EMBO/soda-nel huggingface dataset:
MCL‐1 is a stress sensor that regulates autophagy in a developmentally regulated manner (2010)
Figure 5

Conditional knockout of MCL‐1 in the forebrain activates an exacerbated autophagic response. (A) Control and MCL‐1Δ/Δ mice. (B) Expression levels of various BCL‐2 homologues in the cortices of animals of the indicated genotypes. (C) Brain morphology of MCL‐1Δ/Δ. Brain sections were stained with cresyl violet and imaged using a × 1 objective. (D–G) Cortical neurons around the lesion in MCL‐1Δ/Δ animals are positive for the autophagic marker LC3. Brain sections from animals with the indicated genotypes (P14 for E, G; P7 for H) were stained for LC3 (Green) and the neuronal marker NeuN (in H, red), along with the nuclear stain Hoechst (Blue). Confocal images were taken using × 10 (E) or × 63 (G, H) objectives. Scale bars=200 μm (E); 50 μm (G, H). Quantification of the LC3 staining is shown in D. The total number of cells was determined by counting the Hoechst‐positive nuclei. Data are expressed as percent of LC3‐positive cells in at least three animals per genotype±s.d. *P0.01. (F) Quantification of the total surface area occupied by autophagosomes. Data are expressed as percent of total cortical area covered by autophagosomes in at least 16 EM images per genotype±s.d. P0.005 (I) Representative EM images from MCL‐1+/Δ and MCL‐1Δ/Δ P14 mice. N, nucleus; *, autophagosomal structures; arrowheads, double membrane; arrow, mitochondrion inside a vesicle.
MCL‐1 is a stress sensor that regulates autophagy in a developmentally regulated manner (2010)
Figure 6

Regulation of apoptosis in MCL‐1 conditional knockout mice. (A) Cortices from the indicated genotypes and ages were analysed by western blot for the presence of active caspase‐3, p62 and MCL‐1. Primary cortical neurons treated for 12 h with camptothecin were used as a positive control for active caspase‐3. (B) Active BAX (6A7) and active caspase‐3 were also quantified by staining the cortex of 1‐ to 14‐day‐old MCL‐1Δ/Δ mice. Representative images from at least three sections from each animal were counted. The total number of cells was determined by counting the Hoechst‐positive nuclei. Data are expressed as percent of positive cells for at least three animals per genotype±s.d. *P0.05. (C) Active BAX is present in a subset of LC3‐positive cells. Sections from P7 and P14 MCL‐1Δ/Δ brains were stained for LC3 (Green) and active BAX (6A7; Red). Confocal images were taken using × 63 objective. Arrows point to BAX‐positive, LC3‐negative cells. Representative images are shown. Scale bars=50 μm. (D) Primary cortical neurons of the indicated genotypes were infected with a lentivirus expressing Cre at the time of plating and analysed for the presence of the caspase‐cleaved fragment of the caspase‐3 substrate p130CAS 5 days later. Alternatively (E), the neurons were analysed for cytochrome c release by immunofluorescence. Data are expressed as percent of positive cells for at least three embryos per genotype±s.d.
MCL‐1 is a stress sensor that regulates autophagy in a developmentally regulated manner (2010)
Figure 7

Apoptosis and autophagy are activated in a developmentally regulated manner in MCL‐1‐null animals. (A) Brains from E12.5 Foxg1 Cre MCL‐1 animals were analysed by western blot for the presence of the caspase‐cleaved fragment of the caspase‐3 substrate p130CAS and the autophagic markers LC3 and p62. (B) Autophagy in Foxg1 Cre MCL‐1Δ/Δ mice. Brain sections from E15.5 animals with the indicated genotypes were stained for LC3 (green). Confocal images were taken using × 20 (bottom panel) or × 63 (top and middle panels) objectives. Scale bars=50 μm. (C) Colocalization of LC3 vesicles with the lysosomal marker LAMP1 was analysed by immunohistochemistry in E15.5 wild‐type embryos. Data are expressed as the percentage of LC3 vesicles that are LAMP1‐positive±s.d. About 100 LC3 vesicles were counted per section, with four sections per embryo. VZ, ventricular zone. (D) Primary cortical neurons and neuronal progenitor cells were analysed for the presence of the indicated apoptosis (BIM, Puma) and autophagy (ATG7, ATG5‐ATG12) related proteins as well as Tuj‐1 (neuronal marker), Nestin (neural progenitor marker) and HSC70 (loading control). (E) Primary neurospheres were treated for 8 h with EBSS and analysed by western blot for the presence of MCL‐1 and actin as a loading control. (F) Primary neurons and neurospheres were treated as in E and analysed by western blot for p130CAS, LC3 and actin as a loading control. Alternatively, they were analysed by immunofluorescence for cytochrome c release (G) in the presence of 50 μM zVAD‐FMK to prevent apoptotic cells from detaching from the coverslip. Data are expressed as the average of 3–7 embryos±s.d. *P0.001.
MCL‐1 is a stress sensor that regulates autophagy in a developmentally regulated manner (2010)
Figure 8

Inhibition of autophagy in MCL‐1‐deficient neurons activates apoptosis. (A) Brain morphology of P14 MCL‐1Δ/Δ Beclin‐1+/− mice. Brain sections were stained with cresyl violet and imaged using a × 1 objective. (B–D) Active BAX (6A7; B) and active caspase‐3 (C) were quantified by staining the cortex of 14‐day‐old MCL‐1Δ/Δ and MCL‐1Δ/Δ Beclin‐1+/− mice. The number of BAX‐positive LC3‐negative cells was also quantified. (D) Representative images from at least three sections from each animal were counted. The total number of cells was determined by counting the Hoechst‐positive nuclei. Data are expressed as percent of positive cells for least three animals per genotype±s.d. *P0.05. (E) Survival data from MCL‐1Δ/Δ (n=14) and their MCL‐1Δ/Δ Beclin‐1+/− littermates (n=13). (F) Model for the role of MCL‐1 in regulating both autophagy and apoptosis. See text for details.
Aβ43-producing PS1 FAD mutants cause altered substrate interactions and respond to γ-secretase modulation (2019)
Figure 1

Figure 1. Expression, endoproteolysis and APP processing of Aβ43-generating PS1 FAD mutants

(A) Immunoblot analysis of presenilin expression and endoproteolysis in cell lysates of untransfected HEK293/sw cells endogenously expressing presenilin and single cell clones overexpressing PS1 WT or the indicated PS1 mutants.

(B) Immunoblot analysis of total Aβ secretion and the production of different Aβ species (shown in low and high immunoblot exposures) in conditioned media of cell lines described in (A).

(C) Mass spectrometry analysis of Aβ species immunoprecipitated from conditioned media of cell lines described in (A).
Aβ43-producing PS1 FAD mutants cause altered substrate interactions and respond to γ-secretase modulation (2019)
Figure 2

Figure 2. Aβ43-generating PS1 FAD mutants show altered C99 interactions

(A) Immunoblot analysis of C99-PS1 NTF or full-length (FL) crosslinks (CL) of the indicated mutants. Samples that were not UV-irradiated were loaded to control for specificity.

(B) Changes in the ratio of crosslink efficiencies at positions T48 and L49 compared to corresponding controls (n = 4-5 biological replicates).

(C) Changes in the ratio of crosslink efficiencies at positions K54 and V44 compared to corresponding controls (n = 4-5 biological replicates).

Data information: Data in (B) and (C) are presented as mean ± SD.
Aβ43-producing PS1 FAD mutants cause altered substrate interactions and respond to γ-secretase modulation (2019)
Figure 3

Figure 3. Characterization of RO7019009

(A) Structure of RO7019009.

(B) ELISA analysis of Aβ species in conditioned medium of HEK293/sw cells treated with RO7019009 or vehicle (DMSO). Aβ levels are shown relative to DMSO vehicle control (n = 4 biological replicates).

(C) Dose response curve of RO7019009 in HEK293/sw cells measured using the MSD sandwich-immunoassay (n = 3 biological replicates).

(D) Dose response curves for inhibition of Aβ42 generation and Notch cleavage by RO7019009 in different cell lines. Comparison of in vitro potencies for Aβ42 inhibition in HEK293/sw cells, IC50 = 14 nM (n = 3) (as in (C)) with those in H4/sw cells, IC50 = 5 nM (n = 7) and N2a/sw cells, IC50 = 2 nM (n = 6) together with that of Notch cleavage inhibition in a HEK293-based Notch1 luciferase reporter cell line, IC50 > 10 µM (n = 2) (all n numbers represent biological replicates).

(E) Left panel: Immunoblot analysis of total Aβ in conditioned media of HEK293/sw cells treated with RO7019009 or vehicle (DMSO). Total APPs levels were analyzed to control for normal APP secretion and equal sample loading. Right panel: Quantification of relative Aβ amounts in (E) (n = 2 biological replicates).

(F) Immunoblot analysis of Aβ species in conditioned media of RO7019009 treated HEK293/sw cells after separation by Tris-Bicine urea SDS PAGE; synthetic Aβ peptides were loaded as size markers.

Data information: Data in (B-F) are presented as mean ± SD.
Aβ43-producing PS1 FAD mutants cause altered substrate interactions and respond to γ-secretase modulation (2019)
Figure 4

Figure 4. Modulation of PS1 FAD Mutants with RO7019009

(A-C) ELISA analysis of Aβ species in conditioned media of HEK293/sw cells overexpressing WT or mutant PS1 treated with RO7019009 or vehicle (DMSO) showing the secretion of Aβ38 (A), Aβ43 (B) and Aβ42 (C) compared to total Aβ (Aβ38 + Aβ40 + Aβ42 + Aβ43) (n = 5 biological replicates). Note that in some experiments Aβ38 generation was below the detection limit for some mutants. Data are presented as mean ± SD.
Aβ43-producing PS1 FAD mutants cause altered substrate interactions and respond to γ-secretase modulation (2019)
Figure 5

Figure 5. Modulation of Aβ profiles of PS1 FAD mutants by RO7019009

Mass spectrometry analysis of Aβ species immunoprecipitated from conditioned media of HEK293/sw cells overexpressing WT or mutant PS1 treated with 500 nM RO7019009 or vehicle (DMSO).
Aβ43-producing PS1 FAD mutants cause altered substrate interactions and respond to γ-secretase modulation (2019)
Figure 6

Figure 6. Partial effects of RO7019009 on C99-PS1 crosslink formation

(A) Immunoblot analysis of C99 V44Bpa crosslinking (CL) to PS1 WT or NTFs (circle) of normally endoproteolysed mutants in the presence of vehicle (DMSO) or RO7019009.

(B) Quantitation of crosslink efficiencies of C99 V44Bpa to the WT or mutant NTF in (A).

(C) Immunoblot analysis of C99 V44Bpa crosslinking to full-length (FL) PS1 (triangle) of endoproteolysis-deficient mutants and the corresponding M292D control in the presence of vehicle (DMSO) or RO7019009.

(D) Quantitation of crosslink efficiencies of C99 V44Bpa to the M292 or FAD mutant FL PS1 in (C).

Data information: Data in (B and D) are presented as box plots (Line, median; box, 75th-25th percentiles; whiskers, 1.5x interquartile range (IQR); single points represent outliers (>1.5x IQR away from box)) (n = 7-11 biological replicates).

Expanded view FIGURE LEGENDS
Fundamental physical cellular constraints drive selforganization of tissues (2015)
Figure 1

Figure 1. Polygon distribution of biological tissues compared with Voronoi diagrams. A) Segmentation of a Drosophila prepupa wing imaginal disc epithelium (dWP). B) Adult Biceps (BCA) biopsy stained with anti-collagen VI (green) and anti-Myosin Heavy Chain (red) antibodies. C) Segmentation of the image showed in (B). D) Segmented image from a chicken embryo neural tube epithelium (cNT). E) Polygon distribution of the different biological tissues analysed. Two types of distribution can be distinguished: The Poisson Voronoi polygon distribution for Diagram 1 (D1) and cNT, and the conserved polygon distribution for the rest of image categories. F) Diagram with Voronoi seed distributed randomly in the plane. G) Poisson-Voronoi tessellation of the seed showed in (F). H) Resulting Voronoi diagram after performing four Lloyd´s algorithm iterations to the Diagram 1 (Diagram 5, D5). In all figures data are represented as mean ± SEM. Scale bar, 10 m in (A) and (D); 150 mm in (B).
Fundamental physical cellular constraints drive selforganization of tissues (2015)
Figure 4

Figure 4. Polygon and area distribution of altered tissues. A) Segmentation of a prepupal eye imaginal disc from Drosophila (anterior to the left). The analysed developing photoreceptors are labelled in green. The cells adjacent to the photoreceptors are labelled in blue (these are “cone cells” in the most posterior clusters). B) Segmentation of a prepupa wing imaginal disc epithelium where myosin II levels have been reduced. C) Adult Biceps biopsy from a patient with neurogenic atrophy pathology. Fibres outlined with collagen VI antibody (green). D) Segmentation of the image showed in (C) highlighting two atrophic elongated fibres that present seven neighbours. E) Comparison of polygon distributions of different tissues with altered contractibility, tension or pressure balance within the tissue. Scale bars, 10 m in (A) and (B); 75 mm in (C).
Synaptic dysfunction induced by glycine-alanine dipeptides in C9orf72-ALS/FTD is rescued by SV2 replenishment (2020)
Figure 1

Figure 1: GA50 aggregates are detected in neurites of cortical neurons over time

Primary rat cortical neurons transfected with eGFP-GA50 were examined to determine at which time points preceding cell death aggregates are found in neurites. Two days post-transfection, aggregates formed by eGFP-GA50 (green) are detectable in neurites (SMI-32 staining in red). These aggregates remain localized to neurites at 96 hours (4 days) and 288 hours (8 days). Colocalization is indicated by yellow overlay of colors (right panels). Inset below each image shows enlargement of neurite regions containing aggregates, Representative fields from 60x magnification z-stack confocal images, scale bar indicates 20μm. Inset scale bars indicate 5μm.
Synaptic dysfunction induced by glycine-alanine dipeptides in C9orf72-ALS/FTD is rescued by SV2 replenishment (2020)
Figure 6

Figure 6: Synaptic vesicle component SV2 is reduced in GA aggregate containing neurons

eGFP-GAn dipeptides were expressed in primary cortical or motor neurons for 48 hours, then immunostained for neurofilament (gray) and SV2 (red).

A-B) Representative z-stack confocal images of neurites from eGFP, eGFP-GA50, and eGFP-GA100 expressing cortical (A) or motor (B) neurons. 60x magnification, scale bar indicates 5μm.

C-D) Quantification of SV2 puncta along neurites in cortical neurons (C) or motor neurons (D) by ImageJ manual counting, normalized to eGFP-only expressing cells. (C) Cortical neurons demonstrated a significant GA-length dependent reduction, as well as a reduction in all cells containing GA-repeats greater than 50 (left panel). (D) Motor neurons demonstrated a significant GA-length dependent reduction, as well as a reduction in all cells containing GA-repeats greater than 100 (left panel). Evaluation of neurite length by NeuronJ software revealed no differences in total neurite length per cell in cortical neurons (C) or motor neurons (D) expressing any GA-repeat length compared with eGFP alone (right panels).

Data information: Data presented as mean ± SEM. One-way ANOVA, post-hoc Dunnett's multiple comparison test, **** p < 0.0001, n= 3 independent biological replicates, m=5-7 cells per replicate. Exact p-values can be found in Appendix Table S1.
Synaptic dysfunction induced by glycine-alanine dipeptides in C9orf72-ALS/FTD is rescued by SV2 replenishment (2020)
Figure 7

Figure 7: SV2 is reduced in i3 neurons from a C9orf72-ALS patient

A, B) i3 cortical neurons from a C9orf72-ALS patient and the paired isogenic control were differentiated for 21 days, then were immunostained for neurofilament (red), and SV2 (green) (A) or synaptophysin (green) (B). Shown are representative z-stack confocal images of neurites.

C-D) Quantification of SV2 (C) or Synaptophysin (D) colocalized with neurofilament normalized to neurofilament area. This analysis revealed a significant reduction in SV2 levels in the C9orf72 line compared with the isogenic control, while synaptophysin remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p < 0.05. 15 non-overlapping regions from 3 differentiated wells for each genotype were evaluated.

E) mRNA was Trizol extracted from 3 independent wells of C9-ALS and isogenic i3 cortical neurons. RNA was converted to cDNA using the Superscript First-strand kit, and q-PCR was performed using PowerUp SYBR Green. Measurements were normalized to the housekeeping gene GAPDH and then to isogenic levels. Analysis was performed using the ΔΔCT method. 2ΔΔCT ± SE, is presented. Unpaired t-test, **p < 0.01.

F) Whole cell lysates were generated from 3 independent wells of C9-ALS and isogenic i3 cortical neurons differentiated for 21 days. Lysates were immunoblotted for SV2, and normalized to total protein loading. Quantification band intensities revealed a significant decrease in SV2 protein levels in the C9orf72 lines compared with the isogenic. Data presented as mean ± SEM. Unpaired t-test, *p < 0.05.

G-H) Whole cell lysates were generated from iMNs differentiated for 32 days. Lysates were immunoblotted for SV2 (G) or synaptophysin (H). Loading controls were β-actin or total protein loading. Quantification band intensities normalized to loading controls revealed a trend towards reduction in SV2 protein levels in the C9orf72 cell lines compared with controls, while synaptophysin levels remained unaltered. Data presented as mean ± SEM. Unpaired t-test. Samples from 5 independent control and 4 independent C9orf72 patient lines were used. Exact p-values can be found in Appendix Table S1.
Synaptic dysfunction induced by glycine-alanine dipeptides in C9orf72-ALS/FTD is rescued by SV2 replenishment (2020)
Figure 9

Figure 9: SV2 is specifically reduced in spinal cord lysates of symptomatic transgenic GA149-CFP mice

Spinal cord lysates were generated from fresh frozen tissue of 20-month old mice either expressing GA149-CFP under the Thy1 promoter (Tgx GA149) or wild type controls.

A-C) Lysates were immunoblotted for SV2 (A), synaptophysin (B), and PSD-95 (C). Total protein levels were determined by imaging the Mini-PROTEAN TGX stain-free gel prior to transfer. Quantification of band intensities normalized to total protein loading revealed a significant reduction in SV2 protein levels in the GA-expressing cohort compared with wild type animals, while synaptophysin and PSD-95 levels remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 4 animals per group were examined. Exact p-value can be found in Appendix Table S1.
Synaptic dysfunction induced by glycine-alanine dipeptides in C9orf72-ALS/FTD is rescued by SV2 replenishment (2020)
Figure 10

Figure 10: SV2 is specifically reduced at neuromuscular junctions of symptomatic transgenic GA149-CFP mice

Muscle sections (30μm) were cut from snap frozen tissue of 20-month old GA149 mice (Tgx GA149) or wild type controls. Immunohistochemical staining was performed to assess synaptic protein levels in proximity to NMJs. Sections were immunostained for SV2 or Synaptophysin, and counterstained with neurofilament.

A) Left Panels: Representative images show SV2 levels (green) in proximity to α-bungarotoxin positive NMJs (red) in wild type animals (top) versus GA149 animals (bottom). Right Panels: Representative images show SV2 levels (green) overlapping with neurofilament (blue) at synaptic terminals approaching muscle in wild type animals (top) versus GA149 animals (bottom).

B-C) Quantification of SV2 (B) or Synaptophysin (C) colocalized with neurofilament and normalized to neurofilament area. This analysis revealed a significant reduction in SV2 levels in GA149 animals compared with wild types, while synaptophysin remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 10 non-overlapping regions of muscle from each of 5 animals per group were evaluated. Exact p-values can be found in Appendix Table S1.

TABLES:

Table 1: Motor Neuron Counts by Immunocytochemical Staining

EXPANDED VIEW FIGURE LEGENDS:
Cerebral malaria is associated with differential cytoadherence to brain endothelial cells. (2018)
Figure 3

Figure 3. Inhibition of cytoadherence of IE from CM and UM cases to HDMEC and HBMEC by αICAM-1 and αCD36 antibody and rEPCR

A IE were isolated and binding to HBMEC and HDMEC was determined under flow conditions in the absence and presence of 5 µg/ml αICAM-1 or αCD36 antibody or 50 µg/ml rEPCR. Number of IE bound per mm2 EC surface was determined.

B Using the same data, percentage inhibition by αICAM-1 antibody was calculated relatively to binding in the absence of antibody.

C Using the same data, percentage inhibition by rEPCR was calculated relatively to binding in the absence of inhibitor.

D Using the same data, percentage inhibition by αCD36 antibody was calculated relatively to binding in the absence of antibody.

Data information: (A) shown is the paired analysis between the absence and presence of inhibitor, with number of cases (n) indicated. Statistical significance was determined by two-tailed paired t-test and the P-value is shown, ns is not significant. (B-D) shown are the mean ± 95% CI and no significant differences were determined with a two-tailed unpaired t-test.
Aryl Hydrocarbon Receptor is required for optimal B cell proliferation (2016)
Figure 1

Figure 1. B cell activation via BCR engagement and/or IL-4 up-regulates Ahr expression.

A. qPCR analysis of Ahr expression in B cell subsets purified from C57Bl/6 mice. Ahr expression was normalized to Hprt1. n = 3-5 independent experiments; mean ± SD.

B. qPCR analysis of Ahr expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 4h as indicated. Ahr expression was normalized to Hprt1. Ahr expression among groups was normalized to Medium. n = 4 independent experiments; mean ± SEM; one-way ANOVA, Dunnett’s multiple comparison test.

C. qPCR analysis of Ahr expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 4h with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Ahr expression was normalized to Hprt1. Ahr expression among groups was normalized to Medium. n = 3 independent experiments; mean ± SEM; one-way ANOVA, Tukey’s multiple comparison test.

D. Western blot of protein extracts from splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 4h with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Values above the blots indicate AhR protein quantification obtained by densitometry, normalized to β-actin and compared to Medium sample. Sample A and B indicate 2 independent replicates.

E. Quantification by densitometric analysis of the results shown in C. n = 2 independent experiments; mean ± range.

F. qPCR analysis of Ahr expression in purified splenic B cell subsets isolated from C57Bl/6 mice and cultured as indicated for 4h. Ahr expression was normalized to Hprt1. n = 2 independent experiments; mean ± range.

G. qPCR analysis of Ahr expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for the indicated time points with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Ahr expression was normalized to Hprt1. Ahr expression among groups was normalized to Medium. n = 5 independent experiments; mean ± SEM; one-way ANOVA, Dunnett’s multiple comparison test.