EMBO/soda-nel huggingface dataset:
Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1-dependent TFEB/TFE3 regulation (2021)
Figure 7

Figure 7. Compound heterozygous expression of hVps41 variants fails to rescue α-synuclein-induced neurodegeneration in C. elegans.

A. Graph representing the percentage of adult C. elegans animals with 6 normal dopaminergic neurons. Heterozygous expression with hVPS41WT (strains UA386 or UA387) or compound heterozygous expression of hVPS41 variants (strain UA388) does not yield significant changes in neurodegeneration compared to the Pdat-1::GFP (strain BY250) control at either day 7 or day 10 post-hatching. n=30 Adult worms for each of 3 independent experiments for GFP (total of 90 worms) and n=90 for each independent transgenic strain (30 worms/trial x 3 independent transgenic lines = 270 worms), for each of 3 independent experiments.

B. Representative images of the neuroprotection assay described in (A), where worms express GFP specifically in the anterior 6 DA neurons. Intact DA neurons are indicated with arrowheads. hVPS41 variant backgrounds are expressed as indicated. Scale bar 20µm.

C. Graph representing the percentage of animals with 6 normal dopaminergic neurons in the anterior region of Pdat-1::GFP; Pdat-1:: α-syn (strain UA44) animals with heterozygous expression of hVPS41 variants. Heterozygous expression of hVPS41WT with either variant (strains UA389 or UA390) significantly rescues neurons from α-synuclein-induced degeneration at day 7 whereas compound heterozygous expression (strain UA391) fails to rescue neurodegeneration. At day 10, none of the heterozygous backgrounds significantly rescues α-synuclein-induced neurodegeneration. n=30 Adult worms for each of 3 independent experiments for the α-synuclein (α-syn) strain (total of 90 worms) and n=90 for each independent transgenic strain (30 worms/trial x 3 independent transgenic lines = 270 worms) for each of 3 independent experiments.

D. Representative images of DA neurons from C. elegans expressing Pdat-1::GFP; Pdat-1:: α-syn, with or without hVPS41 variants, as described in (C). Intact DA neurons are indicated with arrowheads and missing neurons are indicated with arrows. Scale bar 20µm.

Data information: Data are represented as mean ± SD. *P < 0.05. One-way ANOVA with Tukey's correction (A and C). Exact p-values are reported in Appendix Table S3.

Expanded View Figure Legends
AAV-mediated delivery of an anti-BACE1 VHH alleviates pathology in an Alzheimer's disease model (2022)
Figure 1

Figure 1: Characterization of anti-BACE1 VHH.

(A) VHHs inhibiting BACE1 were identified using an in vitro APP cleavage assay. VHHs were recombinantly expressed in bacteria, purified and added to the assay at a final concentration of 5 μM. VHH-B9, VHH-10C4 and VHH-4A2 consistently inhibited BACE1 activity, compared to PBS or control VHH (Aβ3 and BCIILP, raised against Aβ peptide and β-lactamase BCII 659/H, respectively). Values are mean ± range, n=2, technical replicates.

(B) The EC50 values (95% confidence interval) for VHH-B9, VHH-10C4 and VHH-4A2 are 99.2 nM (83.8-117.5 nM), 112.5 nM (101.1-125.2 nM) and 788.7 nM (682.7-911.2 nM), respectively. Values are mean ± S.D., n=3, technical replicates.

(C, D) Primary cultured neurons were transduced by Semliki Forest virus (SFV) expressing wild type human APP695 and treated with 3 µM of the indicated VHHs. PBS and anti-GFP VHH were used as controls. (C) Western blot analysis of conditioned media for sAPP⍺ and sAPPβ, as well as cell extracts for full length APP, CTF⍺ and CTFβ. (D) ELISA measurements of Aβ1-40 and Aβ1-42 in conditioned media. Values are mean ± S.E.M., n=3 cultures for each analysis. One-way ANOVA, *p<0.5, **p<0.01, *** p<0.0001.

(E, F) Dose-dependent inhibition of BACE1 in primary cultured neurons by VHH-B9. Cultured neurons were transduced by SFV expressing wild type human APP695 and treated with PBS (control: CT) or decreasing concentrations of VHH-B9, ranging from 10 µM to 0.7 nM. (E) Western blot analysis of conditioned media for sAPP⍺ and sAPPβ, as well as cell extracts for full length APP, CTF⍺ and CTFβ. (F) Conditioned media was analyzed by ELISA to assess levels of Aβ1-40. Values are mean ± S.D., n=3 cultures for each analysis. The EC50 value (95 % confidence interval) was estimated as 85.4 nM (52.5-125.6 nM).
AAV-mediated delivery of an anti-BACE1 VHH alleviates pathology in an Alzheimer's disease model (2022)
Figure 4

Figure 4: BACE1 inhibition in AppNL-G-F mice by AAV-VHH-B9 delivery significantly reduces neuroinflammation.

AAV-VHH-B9 or AVV-GFP were systemically injected into AppNL-G-F mice at a dose of 1x1012 vg when mice were 6 weeks of age. Brains were recovered approximately 12 months post-injection following behavioral testing.

(A) (left) Representative images of anti-GFAP immunostaining in Prefrontal Cortex and Amygdala from AppNL-G-F mice receiving either AAV-VHH-B9 or AAV-GFP; (right) quantification of the GFAP+ cell number in the indicated brain regions across experimental cohorts.

(B) (left) Representative images of anti-IBA1 immunostaining in dentate gyrus and amygdala from AppNL-G-F mice receiving either AAV-VHH-B9 or AAV-GFP; (right) quantification of the IBA1+ cell number in the indicated brain regions across experimental cohorts.

Data information: For both (A) and (B), cell numbers were measured in three slices from each of three independent animals (selected based on their performance in the sociability and preference for social novelty test); each dot represents cells counted in an individual slice. Bars represent mean + SEM. Sample group size is shown in the graph. Statistical analyses were performed with an Unpaired t-test with Welch's correction. (A) *p=0.02, **p=0.001; (B) *p=0.01. Scale bars, 50 µm.
SOX9 predicts progression towards cirrhosis in patients while its loss protects against liver fibrosis (2017)
Figure 1

Figure 1 - SOX9 in human liver fibrosis.

A Liver biopsies showing no (IS0), moderate (IS3), and severe (IS6) fibrosis stained by PSR. Size bar, 300 μm.

B Immunohistochemistry (brown staining) for αSMA and SOX9 on consecutive sections from patients with no (IS0), moderate (IS3), and severe (IS6) fibrosis. Boxed areas are shown to the right at higher magnification. Bile duct (bd). SOX9 detected in HSCs with elongated nuclei (hatched line) next to scar and in hepatocytes with rounded nuclei (arrows). Size bars, 200 µm.

C Immunofluorescence for SOX9 (red) co-stained with CK19, CK7, α1AT, and αSMA (green). Arrowheads show elongated nuclei in αSMA-positive HSCs.

D Immunohistochemistry for SOX9 and EpCAM (brown staining) on consecutive sections from severe (IS6) fibrosis and normal fetal liver at 18 weeks post-conception (wpc). Dual immunofluorescence confirms SOX9 and EpCAM in the same cell in severe fibrosis.

E, F, G Data for SOX9 index for the biliary, HSC/Hep, and total (biliary + HSC/Hep) populations. In worsening categories of liver fibrosis (E). In the initial biopsy plotted against the increment in Ishak fibrosis stage between the initial and follow-up biopsy (F). The lines shows the best fit by ordinal logistic regression analysis. In the initial biopsy categorized by whether patients did or did not progress by at least two stages on follow-up biopsy (G).

H Receiver operator curves for the sensitivity and specificity of the SOX9 index in the initial biopsy as a predictor of non-progressors and progressors.

I-L Plots of SOX9 index (total, HSC/Hep, and biliary) and NI score marking progressors in green and non-progressors in red. The solid line indicates the mean in each graph. The broken line in NI dot plot (L) indicates the threshold (score = 8) below which all NI is categorized as 'mild'.

Data information: Unless otherwise indicated in text, two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. P values are indicated.
SOX9 predicts progression towards cirrhosis in patients while its loss protects against liver fibrosis (2017)
Figure 2

Figure 2 - SOX9 is involved in liver fibrosis in vivo.

A Immunohistochemistry for SOX9 (brown) in wild-type (WT) mice following CCl4 or BDL induced fibrosis. SOX9 positive bile ducts (bd) and hepatocytes (arrowheads) indicated.

B (Right panel) Immunofluorescence for SOX9 (green) and HNF4α (red) in WT fibrotic mice. Arrowheads (orange/yellow staining) indicate SOX9+/HNF4α+ hepatocytes and star (*; red) indicates SOX-/ HNF4α+ hepatocytes. (Left panel) Immunofluorescence for SOX9 (green nucleus) and CK19 (red cytoplasm) in WT fibrotic mice. Arrowheads (green) indicate SOX9+ hepatocytes and star (*) indicates SOX- hepatocytes identified by DAPI staining in Appendix Figure S1.

C Co-localization by immunohistochemistry (top panel) and in situ hybridization (lower panel) for SOX9 (red) and αSMA (brown) in wild-type (WT) mice following CCl4 or BDL induced fibrosis.

D Expression of SOX9 protein by immunoblotting of in vivo activated HSCs extracted from WT mice following CCl4 injections compared to olive oil control.

E Immunohistochemistry for SOX9 (brown) in control (Sox9fl/fl;RosaCre-) or Sox9-null (Sox9fl/fl;RosaCre+) mice following fibrosis. Bile ducts (bd) and hepatocytes (arrowheads) indicated. SOX9 is absent, even in bile ducts in Sox9-null liver. Size bar, 25 μm (A, B and E), 10 μm (C).
SOX9 predicts progression towards cirrhosis in patients while its loss protects against liver fibrosis (2017)
Figure 3

Figure 3 - Loss of Sox9 improves liver fibrosis in vivo.

A-J Representative images and quantification shown for olive oil treated (n=6) or chronic CCl4 (n=5 Sox9fl/fl;RosaCreER-; n=8 Sox9fl/fl;RosaCreER+) induced fibrosis or following sham operation (n=5) or BDL (n=7 Sox9fl/fl;RosaCreER-; n=5 Sox9fl/fl;RosaCreER+)) induced fibrosis.

A Picrosirius red (PSR) staining (collagen deposition in red) counterstained with fast green (top row) and immunohistochemistry for α-SMA (brown staining bottom row; activated HSC / myofibroblast marker) in olive oil treated (left) or chronic CCl4 induced fibrosis (right) in control and Sox9-null mice. Size bar = 200 μm.

B, C Quantification of surface area covered by the PSR staining or α-SMA in control (Cnt) and Sox9-null (Null) in (A).

D PSR staining (red; top row) and immunohistochemistry for α-SMA (brown; middle row) and CK19 (brown; bottom row) in control and Sox9-null mice following sham operation (left) or BDL induced fibrosis (right). Size bar = 500 μm.

E, F Quantification of surface area covered by the PSR staining or α-SMA in (D).

G, H Liver function is improved in Sox9-null mice following CCl4 (G) or BDL (H) induced fibrosis compared to control mice. Reduction in serum alanine aminotransferase (ALT; G, H) and bilirubin (H) down to levels shown in non-fibrotic mice (olive oil treated groups; Oil).

I Sox9-loss improved severity of fibrosis compared to control mice indicated by quantification of bridging fibrosis in PSR sections following CCl4. Olive oil treated mice had no bridging fibrosis, in line with a non-fibrotic liver histology in (A).

J Ductal hyperplasia as quantified by the surface area covered by CK19 positive ducts in (D) is reduced in Sox9-null mice compared to control following BDL. All mice were treated with tamoxifen (Tam) which did not induce ectopic expression of SOX9 in non-fibrotic livers (Figure 2A, E and Appendix Figure S3). All experiments are n≥5 as indicated. Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. P values indicated.
SOX9 predicts progression towards cirrhosis in patients while its loss protects against liver fibrosis (2017)
Figure 4

Figure 4 - Fibrosis in Sox9fl/fl;AlbCre+/- mice.

A-C Representative images shown for olive oil treated (n=5) or chronic CCl4 (n=5) induced fibrosis or following sham operation (n=6 Sox9fl/fl;AlbCre-; n=5 Sox9fl/fl;AlbCre+) or BDL (n=5 Sox9fl/fl;AlbCre-; n=8 Sox9fl/fl;AlbCre+) induced fibrosis. SOX9 immunohistochemistry (brown; A), in situ hybridization for Sox9 (brown) and α-Sma (red) and collagen deposition by PSR staining (red; B) in control (Sox9fl/fl;AlbCre-) or Sox9-null (Sox9fl/fl;AlbCre+) mice following fibrosis. Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl/fl;AlbCre+ mice (A).

D, E. Quantification of surface area covered by the PSR staining in (C).

F Expression of SOX9 protein by immunohistochemistry in quiescent (Q) and activated (A) HSCs extracted from Sox9fl/fl;AlbCre+ mice.

G Individual fluorescent channels showing localization and expression of α-SMA (left panel), SOX9 (middle panel) and composite image for α-SMA (green) and SOX9 (red) in right panel in activated Sox9fl/fl;AlbCre+ HSCs (genotyping shown in Appendix Figure S9). Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. Size bars = 100 μm (A), 10 μm (B) 200 μm (C), 25 μm (G).
SOX9 predicts progression towards cirrhosis in patients while its loss protects against liver fibrosis (2017)
Figure 5

Figure 5 - Ly6Clow pro-resolution macrophages predominate in Sox9-null animals.

A, B Flow cytometry of CD45+CD11B+ cells in control (Sox9fl/fl;RosaCre-; A) and Sox9-null (Sox9fl/fl;RosaCre+; B) livers stained for Ly6C and MHCII (far left) to identify four populations of cells with differences in their expression of MerTK, CD64, F4/80 and MHCII.

C Graphical representation of Ly6C and F4/80 staining in macrophages from population 3 and 4.

D, E Graphical representation of cell numbers in the four Ly6C and MHCII cell populations in control and Sox9-null livers. Data are representative of four independent experiments.

F F4/80 immunohistochemistry (brown staining) in control and Sox9-null mice livers following 4-weeks CCl4 induced fibrosis. Size bar = 100μm. Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. P values indicated. All experiments are n=4.
SOX9 predicts progression towards cirrhosis in patients while its loss protects against liver fibrosis (2017)
Figure 6

Figure 6 - YAP signaling regulates SOX9.

A, B Immunofluorescence of activated mouse HSCs cultured on plastic (A) or hydrogels (A, B). Individual fluorescent channels showing localization and expression of SOX9 (left), YAP (middle) and composite image for SOX9 (red) and YAP (green) in right panel. (B) Individual and composite images for αSMA (green) and TAZ (red) or TEAD (red). Size bar, 25μm.

C Quantified decrease in SOX9 protein levels following inhibition of YAP using verteporfin (VP) in activated mouse HSCs expressed relative to DMSO control (n=3).

D Immunohistochemistry for SOX9 (brown) following CCl4 or BDL induced fibrosis in mice treated with DMSO (CCl4 n=4, BDL n=7) or verteporfin (CCl4 n=3, BDL n=5).

E, F quantification of SOX9 cell numbers in the HSC/Hep population. Size bar, 50 μm.

Data information: Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. P values are indicated.
Cyclin B1 scaffolds MAD1 at the kinetochore corona to activate the mitotic checkpoint (2020)
Figure 1

Figure 1. Cyclin B1:MAD1 interaction helps both proteins to localise to unattached kinetochores.

A. Endogenous Cyclin B1-YFP localisation during mitosis live in RPE1 cells (still from Movie EV1).

B,C. Immunofluorescence images (B) and quantifications (C) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC. Each dot represents a kinetochore, data is from 40 kinetochore pairs (13 cells, max 5 kinetochore pairs/cell).

D,E. Quantification of relative kinetochore intensities of Cyclin B1 and MAD1 in nocodazole-arrested cells (noco) treated with the MPS1 inhibitors, AZ-3146 (5μM) or Reversine (500nM), either before (D) or after (E) mitotic entry.

F. Immunofluorescence images of LacI-MAD1 and Cyclin B1 in U2OS cells containing a LacO arrays on chromosome 1 .

G. Live imaging of Cyclin B1-mCherry (CycB1-mCh) in LacO-U2OS cells transfected with LacI-MAD1-FL (full length: aa1-718) or various LacI-MAD1 truncations (amino acid numbers indicated).

H,I. Immunofluorescence images (H) and quantifications (I) of Cyclin B1 and MAD1 kinetochore levels in control (MAD1-WT) or MAD1β HeLa cells (two independent clones: C13 or C24) treated with nocodazole.

J,K. Immunofluorescence images (J) and quantification (K) of Cyclin B1 and MAD1 kinetochore localisation in doxycycline-inducible MAD1α and β knockouts treated with or without dox for 10 days and then arrested in nocodazole. Cells were selected that had full MAD1 knockout in the doxycycline treatment (this constituted approximately 30% of cells).

L. Relative kinetochore volumes occupied by Cyclin B1 and MAD1 (relative to CenpC) in nocodazole-arrested MAD1α and MAD1β cells (calculated from experiments shown in (H,I).

Data information: For all graphs, each dot represents a cell, except panel 1C where dots represent individual kinetochores. The horizontal lines in the graphs indicate the median and vertical bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). All graphs display data that is relative to the controls, which are displayed on the left side of each graph and normalised to 1. The mean level of the normalised controls is indicated by the dotted lines. D, E, I and L show 30 cells from 3 experiments and K shows 40 cells from 4 experiments. Scale bars = 5µM.
Cyclin B1 scaffolds MAD1 at the kinetochore corona to activate the mitotic checkpoint (2020)
Figure 2

Figure 2. Cyclin B1 and MAD1 interact directly through an N-terminal acidic patch on MAD1.

A. Elution profiles and SDS-PAGE for SEC runs on the indicated column of the Cyclin B1:CDK1 complex (blue profile), MBP-MAD1:MAD2 (red), and their combination (green). Note that the same Cyclin B1:CDK1 elution profile and SDS-PAGE is also displayed as reference in panels D and F to improve clarity. For the same reason, the MBP-MAD1:MAD2 elution profile and SDS-PAGE is also displayed in panels 2A-C.

B. Elution profiles and SDS-PAGE for SEC runs on the indicated column of CDK1 (blue), MBP-MAD1:MAD2 (red), and their combination (green).

C. Elution profiles and SDS-PAGE for SEC runs on the indicated column of Cyclin B1 (blue), MBP-MAD1:MAD2 (red), and their combination (green).

D. Elution profiles and SDS-PAGE for SEC runs of the Cyclin B1:CDK1 complex (blue), MBP-MAD1∆93:MAD2 (green), and their combination (red).

E. Elution profiles and SDS-PAGE for SEC runs of the Cyclin B1:CDK1 complex (blue), MBP-MAD11-92-SNAP (red), and their combination (green).

F. Elution profiles and SDS-PAGE for SEC runs of Cyclin B1:CDK1 (blue), MBP-MAD13EK:MAD2 (E52K, E53K, and E56K mutations; red), and their combination (green). In A-D, individual potential binding partners were combined at a concentration of 5 µM.

G. Alignment of the N-terminal region of Cyclin B1 that contains the MAD1 binding region. Numbering refers to the human MAD1 sequence. *conserved, negatively charged residues in MAD1 (E52K, E53K, E56K) required for MAD1:Cyclin B1 interaction.
Cyclin B1 scaffolds MAD1 at the kinetochore corona to activate the mitotic checkpoint (2020)
Figure 3

Figure 3. Cyclin B scaffolds MAD1 at the corona

A. Western blot analysis of indicated vsv-MAD1-WT or 3EK HeLa clones treated with or without doxycycline for 10 days.

B. Immunofluorescence images showing MAD1 and ZW10 kinetochore levels in nocodazole-arrested MAD1-WT-C13 AND 3EK-C14 just after nuclear envelope breakdown (early prometaphase) or later in mitosis when the chromatin is condensed (late prometaphase). Note, that early and late prometaphase was defined based on the level of chromatin condensation.

C,D. Relative kinetochore volumes occupied by MAD1 (C) and ZW10 (D) (relative to CenpC) in MAD1-WT and -3EK cells in early and late prometaphase.

E. Quantification of MAD1 kinetochore intensity from indicated MAD1-WT and 3EK clones treated as in (B).

Data information: For all graphs, each dot represents a cell, horizontal lines indicate the median and vertical bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). All graphs display data that is relative to HeLa early prometaphase controls, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines. 30 cells from 3 experiments. Scale bars = 5µM.
Cyclin B1 scaffolds MAD1 at the kinetochore corona to activate the mitotic checkpoint (2020)
Figure 4

Figure 4. Inhibiting Cyclin B1:MAD1 interaction weakens the SAC.

A-C. Immunofluorescence images (A) and quantifications (B,C) of relative MAD1 and MAD2 kinetochore levels in indicated cells lines arrested in nocodazole and treated with/without AZ-3146 for 30 min to inhibit MPS1. In all kinetochore intensity graphs, each dot represents a cell, horizontal lines indicate the median and vertical bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). All kinetochore intensity graphs display data that is relative to WT C11 controls in nocodazole, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines. 30 cells from 3 experiments. Scale bars = 5µM.

D. Duration of mitotic arrest in indicated cell lines arrested in nocodazole and then treated with 1.25 µM AZ-3146. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.
Cyclin B1 scaffolds MAD1 at the kinetochore corona to activate the mitotic checkpoint (2020)
Figure 5

Figure 5. Cyclin B1 scaffolds MAD1 at the corona to allow the SAC to tolerate MPS1 inhibition.

A. Immunofluorescence images of MAD1 and MAD1-pT716 kinetochore levels in nocodazole-arrested RPE1 cells treated with or without AZ-3146. Scale bars = 5µM.

B. Electron micrographs of rotary-shadowed MAD193-718:MAD2 (top row), MBP-MAD1:MAD2 (middle row), and MBP-MAD1:MAD2 in complex with GST-CDK1:Cyclin B1 (bottom row). Scale bar = 50 nm.

C. Quantifications (top) and corresponding immunofluorescence images (underneath) of relative kinetochore MAD1 and MAD1-pT716 levels in nocodazole-arrested of MAD1-WT-C14 and MAD1-3EK-C12 cells treated with different doses of AZ-3146 for 30 min. MG132 was included at the time of AZ-3146 addition to prevent mitotic exit. Each dot represents a cell, horizontal lines indicate the median and error bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). 30 cells from 3 experiments. Both kinetochore intensity graphs display data that is relative to the WT-C14 untreated controls, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines.

D. Duration of mitotic arrest in MAD1-WT C14 or MAD1-3EK C12 cells arrested in nocodazole and then treated with indicated concentrations of AZ-3146. Note, the 1.25 µM AZ-3146 data is displayed in Figure 4D, but also included here to allow comparison with other drug doses. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.