EMBO/soda-nel huggingface dataset:
Global and precise identification of functional miRNA targets in mESCs by integrative analysis (2022)
Figure 2

Figure 2. Characterization of predicted miRNA interactions

Cumulative distribution function of differential ribosome occupancy as detected by ribosome profiling in miRNA_KO mutants versus WT. Of 759 predicted miRNA target genes, 728 (96%) were detectable in the ribosome profiling data and appear in the plot.

Boxplot of number of Argonaute2 (AGO2) HEAP peaks for genes grouped by the number of seed match-based miRNA response elements (MRE) in their 3' untranslated regions (UTRs). "Outliers" with more than 15 peaks are not shown.

Number of genes with different numbers of filtered MREs/interactions.

Differential ribosome occupancy in miRNA_KO mutants versus WT of genes grouped by MRE/interaction counts. Groups (colors) correspond to Fig 2C. Shown y-axis range has been limited and some outliers are hidden.

Overlap of candidate miRNA targets with previously reported miRNA targets as collected by the miRTarBase database. Selected genes described in the literature are labeled.

Top: Immunoblot analysis of CIC in WT, Drosha_KO, Dicer_KO and Ago2&1_KO mESCs. TUBULIN was used as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of CIC intensity, normalized to TUBULIN and relative to the WT sample in three biological replicates.

Data information: In A, student's t-test was applied to assess the significance for the increase in ribosome occupancy of the 728/759 miRNA targets (p-value<1e51 for every mutant). Box plots in B and D indicate median as the central band, 25-75%-intervals as box and 1.5 times the interquartile range as the whiskers. Group sizes in B, are 27, 103, 773, 2566, 3293 (from left to right; genes with non-detectable HEAP levels are excluded from this plot), group sizes for D are indicated in panel C. Bar graph in Fig 2F shows mean intensity of CIC signal ± SD normalized to TUBULIN. Values are relative to WT, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05.
Global and precise identification of functional miRNA targets in mESCs by integrative analysis (2022)
Figure 4

Figure 4. Tfap4 is a key regulator of gene expression in mESCs

Immunoblot validation of siPOOL-mediated knock down of Tfap4. TFAP4 levels were compared between untreated WT versus miR-290-295_KO cells treated with a negative control and a Tfap4-targeted siPOOL. Immunoblots were stained with Coomassie blue dye as a loading control. Blot is a representative image of two biological replicates.

Scatterplot of DEG in miR-290-295_KO control versus siPOOL-Tfap4. SiPOOL experiments were performed in miR-290-295_KO cells and DE was assessed relative to a negative control siPOOL transfection. Only genes that are statistically significantly differentially expressed in miR-290-295_KO are shown. Genes predicted to be targeted by miR-290-295 are marked in blue. Genes are defined as rescued (orange) if the log2FoldChange-ratio between miR-290-295_KO control and siPOOL-Tfap4 is in the range [-2, -1/2]. Tfap4 is marked by a red dot.

Gene ontology analysis of 121 putative TFAP4 target genes using ClueGO. Only members of the top 5 terms (indicated by different colors) are shown. Colors and edges indicate associated terms.

Representative images of NANOG (red) and OCT4 (green) immunofluorescence staining in WT, Drosha_KO and Drosha & Tfap4_KO mESCs. Scale bar = 20 μm.

Quantification of NANOG fluorescence signal intensity in WT, Drosha_KO and Drosha & Tfap4_KO mESCs show no significant rescue of NANOG intensity in Drosha & Tfap4_KO

Data information: In C, statistical significance for GO terms was determined by ClueGO and is indicated in the figure. In E, box plots show nuclear intensity of NANOG in ~300 cells per genotype. P-values were generated using ordinary one-way ANOVA. ****p-value<0.0001. In panel E, the box plot's central band represents the median relative intensity for each genotype. The boxes represent the interquartile range and the whiskers represent the maximum and minimum relative intensity values.

Expanded View Figure Legends
Intrathecal activation of CD8+ memory T cells in IgG4-related disease of the brain parenchyma (2021)
Figure 1

Figure 1. Comparative cerebrospinal fluid single-cell profiling of an inflammatory pseudotumor.

a. Uniform Manifold Approximation and Projection (UMAP) of sequenced single cells from inflammatory pseudotumor (IPT) CSF (n=1 sample, n=4,324 single cells, left), control CSF (n=6 samples, n=15,467 single cells, middle) and multiple sclerosis (MS) patients-derived CSF (n=6, n=18,412 cells, right). Indicated immune cell subsets as identified by Seurat v4 reference (ref) mapping.

b. Stacked bar chart of relative B cell abundances as identified by Seurat v4 reference mapping in inflammatory pseudotumor (IPT) CSF, control CSF, and MS patient-derived CSF.

c. Circle plots representing the relative abundance of B cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF, control CSF, and MS patient-derived CSF.

d. Pseudotime analysis of intrathecal B cell subsets in inflammatory pseudotumor CSF with the naïve B cell cluster, identified by canonical markers, as root node. Percentage of cycling naïve B cells and IgG4 B cells depicted in pie charts.

e. Retrospective immunohistochemistry DAB staining of IgG4 and IgG on archival temporal lobe resection tissue. As suggested by the consensus statement on the pathology of IgG4-RD (Deshpande et al, 2012), three 40x fields with the highest number of IgG4+ and IgG+ cells were selected, counted and averaged within these fields. Cell counts as indicated.

f. C-X-C motif chemokine 13 (CXCL13) concentrations measured by ELISA from inflammatory pseudotumor (IPT) CSF, control CSF, and MS patient-derived CSF. Individual values, mean ± SEM; n=4 experiment repeats with technical replicates.

g. Stacked bar chart depicting mean relative expression levels of T helper cell-associated indicated cytokines in T cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF as in (a). TEM, T effector memory; TCM, T central memory; Treg, regulatory T cell.

h. Violin plot depicting relative expression levels of IL4R (left) and IL10RA (right) in B cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF.

Data information: b-c. Cell subsets as indicated by the legend on the right.
Vasculotide reduces endothelial permeability and tumor cell extravasation in the absence of binding to or agonistic activation of Tie2. (2015)
Figure 1

Vasculotide and Ang1 treatments counteract thrombin induction of trans-endothelium macromolecular permeability and tumor cell migration in vitro

A–E Microvascular leak and tumor cell extravasation were modeled in vitro using modified Boyden chamber experiments where lung HMVECs (A–C) and dermal HMVECs (D–E) were grown to 100% confluence over 8-μm-pore insert membranes. ECs were first treated with Vasculotide (VT), PBS (vehicle/negative control), Ang1 (positive control), PEG-Cys (polyethylene glycol backbone), T7c (non-PEGylated CHHHRHSF peptides), or VT in the presence of 100-fold molar excess of T7c. The concentrations used, 10–20 ng/mL VT and 200–400 ng/mL Ang1, are estimated molar equivalents (0.71–1.43 nM). Thirty minutes later, ECs were stimulated with thrombin, 0.1% BSA (vehicle/negative control), or EDTA (positive control). Another 30mins later, the amount of FITC-dextran diffusion into the lower chambers (A, D) provides a measure of endothelial permeability. CMTPX-labeled tumor cells (TCs) were then dispensed into inserts, and the amount of TC fluorescence emitting from the underside of insert membranes after 20 h (B–C) or 28 h (E) reflects the efficiency of trans-endothelial TC migration. Representative fluorescent images (10×) of membrane undersides are shown in (C), where DAPI-stained nuclei of ECs/TCs are shown in blue, ‘extravasated’ CMTPX-labeled TCs are shown in red, and the # of CMTPX+ pixels are shown numerically in red. Means ± SEM are shown (A,B,D,E). Three experiments (twice with HMVEC-LBl and once with HMVEC-DBl) were run with n = 3–5 inserts (independent biological replicates) per group and analyzed by two-sampled unpaired t-tests.
Vasculotide reduces endothelial permeability and tumor cell extravasation in the absence of binding to or agonistic activation of Tie2. (2015)
Figure 4

Endothelial cell-activating cytokines produced by Tie2− tumor cells may influence the anti-permeability efficacy of Vasculotide and Ang1

A, B In modified Boyden chamber experiments, confluent dermal HMVECs were stimulated with tumor cell-conditioned media (TC-CM)—that is, media conditioned by either LM2-4luc, SN12luc, or HT29luc tumor cells—and treated with 20 ng/mL Vasculotide vs. 400 ng/mL Ang1 (positive control) vs. PBS (negative control). Meta-analyses of treatment effects on dextran permeability (A) and tumor cell migration (B), where each datapoint represents the mean of an independent experiment, averaged over 2–3 biological replicates (inserts) per treatment group and normalized internally within that same experiment. Statistical significance of overall Ang1 or VT treatment effects was determined by paired t-tests, n = 11 (A) or 6 (B) experimental replicates.

C Representative fluorescent images (10×) of ‘extravasated’ CMTPX-labeled TCs (red) fixed on membrane undersides.

D LM2-4luc produced the highest levels of VEGF and IL-8, but lower levels of IL-6, compared to other TC types; ***P < 0.001 and *P < 0.05 (unpaired t-tests).
Vasculotide reduces endothelial permeability and tumor cell extravasation in the absence of binding to or agonistic activation of Tie2. (2015)
Figure 5

Vasculotide delays the dissemination of spontaneous lung metastases from orthotopic breast cancer (LM2-4luc) xenografts without having effect on primary tumors

A–C Vasculotide (VT) did not alter the growth kinetics of orthotopic LM2-4luc tumors; n = 8 mice per group. Sunitinib (SU) treatment significantly lowered primary tumor burden as measured by volume (B: one-way ANOVA, P < 0.01) or bioluminescent activity (C: one-way ANOVA, P < 0.001).

D VT treatment did not alter the mean thickness of the viable peripheral regions of primary tumors, as quantified by H&E histological staining.

E VT treatment did not alter the mean thickness of the CAIX+ hypoxic edge between the viable periphery and necrotic core of a primary tumor; IHC control and sample images taken at 5× are shown.

F Ex vivo bioluminescent imaging suggests that VT treatment lowered the proportion of mice with metastatic involvement in the lungs, without the corresponding effect on lymphatic metastases. Representative bioluminescent vs. photographic images of dissected lungs in a 24-well plate are shown.

G Human LM2-4luc cells can be identified on histological sections of mouse lung tissue by IHC staining of human vimentin, a cytoskeletal protein. Size distribution analysis show VT monotherapy reducing the occurence of bigger metastatic nodules.

Data information: Means ± SD are depicted in (A). Means ± SEM and P values from t-tests are depicted in (B–E and G).
Vasculotide reduces endothelial permeability and tumor cell extravasation in the absence of binding to or agonistic activation of Tie2. (2015)
Figure 7

In vitro Vasculotide vs. Ang1 stimulations: effects on Tie2 receptor phosphorylation in primary endothelial cells—HUVECs (A-E) and HMVEC-DBls (F)

A–F Primary human umbilical vein endothelial cells (HUVECs) or primary human dermal microvascular endothelial cells (HMVEC-DBls) were stimulated at 80% confluency with Vasculotide (treatment), Ang1 (positive control; ligand of Tie2), PBS (vehicle control), or VEGF (negative control; non-ligand of Tie2). Tie2 was immunoprecipitated from cell lysates and immunoblotted for total Tie2 or phosphotyrosine. Vasculotide treatment did not lead to increases in the pTie2/Tie2 ratio in HUVECs after 45-min (A–D) or 15-min (E) treatments, with (E) or without (A–D) serum/supplement starvation. Vasculotide also did not activate Tie2 receptors in HMVEC-DBls, whether in the presence or absence of serum, growth factor, and cytokine supplements (F).

Source data are available online for this figure.
Vasculotide reduces endothelial permeability and tumor cell extravasation in the absence of binding to or agonistic activation of Tie2. (2015)
Figure 8

In vivo Vasculotide treatments: effects on Tie2 receptor phosphorylation in vascular-rich organs (liver, lungs, and kidneys)

Healthy non-tumor-bearing mice (n = 8–9 per group) were treated for 1 week with vehicle (Veh); Vasculotide 250 ng/2 days IP (VT); sunitinib 60 mg/kg/day PO (SU); or VT + SU. Tie2 immunoprecipitates from lung, kidney, or liver homogenates were pooled from two mice per treatment group and immunoblotted with an anti-Tie2 antibody (‘Tie2’) or an anti-phosphotyrosine antibody (‘p-Tie2’).

Densitometry analysis of immunoblots from (A). pTie2/Tie2 ratios were internally normalized to the vehicle group within each blot. Means ± SEM and P values (unpaired t-tests) are depicted.

Source data are available online for this figure.
Vasculotide reduces endothelial permeability and tumor cell extravasation in the absence of binding to or agonistic activation of Tie2. (2015)
Figure 9

Binding of Vasculotide vs. Ang1 variants to the extracellular domain of Tie2

A–B Pull-down assay. Input ligands—rhAng1 (A) and VT (B)—were incubated with either protein A sepharose beads only, bead-conjugated mouse Tie2-Fc (mTie2), or bead-conjugated human Tie2-Fc (hTie2). The ‘bound’ fraction shows the total amount pulled down by beads. The ‘unbound’ fraction is 7% of residual/free ligands remaining in the supernatant.

C Quantitative kinetics analysis by SPR. All analytes were run at increasing concentrations of 0.625, 1.25, 2.5, 5, and 10 nM, at 37°C and pH 7.4. The positive controls—rhAng1, BowAng1, COMP-Ang1—all bound strongly to immobilized hTie2-Fc. There was no detectable binding of VT, or the negative control, rhVEGF, to immobilized hTie2-Fc.

Source data are available online for this figure.
Targeting USP8 inhibits Cancer Progression and Prevents EV-TβRII-Induced Exhaustion of CD8+ T cells (2022)
Figure 2

Figure 2. USP8 is a critical component of TGF-β signalling
a, Heatmap of USP8-correlated genes in a comparison of breast cancer patients with USP8-high (n = 38) versus with USP8-low (n = 39).

b, Pre-ranked gene set enrichment analysis (GSEA) in USP8-high (n = 38) versus USP8-low (n = 39) breast cancer patients.

c, Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice (n = 10). qRT-PCR analysis of bone metastatic cells showing the correlations between USP8 and Snai1 or Snai2 (Slug) in cells isolated from mutiple metastatic nodules (n = 21) from mice.

d, 3D spheroid invasion assay of MCF10A-Ras cells showing USP8 wt increased TGF-β-induced invasion. Relative invasion was quantified and shown in the right panel.

e, MCF10A-Ras cells stably expressing control vector (Co.vec), USP8 wt or USP8 cs were injected into the blood circulation of 48-hpf zebrafish embryos. SB431542 (5 μM) was added to the zebrafish environment. Representative images of zebrafish at 5 dpi (left panel); the experimental metastatic areas (middle panel) and percentage of embryos showing metastasis (right panel) are shown, n = 20.

f, Diagram of DUB cDNA screening data of TGF-β-induced SMAD3/ SMAD4-dependent CAGA12-Luc transcriptional reporter in HEK293T cells. The X- and Y-axes are the relative luciferase activity in two replicates.

g, CAGA12-Luc transcriptional response of HEK293T cells transfected with USP8 wt/cs or shUSP8 (#1 and #2) as indicated and treated with TGF-β (1 ng/ml) overnight.

h, Heatmap of TGF-β target genes by qRT-PCR analysis in control or MCF10A-RAS cells stably depleting USP8 (#1 and #2 shRNA) or expressing USP8 wt/cs and treated with or without TGF-β (2.5 ng/ml) for 8 hrs. Appendix Table S2 for details.

i, Immunoblot analysis of total cell lysates, immunoprecipitates derived from control and USP8 stably depleted (#1 and #2 shRNA) (left panel) and USP8 wt/cs over-expressed (right panel) MDA-MB-231 cells treated with TGF-β (2.5 ng/ml) for indicated time points.

j, Immunofluorescence and 4, 6-diamidino-2-phenylindole (DAPI) staining of HaCaT cells infected with control or USP8 wt/cs and treated with TGF-β (2.5 ng/ml) and SB431542 (10 μM) for 72 hrs. Scale bar, 20 μm.

k, Immunoblot analysis of cell lysate of control and USP8 stably depleted-MCF10AR-RAS (MII) cells treated with TGF-β (5 ng/ml) for 48 hrs (left); Immunoblot analysis of cell lysate of control and USP8 wt or USP8 cs stably expressed-MCF10AR-RAS (MII) cells treated with TGF-β (5 ng/ml) or SB431542 (10 μM) for 48 hrs (right).
Data information: *p < 0.05 (two-tailed Student's t test (d, e (middle), g) or two-way ANOVA (c, e (right), f)). Data are representative of at least three independent experiments and shown as means + SD (d (right), g); Data are shown as means ± SD (e (right)).
Targeting USP8 inhibits Cancer Progression and Prevents EV-TβRII-Induced Exhaustion of CD8+ T cells (2022)
Figure 4

Figure 4. Gain of USP8 function promotes the secretion of EV-TβRII during metastasis

a, Comparison of the pre-treatment levels of USP8 mRNA in patient-derived tissue samples by qRT-PCR (left panel), enzyme activity of USP8 in patient-derived tissue samples by Ub-VME (middle panel) and TβRII+ crEVs level in the plasma from breast cancer patients by ELISA (right panel) between breast cancer patients with or without clinical response to PTX. R, responders; n = 16; NR, non-responders; n = 14.

b, Pearson correlation of the USP8 mRNA or USP8 enzyme activity in patient-derived tissue samples with the EV-TβRII levels in the plasma from breast cancer patients without clinical response to PTX as in a (NR, n = 14).

c, Objective response rate (ORR) for patients as in a (n = 30) with high (>0.95 ng/ml) and low (<0.95 ng/ml) pre-treatment levels of circulating EV-TβRII.

d, Pearson correlation of the percentage of IFN-γ+ of CD8+ cells to the TβRII+ crEV level in the plasma from breast cancer patients without clinical response to PTX as in a (NR, n = 14).

e, TβRII+ crEVs (left panel), the USP8 enzyme activity (midde panel) and USP8 mRNA (right panel) from breast cancer patients with or without clinical response to combined therapy using anti-PD-L1 (Atezolizumab) and paclitaxel. R, responders; n = 10 (left panel), n = 5 (middle and right panels, only 5 patient-derived tissue samples obtained). NR, non-responders; n = 10 (left panel), n = 6 (middle and right panels, only 5 patient-derived tissue samples obtained).

f-h, Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice. Bioluminescent imaging (BLI) of representative mice from each group at week 6 injected with control (n = 9) or MDA-MB-231 cells stably ovexpressed with USP8-WT (n = 7) or USP8-CS (n = 9). BLI images are shown (f (left panel)). BLI signal of each mice (f (middle panel)) and number of bone metastasis (f (right panel)) of every mouse, and the percentage of bone metastasis free mice survival in each experimental group followed in time (g). Fold change of circulating EV-TβRII by ELISA analysis in plasma samples of mice in each experimental group at week 6 (h).

i-l, Experimental procedure in vivo: nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) (i). Normalized BLI signals (left panel) and representative mice (right panel) from each group at the indicated times (j). Immunoblot analysis of the circulating EVs from plasma in mice (k). ELISA analysis of circulating exosomal TβRII level in plasma samples of mice at the indicated times (l).
Data information: *p < 0.05 (two-tailed Student's t test (a, c, e, f, h, l), pearson correlation test (b, d), two-way ANOVA (g, j (left))). Data are shown as mean + SD (l) or as means ± SD (a, e, f, h, j (left)).
Targeting USP8 inhibits Cancer Progression and Prevents EV-TβRII-Induced Exhaustion of CD8+ T cells (2022)
Figure 5

Figure 5. The TβRII levels in crEVs are dependent on deubiquitination by USP8

a, Gene signatures indicating the Golgi-associated vesicle biogenesis- and membrane trafficking-related genes are significantly enriched in USP8-high (n = 38) patients versus USP8-low (n = 39) expressing patients; shown by pre-ranked gene-set enrichment analysis (GSEA).

b, Heatmap shows the up-regulated genes upon USP8 over-expression and the down-regulated genes upon USP8 depletion when compared with control MDA-MB-231 cells by RNA-seq analysis (left panel). qRT-PCR analysis of control or MDA-MB-231 cells stably over-expressing USP8 (USP8 OE) or USP8 shRNA (right panel).

c, Go enrichment analysis of significant altered gene sets in USP8 over-expressed MDA-MB-231 cells versus control.

d, Go enrichment analysis of significant up- and down-regulated gene sets in control versus USP8-depleted MDA-MB-231 cells.

e, FACS analysis of EVs from 4T1 and MDA-MB-231 cells infected with lentivirus encoding control shRNA (Co.sh) or USP8 shRNA.

f, Immunoblot analysis of EVs from MDA-MB-231 cells infected with lentivirus encoding control shRNA (Co.sh) or USP8 shRNA (#1 & #2).

g, Immunoblot analysis of the EVs derived from MDA-MB-231 cells ectopically expressing USP8 wt/cs.

h, Experimental procedure in vivo: BALB/c mice were nipple injected with 4T1-Luc cells (5 × 105 cells per mouse) expressing control shRNA or doxycycline-inducible shRNA targeting USP8 (shUSP8) and tumors were grown for 3 weeks, followed by the administration of doxycycline for 3 weeks (n = 6 for each group).

i, Quantification of the percentage of TβRII+ crEVs in plasma samples from mice at day 24.

j, Immunoblot analysis of tumor and the TEVs (left panel) and quantification of the percentage of TβRII+ EVs (right panel) in plasma samples from mice at day 42.

k, Normalized photon ﬂux at the indicated times.

l, Representative BLI signals of primary tumor in each group at day 42 after implantation of 4T1 cells (2 × 105 cells per mouse, n = 6 per group).

m, Normalized BLI signals (left panel) and bioluminescent images (right panel) of the lung metastasis nodules. Scale bars, 2 mm.

n, Percentage of CD8+ T cells of tumor-draining lymph nodes (TDLN) at day 42.
Data information: *p < 0.05 (two-tailed Student's t test (e, i, j (right), m (left), n) or two-way ANOVA (k). Data are representative of at least three independent experiments and shown as mean + SD (e); Data are shown as means ± SD (i, j (right), m (left), n).
A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast (2022)
Figure 1

Figure 1: Utilising an auxin-inducible degron to reduce levels of endogenous biotinylation in TurboID-expressing cells.

(A) The abundance of endogenously biotinylated proteins (orange circles) relative to the abundance of all yeast proteins (black circles) as determined by (Nagaraj et al, 2012).

(B) A streptavidin blot of a control strain showing the running pattern of the endogenously biotinylated proteins highlighted in (A), and labelled according to (Pirner & Stolz, 2006).

(C) Schematic of the EMC showing Emc6 N-terminally tagged with BioID2-HA or TurboID-HA.

(D) Western blot analysis of a strain expressing TurboID-HA-Emc6, Bpl1-AID*-9myc and OsTIR1 which was grown overnight in SD media with or without auxin. The cells were then diluted into fresh SD media and grown to mid logarithmic phase (~4hours) with or without auxin (respectively). When required, biotin was added either 30min before harvesting, or for the entire 4hours. H3 (histone H3) is used as a loading control.

(E) Schematic of the growth conditions used in (D) which were selected for the remaining ABOLISH experiments.