D positions for centromeres of normally segregating chromosomes as black dots, and centromeres of laggard-producing chromosome as red dots in a side view along the spindle equator. One of the centromeres is left behind at anaphase (blue arrowhead), but will segregate equationally. Tracks of individual centromeres are color-coded according to the speed, as indicated by the colour bar. Time is shown relative to anaphase II onset (min:sec). Scale bars, 5 μm.
A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1 (2006)
figf1

Amounts of ATG16L1 protein are influenced neither by the rs2241880 genotype nor by intestinal inflammation. (a) Expression of ATG16L1 in a set of different tissues as detected by RT-PCR (IEC, intestinal epithelial cells). The corresponding β-actin control (with a 518-bp amplicon size) is given below. (b) Amounts of ATG16L1 in colonic mucosal biopsy specimens. Proteins (15 μg) from rectal mucosal biopsies of Crohn disease patients and normal controls (N) were separated by denaturing SDS-PAGE, transferred onto polyvinylidine fluoride (PVDF) membranes and probed for the presence of ATG16L1 using a specific primary antibody and horseradish peroxidase–coupled secondary antibody. The abundant 68.2-kDa band corresponds to the predicted molecular weight of the isoform encoded by GenBank AY398617 (UniProt accession number Q676U5). This major protein form was used for three-dimensional modeling of the ATG16L1 protein (Fig. 2). The 48.5-kDa band corresponds to GenBank EF079890, which contains exon 9 (harboring rs2241880) in the same reading frame as the major form. (c–i) Representative immunohistochemistry results (out of six normal controls and six individuals with Crohn disease) from colonic mucosal tissue samples from normal controls for genotype AA (c) and GG (f) and individuals with Crohn disease for genotype AA (d) and GG (g) are shown to demonstrate the cellular localization of the ATG16L1 protein. Cellular expression patterns in mononuclear cells (h) and intestinal epithelial cells (i) of a Crohn disease sample (rs2241880AA) are shown at higher magnification. e shows a Crohn disease sample without the primary antibody, used as a control. Bar represents 10 μm in c–g.
Human Fis1 regulates mitochondrial dynamics through inhibition of the fusion machinery (2019)
Figure 1

Figure 1. Drp1 is not required for hFis1-induced mitochondrial fragmentation

A. Confocal images of mitochondrial morphology in wild-type (WT) and Drp1-/- 293T cells transfected with empty vector (left panel) and Myc-hFis1 (right panel), stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).

B. Quantitative analyses of fragmented mitochondria size (mean area (μm2) per mitochondrion) after Myc-hFis1 overexpression in WT and Drp1-/- 293T cells using Image J software (Particle analysis) in three independent experiments. In each cell, only dispersed individual mitochondria were analyzed. The total number of mitochondria (mito) analyzed is indicated for each condition.

C. Quantitative analysis of mean mitochondria number per cell in WT and Drp1-/- 293T cells overexpressing Myc-hFis1 using Image J software (Particle analysis) in three independent experiments.

D. Schematic representation of hFis1 mutants used in this experiment.

E. Percentages of cells with indicated mitochondrial morphologies in WT and Drp1-/- 293T cells transfected with empty vector (control), Myc-hFis1 and either Myc- or GFP-tagged mutants as indicated in three independent experiments.

Data information: Data are expressed as means ± s.e.m. and were statistically analyzed by Student's t‐test. n represents the number of cells analyzed (B, C, and E).