EMBO/soda-nel huggingface dataset:
The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates. (2012)
Figure 6

SseL deubiquitinates <italic>Salmonella</italic>- and puromycin-induced ALIS.

(A) Quantification of large (>2 µm), ubiquitinated aggresome-like induced structures (ALIS) in macrophages. Primary bone marrow-derived macrophages (BMM) were infected with the indicated GFP-expressing S. Typhimurium strains for 10 h, immunolabelled for ubiquitin and analysed by confocal microscopy. A minimum of 50 cells were counted for each bacterial infection per experiment and values are the mean ± SEM of 3 independent experiments. Uninfected cells (uninf) were from the same wells as infected and therefore were exposed to extracellular bacteria. * p<0.05; ** p<0.01. (B) Quantification of the number of puromycin-induced ubiquitin inclusions in cells. HeLa cells were transfected with a vector expressing myc-SseL or myc-SseLC/A for 16 h followed by treatment with puromycin (5 µg/ml) for 4 h and immunolabelled with anti-myc, anti-p62 and anti-ubiquitin. 50 individual cells were counted per condition in each experiment. Values are the means ± SEM of at least 3 independent experiments. ** p<0.01. (C) Single confocal sections of HeLa cells transfected with a vector expressing myc-SseL for 16 h followed by treatment with puromycin (5 µg/ml) for 4 h and immunolabelled with anti-myc (blue), anti-p62 (green) and anti-ubiquitin (Ub, red) (scale bars, 5 µm). The lower panels show a merged image of p62, ubiquitin and myc-SseL. Arrows indicate ubiquitin and p62 aggregates present in untransfected cells and arrowheads indicate cells expressing myc-SseL.
The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates. (2012)
Figure 7

SseL interacts with p62 and autophagic substrates.

(A and B) Co-immunoprecipitation from RAW264.7 macrophages infected with ΔsseL mutant bacteria expressing SseL-HA or SseLC/A-HA for 10 h. (A) Infectedcell lysates and immunoprecipated fractions were probed with anti-HA, anti-ubiquitin and anti-p62 antibodies. (B) At 7 h post-infection, infected cells were subjected to mock, 3-MA or starvation (Stv) treatments for 3 h before harvesting and processing as described in (A). Cell lysates and immunoprecipiations were probed with anti-HA and anti-ubiquitin antibodies.
The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates. (2012)
Figure 8

SseL reduces autophagic flux and can promote bacterial replication in macrophages.

(A) Lipidated LC3B (LC3-II) levels in RAW264.7 macrophages infected with the indicated strains of S. Typhimurium for 12 h. Cells were left untreated (−) or subjected to 10 mM NH4Cl treatment (+) for 2 h before harvesting. (B) Quantification of relative LC3 II levels in infected cells. Denistometry of bands was analysed using Image J software. The ratio of LC3-II/tubulin signal intensity was normalized to 1 for wild-type untreated infections. The graph shows the mean relative LC3 II/tubulin ratios ± SEM for 3 independent experiments. (C) RAW264.7 or (D) murine bone marrow-derived primary macrophages were infected with the indicated strains of S. Typhimurium for (C) 10 h or (D) 16 h and bacterial replication was monitored using flow cytometry. Results are represented as fold replication normalized to wt; values are the means ± SEM of at least 3 independent experiments. * p<0.05; ** p<0.01.
Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning & reduces neurodegeneration (2020)
Figure 1

Figure 1: Arginine in hnRNPA2 LC is required for the interaction with granule component hnRNPF PLD. See also Appendix Figures S1-S3.

A) hnRNPA2 LC (AlexaFluor488-tagged, green) undergoes LLPS, while hnRNPF PLD (AlexaFluor555-tagged, red) does not. However, hnRNPF PLD partitions into hnRNPA2 LC droplets when mixed at a 1:1 ratio. Conditions: 20 µM indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm.

B) At 300 µM, FUS LC (AlexaFluor488-tagged, green) undergoes LLPS, but at 200 µM hnRNPF PLD (AlexaFluor555-tagged, red) still does not undergo LLPS. When mixed at 300 µM FUS LC and 200 µM hnRNPF PLD, hnRNPF PLD does not partition into FUS LC droplets. Conditions: 300 µM FUS and 200 µM hnRNPF PLD (~1% fluorescently tagged), 20 mM MES pH 5.5, 150 mM NaCl, 150 mM urea. Scale bar: 10 µm.

C) While FUS LC and hnRNPF PLD both have a small negative predicted net charge at neutral pH, hnRNPA2 LC has a predicted +4 net positive charge, due to the 9 positively charged residues (8 arginine, 1 lysine) and 5 negatively charged residues.

D) Removal of the charged residues from hnRNPA2 LC (hnRNPA2 LCCD) prevents partitioning of hnRNPF PLD into the hnRNPA2 LC phase. Addition of hnRNPA2 LC-like charged residue patterning to FUS LC (FUS LCCE) allows the partitioning of hnRNPF PLD at 40 µM. Conditions: protein concentration indicated next to image (20 µM hnRNPA2 LC and hnRNPA2 LCCD, 40 µM FUS LCCE, hnRNPF PLD concentration matches other protein in mixture (either 20 or 40 µM)) (all ~1% fluorescently tagged), 20 mM MES pH 5.5 50 mM NaCl, 150 mM urea. Scale bar: 10 µm.

E) Substitution of all arginines in hnRNPA2 LC with lysine prevents the partitioning of hnRNPF PLD into hnRNPA2 LCRK droplets. Removing all charged residues except for arginine from hnRNPA2 LC (hnRNPA2 LCCD,R) allows partitioning of hnRNPF PLD into droplets, indicating arginine in hnRNPA2 LC is required and necessary for hnRNPF partitioning. hnRNPA2 LCRK does not phase separate much as hnRNPA2 LC at these conditions, see Figure S1J for quantification of phase separation of variants. Conditions: 20 µM proteins, 20 mM MES pH 5.5 50 mM NaCl, 150 mM urea. Scale bar: 10 µm.

F) Quantification of phase separation of hnRNPA2 LC constructs used to determine the residue types important for hnRNPF PLD partitioning. hnRNPA2 LCNS (purple) has similar phase separation to hnRNPA2 LC. hnRNPA2 LCCD (red) is consistently phase separated with ~5 µM protein remaining in the supernatant at all salt conditions tested. Adding back arginines to hnRNPA2 LC no charge (hnRNPA2 LCCD,R, green) brings phase separation as a function of salt to similar levels as hnRNPA2 LC. Changing all the arginine residues to lysine (removing the π-character but maintaining positive charge, hnRNPA2 LCRK) also removes the salt dependence of phase separation but has reduced phase separation overall. Conditions: 20 µM of each protein, pH 5.5 MES, NaCl concentration as indicated, 25° C. Error bars are standard deviation of three replicates.
Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning & reduces neurodegeneration (2020)
Figure 2

Figure 2: Transport granule component TOG D1 interacts weakly with hnRNPA2 LC. See also Appendix Figures S4-S6.

A) AlexaFluor488-tagged (green) hnRNPA2 LC undergoes LLPS, while AlexaFluor555-tagged (red) TOG D1 does not. However, TOG D1 partitions into hnRNPA2 LC droplets when mixed at a 1:1 ratio. Conditions: 20 µM indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. hnRNPA2 LC control duplicated from Figure 1A as hnRNPF PLD and TOG D1 samples were made concurrently.

B) Similar to hnRNPF PLD, AlexaFluor555-tagged TOG D1 does not undergo LLPS at 300 µM or partition into AlexaFluor488-tagged FUS LC droplets with both proteins at 300 µM. Conditions: 300 µM proteins (~1% fluorescently tagged), 20 mM MES pH 5.5 150 mM NaCl, 150 mM urea. Scale bar: 10 µm.

C-D) TOG D1 homology structure with Γ2 values from PRE experiments for hnRNPA2 LC (c) S285C and (d) S329C. Amino acids are colored based on Γ2 value: red corresponds to Γ2 > 2, orange to 2 > Γ2 > 1, yellow to 1 > Γ2 > 0.5.

E) AlexaFluor555-tagged hnRNPF PLD or FL and AlexaFluor405-tagged TOG D1 partition simultaneously into AlexaFluor488-tagged hnRNPA2 LC droplets. Conditions: 20 µM of each indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 20 µm.
Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning & reduces neurodegeneration (2020)
Figure 3

Figure 3: Tyrosine phosphorylation of hnRNPA2 LC alters LLPS and prevents partitioning of hnRNPF PLD or TOG D1. See also Appendix Figures S7-S8.

A) Concentration of protein remaining in the supernatant after centrifugation, which is an inverse measure of phase separation, changes with salt concentration. hnRNPA2 LC shows low phase separation (high protein remaining in the supernatant) at low salt conditions and near complete phase separation (no protein remaining in the supernatant) at high salt concentrations. In contrast, tyrosine phosphorylated hnRNPA2 LC shows higher phase separation (less protein remaining in the supernatant) at low salt concentrations than at high salt concentrations. Conditions: 20 µM protein, 20 mM MES pH 5.5 150 mM urea, salt concentration as indicated, 25˚C. Error bars are standard deviation of three replicates.

B) While hnRNPA2 LC shows no droplets in low salt conditions and droplets in high salt conditions, tyrosine phosphorylated hnRNPA2 LC shows droplets in all salt concentrations tested, although more droplets are present at low salt conditions. Conditions: 20 µM proteins, 20 mM MES pH 5.5 150 mM urea with 0 mM, 50 mM, or 500 mM NaCl as indicated. Scale bar: 10 µm.

C) Fluorescence micrographs showing that although TOG D1 and hnRNPF PLD partition into hnRNPA2 LC droplets (rows 1 and 3), they are unable to partition into tyrosine phosphorylated hnRNPA2 LC droplets (rows 2 and 4). Conditions: 20 µM proteins (~1% fluorescently labeled), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm.

D) Phosphomimic variants containing 5 or 12 serine to glutamate substitutions (hnRNPA2 LC5E and hnRNPA2 LC12E, respectively) both allow partitioning of TOG D1 and hnRNPF PLD, even though hnRNPA2 LC12E barely phase separates at these conditions, indicating that increased negative charge is insufficient to prevent partitioning of TOG D1 and hnRNPF PLD. See Appendix Figure S7B for quantification of LLPS of hnRNPA2 LC5E and hnRNPA2 LC12E. Conditions: 20 µM proteins (~1% fluorescently labeled), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm.
Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning & reduces neurodegeneration (2020)
Figure 4

Figure 4: Tyrosine phosphorylation reduces contacts and aggregation of disease mutants. See also Appendix Figure S9.

A) After cleavage of a C-terminal maltose binding protein solubility tag, hnRNPA2 FL WT undergoes LLPS and disease mutants D290V and P298L aggregate. In contrast, phosphorylated hnRNPA2 FL WT and P298L do not undergo LLPS or aggregation in the time frame tested. Phosphorylated hnRNPA2 FL D290V can form some structures resembling liquid droplets but does not form amorphous aggregates in the time frame tested. Conditions: 20 µM proteins, 20 mM Tris pH 7.4, 50 mM NaCl. Scale bar: 10 µm

B) PLAAC analysis (Lancaster et al., 2014) of hnRNPA2 (black), changing all tyrosine residues in hnRNPA2 LC to glutamate (17 YE, red), 5 serine to glutamate phosphomimic mutations (5 SE, blue), all serine (12 SE, purple), and all tyrosine to glutamine (17 YQ) shows that hnRNPA2 LC prion-like character reduces slightly with serine phosphomimic mutations, but decreases drastically with tyrosine phosphomimic mutations.

C) As the number of phosphorylation events increases, the radius of gyration (Rg) of hnRNPA2 LC increases in coarse-grained simulations. Rg uncertainty bars represent S.E.M. from 10 different simulations with the same number of phosphorylated tyrosines randomly placed in different positions.

D) Coarse grained simulations of WT show that compared to the unphosphorylated form, phosphotyrosine hnRNPA2 LC forms fewer contacts with itself.
Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning & reduces neurodegeneration (2020)
Figure 5

Figure 5: Stress alters HRPA-1 assembly in vivo. See also Appendix Figure S10, S12 and Appendix Tables S1-S2.

A) mScarlet was added between the RNA recognition motifs (RRMs) and LC of HRPA-1 and the third exon was replaced with the corresponding human sequence; disease mutation D290V was introduced.

B) Glutamatergic touch neurons ALM and PLM expressing mec-4 were scored for number of spots in processes.

C) Representative images of mec-4 neurons with and without HRPA-1mScarlet spots. (various HRPA-1mScarlet containing genotypes.)

D) After 22 hours of paraquat induced oxidative stress, both HRPA-1mScarlet and HRPA-1HsLCWTmScarlet assemble more than when unstressed (ANOVA p<0.0001). No significant differences were found within the no stress (p=0.8) stress groups (p=0.45) by ANOVA, indicating that there is no difference in assembly between HRPA-1mScarlet and HRPA-1HsLCWTmScarlet. N = 12 animals per genotype.

E) Fyn expression constructs including activated Fyn (Fyn*, Y531F) and empty control Fynempty.

F) There is no significant difference in number of spots in HRPA-1HsLCWTmScarlet lines with or without Fyn, although there seems to be a trend of reduced spots in HRPA-1HsLCD290VmScarlet lines in animals also expressing Fyn*; this difference is not statistically significant (ANOVA). N = 12 animals per genotype.

G) After exposure to 22 hours paraquat induced oxidative stress there is no significant difference between HRPA-1HsLCWTmScarlet lines with or without Fyn, although there seems to be a trend of reduced spots in HRPA-1HsLCD290VmScarlet lines in animals also expressing Fyn*; this difference is not statistically significant (ANOVA). N = 12 animals per genotype.

Data information: The mean with S.E.M. is reported. Three independent trials for all determinations were performed, with experimenter blinded to genotype for each trial.
Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning & reduces neurodegeneration (2020)
Figure 6

Figure 6: hnRNPA2-D290V causes C. elegans neurodegeneration, which is rescued by

tdp-1 deletion or overexpression of active Fyn kinase. See also Appendix Figures S11-S12, and Appendix Tables S1-S2.

A) Top: schematic depicting hrpa-1 gene and a loss-of-function deletion allele (tm781), referred to as hrpa-1(∆). Bottom: to create a chimeric protein (HRPA-1HsLC), the third coding exon (blue) was replaced with a codon-optimized sequence encoding the majority of the human LC.

B) After 22 hours of paraquat induced oxidative stress, hrpa-1(∆) animals showed increased degeneration of tail glutamatergic (phasmid) neurons, based on defective dye uptake. Defective dye uptake indicates either neuron process degeneration or death. This defect is rescued by introduction of hrpa-1HsLCWT, but not hrpa-1HsLCD290V or hrpa-1HsLCempty. N= 6-12 animals/genotype/trial, significance from two tailed t-test.

C) The C. elegans ortholog of TDP-43, tdp-1, contains 7 exons. A new deletion allele, tgx58, deletes the entire coding region of tdp-1. A second deletion allele, ok803, removes part of the 4th exon and the remaining three exons.

D) Loss of C. elegans TDP-43 ortholog, tdp-1, rescues glutamatergic tail/phasmid neurodegeneration caused by hrpa-1HsLCD290V after 22 hours paraquat induced oxidative stress, but not hrpa-1(∆). N=4-12 animals/genotype/trial, significance from two tailed t-test; tdp-1∆=tdp-1(tgx58).

E) Loss of C. elegans TDP-43 ortholog, tdp-1, rescues glutamatergic tail/phasmid neurodegeneration caused by hrpa-1HsLCD290V after 22 hours paraquat induced oxidative stress, but not hrpa-1(∆). N=4-12 animals/genotype/trial, significance from two tailed t-test; tdp-1∆=tdp-1(ok803).

F) A constitutively activated Y531F Fyn (Fyn*) construct was expressed in the glutamatergic sensory tail (phasmid) neurons using the osm-10 promoter. An empty construct removing the beginning of Fyn and causing a frame shift mutation was constructed similarly.

G) Expression of Fyn* in sensory glutamatergic neurons (osm-10 promoter, unc-54 3'UTR) reduces glutamatergic tail/phasmid neuron degeneration in hrpa-1HsLCD290V animals after 22 hours paraquat induced oxidative stress. N=9-12 animals/genotype/trial, significance from two tailed t-test.

H) Expression of an empty Fyn control (Fynempty) in sensory glutamatergic neurons (osm-10 promoter, unc-54 3'UTR) does not alter glutamatergic phasmid neuron degeneration in hrpa-1HsLCD290V animals after 22 hours paraquat induced oxidative stress. N=9-12 animals/genotype/trial.

Data information: The mean with SEM is reported. Three independent trials for all determinations were performed, with experimenter blinded to genotype for each trial. In panels B, D, E, G, H, hrpa-1HsLCWT, hrpa-1HsLCD290V, and hrpa-1HsLCempty were maintained on a hrpa-1(∆)/tmC25[tmIs1241] background, but transgenes were tested in animals homozygous for hrpa-1(∆). WT is N2, the standard lab strain, while "+" is the N2 hrpa-1 allele maintained over tmC25[tmIs1241] and assayed as a homozygote.
Redistribution of EZH2 promotes malignant phenotypes by rewiring developmental programs (2019)
Figure 1

Figure 1: De novo transformation drives extensive redistribution of EZH2

A. Schematic representation of the fibroblast-based model of cancer development used in this study. Red strikethrough represents inhibition of p53 and pRB by SV40 large and small T-antigens.

B. Quantification by western-blot analysis of EZH2 and H3K27me3 levels at each stage of de novo transformation. UT: untransformed, PN: pre-neoplastic, TR: transformed. Histone H3 is used as a loading control. The graph on the right displays the H3 normalized densitometric values of western-blot band from untreated and DMSO treated cells. Values represent mean ± SEM from three biological replicates. One asterisk indicates p-value < 0.05 (one-tailed unpaired Student's t-test). ns: non-significant.

C. Venn diagrams showing the overlap between H3K27me3-associated EZH2 binding sites detected at each stage of de novo transformation.

D. Volcano plot showing the relative enrichment of EZH2 binding at all sites detected in either untransformed or transformed cells. Numbers within the volcano plot indicate differential binding sites with a p-value ≤1e-20 and a fold-change in normalised tag count ≥1.5. Enrichment significance calculated based on a Poisson distribution using the 'getDifferentialPeaks' function in homer (see: methods). P-values of > 1e-100 were set to 1e-100 for display reasons. Tracks on the right show representative high- and low-magnitude EZH2 differential peaks. ChIP-seq signal normalised to sequencing depth is shown. Tracks are scaled to be of the same height to make samples comparable. Blue bars represent regions called as an EZH2 binding site. UT: untransformed, TR: transformed.

E. Neural-related GSEA gene signatures (left) enriched amongst genes with a high-magnitude, differential EZH2 binding site at their promoter (±5kb transcription start site (TSS)). Gene signatures are derived from GSEA Curated and Gene Ontology gene sets. The following gene signature names were modified from their original molecular signature database descriptor for clarity: 'CpG high promoters marked with H3K27me3 in the brain' - 'MEISSNER_BRAIN_HCP_WITH_H3K27ME3', 'CpG high promoters marked with H3K27me3 and H3K4me3 in the brain' - 'MEISSNER_BRAIN_HCP_WITH_H3K4ME3_AND_H3K27ME3', 'CpG high promoters marked with H3K27me3 in neural progenitor cells' - 'MIKKELSEN_NPC_HCP_WITH_H3K27ME3'.

F. Functional classification of differential site-associated genes (right) (total genes: 37) that are found in the gene signatures 'Central Nervous System Development' and 'Neurogenesis'.
Redistribution of EZH2 promotes malignant phenotypes by rewiring developmental programs (2019)
Figure 2

Figure 2: Redistribution of EZH2 leads to misregulation of key developmental regulators

A. Western-blot analysis of H3K27me3 levels after treatment of cells with an EZH2 inhibitor (EZH2i) or DMSO as control for 12 days. Histone H3 is used as a loading control. Values represent H3-normalized densitometric values of the H3K27me3 bands, and are expressed relative to the relevant DMSO control. UT: untransformed, PN: pre-neoplastic, TR: transformed.

B. Quantification of differentially expressed genes (false discovery rate (FDR) ≤ 0.01, Log2FC ≥ 1/≤-1 and maximal transcripts per million (maxTPM) ≥ 1) detected by RNA-seq in each cellular state upon EZH2i treatment. UT: untransformed, PN: pre-neoplastic, TR: transformed.

C. Venn diagrams showing the overlap between genes upregulated (FDR ≤ 0.01, Log2FC ≥ 1 and maxTPM ≥ 1) by EZH2i treatment in each cellular state.

D. Schematic representation of the multi-step filtering strategy employed to identify functionally important changes in EZH2 distribution induced by neoplastic transformation. Blue and red numbers represent the number of peaks/genes present after each filtering step in untransformed and transformed cells, respectively. UT: untransformed, TR: transformed.

E. Heatmap showing the relative expression of genes identified by the multi-step filtering strategy in the indicated cellular states. UT: untransformed, PN: pre-neoplastic, TR: transformed. Colours represent log2-transformed, row-normalised TPM values.
Redistribution of EZH2 promotes malignant phenotypes by rewiring developmental programs (2019)
Figure 5

Figure 5: Re-expression of EMX2 inhibits the tumorigenicity of glioblastoma cells

A. Proliferation assay examining the effect of EMX2 ectopic expression in U-87 MG and DBTRG-05MG GBM cells, RFP is used as a control. Values represent mean ± SEM from three biological replicates. The fold change in cell number after eight days of proliferation whilst expressing the exogenous protein is shown relative to an uninduced control. Two asterisks indicate p-value < 0.01 (one-tailed unpaired Student's t-test). Similar results were reported in [103] using other GBM cell lines.

B. Transplantation assay comparing the growth kinetics of subcutaneous DBTRG-05MG-induced tumours expressing EMX2 at levels comparable to those expressed in normal cells, or expressing RFP as a control. Values represent mean ± SEM from six tumours. Three asterisks indicate p-value < 0.001 (two-tailed single sample Student's t-test). Similar results were obtained in an independent experiment using a distinct batch of transduced cells.

C. MRI scans showing representative brain tumours six weeks post-injection of DBTRG-05MG cells expressing EMX2 or RFP. Axial anatomical scan and coronal signal intensity map indicate location of tumour in RFP control (yellow arrow). Contrast enhancement in post-contrast images indicates tumour blood brain barrier breakdown. Signal was not present in pre-contrast images. Images show the same MRI slice position between mice. Scale bar: 2mm.

D. Quantification of brain tumour size in mice injected with EMX2- or RFP- expressing DBTRG-05MG cells. Values represent mean ± SEM from four control and three EMX2 tumours. Five mice per conditions were injected but three (one for RFP, and two for EMX2) had to be excluded from the study due to complications from the procedure. Three asterisks indicate p-value < 0.001 (two-tailed unpaired Student's t-test).