EMBO/soda-nel huggingface dataset:
RNF43 truncations trap CK1 to drive niche-independent self-renewal in cancer (2020)
Figure 3

Fig 3. Oncogenic RNF43 variants trap components of the β-catenin destruction complex at the plasma membrane

A. Volcano plot showing proteins enriched after streptavidin pull-down of biotin-treated HEK293 cells stably expressing dox-inducible RNF43-BirA*. Striped line demarcates the empirical 0.01 False discovery rate (FDR) cut off. Significantly enriched Wnt/β-catenin pathway components are highlighted in red and RNF43 in orange.

B. Western blot analysis of endogenous destruction complex components that co-precipitated with the indicated RNF43 cancer variants expressed in HEK293T cells. Oncogenic RNF43 truncations are indicated in red.

C-D. Confocal microscopy analysis of the subcellular localization of Axin1-GFP (C) and endogenous CK1α (D) upon expression of WT or oncogenic RNF43 (R519X). Scale bars represent 10 μm. RNF43 is visualised by Flag-staining. Asterisks indicate RNF43-expressing cells.

E. β-catenin-mediated reporter activity in HEK293T cells expressing non-mutated RNF43 R519X (SLSS) or the ALAA, DLDD and ∆486-89 mutants. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.

Data information: IP; immunoprecipitation, IB; immunoblot, WT; wild-type.
RNF43 truncations trap CK1 to drive niche-independent self-renewal in cancer (2020)
Figure 4

Fig 4. Onco-RNF43/TP53KO organoids display an oncogenic transcriptional profile that drives self-renewal and niche-independent growth

A. Bright-field microscopy images of WT, TP53KO and onco-RNF43/TP53KO human colon organoid lines grown in medium with high Wnt/Rspo (20% conditioned medium (CM)) or without Wnt/Rspo (20, 2 or 0.2% CM). Images were taken 6 days after splitting. Scale bars represent 1000 μm. Non-cystic, non-proliferative organoids are indicated with red asterisks.

B. Bar plot showing the enrichment scores of significantly enriched MSigDB hallmark gene sets in onco-RNF43/TP53KO compared to TP53KO organoids (FDR < 0.05).

C. Bright-field microscopy and fluorescence microscopy pictures of WT, TP53KO and onco-RNF43/TP53KO human colon organoid lines grown in two different media and transduced with the TOP-GFP reporter. Images were taken 6 days after splitting. Scale bars represent 1000 μm.

D. Gene Set Enrichment Analysis of onco-RNF43/P53KO compared to TP53KO organoids in medium without Wnt/low Rspo (0.2%). Significantly changed intestinal cell-type gene sets from Haber et al., 2017() are shown (FDR < 0.05).
RNF43 truncations trap CK1 to drive niche-independent self-renewal in cancer (2020)
Figure 5

Fig 5. Onco-RNF43 confers reduced sensitivity of colon organoids to Porcupine inhibitors

A. Top: Schematic experimental set up of the clonogenic assay. Bottom: Bright-field microscopy images of WT, TP53KO and onco-RNF43/TP53KO human colon organoid lines at day 2 and 14. Scale bars represent 1000 μm.

B. Relative outgrowth of WT, TP53KO and onco-RNF43/TP53KO organoid lines treated with the PORCN inhibitor C59 (1 μM) for 7 days. Graph shows the number of organoids at 5 days after splitting (day 14) relative to the number of organoids at day 2. Error bars represent ±s.d. of the mean of n = 3 experiments.

C. Model for mode of action RNF43 LOF and onco-RNF43 variants. (Left) RNF43 performs a bifunctional tumor suppressor role by (I) targeting Wnt receptors for endocytosis and lysosomal degradation, and (II) by transiently interacting with the destruction complex to reconstitute its activity in the cytosol and re-establish Wnt pathway inhibition. This second suppressor role involves CK1-mediated phosphorylation and an unknown molecular activity of the flexible cytosolic tail of RNF43. (Middle) Classical loss-of-function (LOF) mutations prevent RNF43 function at the plasma membrane, leading to Wnt receptor overexpression and, consequently, hypersensitivity of cancer cells to Wnt. (Right) Onco-RNF43 truncated variants are stably expressed at the plasma membrane and retain E3 ligase-dependent Wnt receptor downregulating activity. However, due to loss of the cytosolic tail, these mutants selectively trap CK1 and Axin1 at the plasma membrane, which prevents destruction complex assembly and drives uncontrolled β-catenin-mediated transcription of target genes.

Data information: WT; wild-type, LOF; loss-of-function, β-cat; β-catenin.

Extended View Figures
Fusion of Lysosomes with Secretory organelles leads to Uncontrolled Exocytosis in the Lysosomal Storage disease Mucolipidosis Type IV (2015)
Figure 1

Figure 1: Impaired Ca2+-regulated exocytosis in Trpml1-/- pancreatic acini. (a, b) Isolated acini from wild-type (WT) and Trpml1-/- mice (ML1-/-) were used to determine the frequency of Ca2+ oscillations in response to stimulation with 3 and 10 pM CCK (a) and the response to high concentrations of CCK (b). The average frequency from multiple experiments is given in the columns as mean±s.e.m in (a). The traces and columns in (b) show the mean±s.e.m of 3 experiments each includes 4-6 acini composed of 6-10 cells. (c-e) pancreatic acini were used to measure exocytosis in response to stimulation with the indicated CCK concentrations. (c) shows exocytosis during the first 5 min of stimulation, (d) shows the two phases, first rapid and second sustained phase, of exocytosis and (e) shows exocytosis at the indicated CCK concentrations measured during 30 min of stimulation. The results are averages of 3-4 experiments with each mouse line. denotes p<0.01 or better and # denotes p<0.05 or better.
Fusion of Lysosomes with Secretory organelles leads to Uncontrolled Exocytosis in the Lysosomal Storage disease Mucolipidosis Type IV (2015)
Figure 2

Figure 2: Impaired secretory granules size and association with lysosomes in Trpml1-/- pancreatic acini: Panel (a) shows example TEM ultrastructure images obtained from male and female mice pancreas and the columns shows the granules size distribution. Granules size was analyzed in at least 10 images from 3 wild-type and 5 Trpml1-/- mice. (b) Fixed acini were co-stained with Amylase (granules) and LAMP1 (lysosomes) and the fold overlap was calculated from 14 and 11 images obtained from 3 wild-type and 3 Trpml1-/- mice, respectively. (c) Live wild-type and Trpml1-/- acini were labeled with mitotracker (green) and lysotracker (red). The panels show the merged green, red and DIC images. The large vesicles are marked by white arrows and shown at higher magnification in the inserts. The overlap between lysosomes and granules was determined by ImageJ (see methods). The results are from 14 wild-type and 30 Trpml1-/- acini each comprising 3-7 cells and obtained from 3 wild-type and 4 Trpml1-/- mice. (d) Co-IP of lysosomal LAMP1 and granules VAMP8 and Rab27b. Note the increased lysosomal/granules interaction. (e) Enhanced exocytosis of the lysosomal acid phosphatase by Trpml1-/- acini stimulated with 100 pM CCK. The results are the mean±s.e.m of 3 experiments with acini obtained from 3 wild-type and 3 Trpml1-/- mice.
Fusion of Lysosomes with Secretory organelles leads to Uncontrolled Exocytosis in the Lysosomal Storage disease Mucolipidosis Type IV (2015)
Figure 3

Figure 3: Trpml1-/- mice develop chronic pancreatitis. (a, b) Plasma amylase (a) and pancreatic MPO (b) in 3 mice of each line. (c) Edema was evaluated in pancreas of mice treated with PBS or caerulein by H&E staining. Shown are examples of images and the average damage in the columns, which show the mean±s.e.m of 4 experiments with each mouse line. (d) Intracellular trypsin activity was measured in isolated pancreatic acini before and after stimulation with 10 nM CCK for 30 min. Shown are example images and the averages. (e) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained for the lipid ceramide. The figure shows example images before stimulation and the averaged fluorescence intensity. (f) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained with the autophagy marker LC3. The figure shows example images before stimulation and the columns depict the number of autophagosomes as determined by ImageJ. Results in (c-f) are given as the mean±s.e.m of 4 experiments from 4 mice of each line and at least 10 acini in each experiment.
Fusion of Lysosomes with Secretory organelles leads to Uncontrolled Exocytosis in the Lysosomal Storage disease Mucolipidosis Type IV (2015)
Figure 4

Figure 4: Normal Ca2+-stimulated fluid secretion but augmented cAMP-stimulated exocytosis in Trpml1-/- mice salivary glands. (a) Isolated acini from wild-type (black) and Trpml1-/- mice (red) loaded with Fura2 were used to determine [Ca2+]i in response to stimulation with 100 µM carbachol. The traces are the mean±s.e.m. of 6 acinar clusters. Similar results were obtained in 3 experiments. (b, c) Saliva was collected from wild-type (black) and Trpml1-/- mice (red) injected I.P. with 10 mg/kg pilocarpine (b) or 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol (c) over 20-40 min to stimulate fluid secretion by pilocarpine and amylase secretion by isoproterenol. Each condition used 4-5 mice from each like and the results are given as the mean±s.e.m. (d) the saliva collected in (c) was used to determine the amount of secreted amylase. (e, f) Isolated acini from the parotid glands of wild-type (black) or Trpml1-/- mice (red) were used to measure amylase secretion in response to 50 nM isoproterenol for the indicated times (e) and the indicated isoproterenol concentrations during 30 min stimulation (f). The results are the mean±s.e.m obtained from two mice of each line.
Fusion of Lysosomes with Secretory organelles leads to Uncontrolled Exocytosis in the Lysosomal Storage disease Mucolipidosis Type IV (2015)
Figure 5

Figure 5: Enlarged salivary glands secretory granules, association of lysosomes and secretory granules, and secretion of the lysosomal acid phosphatase in the Trpml1-/- mice: (a) shows example TEM images and (b) shows the granules size distribution in parotid glands determined from 10 images obtained from 3 wild-type mice and 14 images obtained from 4 Trpml1-/- mice. (c) Co-IP of the lysosomal LAMP1 and the secretory granules VAMP8 and Rab27b. Input control is actin. (d) Acini isolated from the parotid glands of wild-type and Trpml1-/- mice were incubated with mitotracker (green) and lysotracker. The large vesicles in the secretory granules/lysosomal area are marked by white arrows. The average number of enlarged profiles is given in the columns and obtained from 3 acinar preparations of each line. (e) Shown is the time course (left panel) and averaged 10 min secretion (columns) of the lysosomal marker acid phosphatase in the saliva collected from wild-type (black) and Trpml1-/- mice (red) stimulated with 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol. The mean±s.e.m is from 4 mice of each line.
Fusion of Lysosomes with Secretory organelles leads to Uncontrolled Exocytosis in the Lysosomal Storage disease Mucolipidosis Type IV (2015)
Figure 6

Figure 6: mEPSCs and [Ca2+]i in brain slices. (a, b) example traces of mEPSCs recorded from wild-type (a) and Trpml1-/- (b) mice brain slices as detailed in Methods. The lower traces in each panel show the portion of the traces marked by dotted boxes in an expanded time scale. (c) histogram distribution of the events at each mEPSC. The lower columns show the distribution of the higher amplitude mEPSCs. (d) the frequency of mEPSCs in each line. The results in (c, b) are the mean±s.e.m of 8 experiments each obtained from 4 mice of each line. (e) the mean±s.e.m of the Ca2+ signal in response to depolarization with 30 mM KCl of 6 and 5 experiments with wild-type and Trpml1-/- slices, respectively.
Fusion of Lysosomes with Secretory organelles leads to Uncontrolled Exocytosis in the Lysosomal Storage disease Mucolipidosis Type IV (2015)
Figure 7

Figure 7: Impaired synaptic vesicle size, number of PSD and exocytosis in cortical neurons. (a) example TEM images of synapses in wild-type and Trpml1-/- mice brain cortex. (b) the distribution of synaptic vesicle size determined in 10 and 14 images obtained from 3 wild-type and 3 Trpml1-/- mice, respectively. (c) example TEM images at low magnification of wild-type and Trpml1-/- mice brain cortical slices with the dark PSD profiles marked by white arrows. (d) average number of PSD/µm2 analyzed in at least 10 images obtained from 3 mice of each line. (e) Glutamate levels and secretion by cultured cerebral cortical neurons. Total glutamate measured in non-stimulated wild-type (black) and Trpml1-/- (green) neurons and neurons stimulated by depolarization with 30 mM KCl (blue and red). (f) Rescue of glutamate levels and secretion by expression of TRPML1 in cultured cerebral cortical neurons. Total glutamate measured in non-stimulated Trpml1-/- neurons (green) and Trpml1-/- neurons transfected with TRPML1 (black) and untransfected (red) and TRPML1-transfected (clue) Trpml1-/- neurons stimulated by depolarization with 30 mM KCl. (g) averaged Co-IP of SNAP25 and VAMP2 in three brain regions of wild-type and Trpml1-/- mice. The images of all blots are given in Extended view Figure E4.
A unique bacterial tactic to circumvent cell-death crosstalk induced by blockade of caspase-8. (2020)
Figure 1

Figure 1. Shigella OspD3 inhibits lytic cell death

(A) HT29 cells were infected with Shigella WT, S325, or ospD deletion mutants, and incubated for 8 h. Aliquots of cellular supernatants were subjected to cytotoxicity assays. *P<0.05 (one-way ANOVA).

(B) HT29 cells were infected with Shigella WT or ∆ospD3 and incubated for 8 h. Infected cells were fixed and subjected to TUNEL and PI staining. Percentages of positive cells (TUNEL, green; PI, red) are shown in graph at right. The nuclei were stained with DAPI (blue). Scale bar: 100 μm. n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).

(C) HT29 cells were infected with Shigella WT, S325, or ∆ospD3 and incubated for 8 h. Infected cells were subjected to Giemsa staining. Arrows indicate cells in which the cytoplasm disappeared. Scale bar: 20 μm.

Data information: Graphs in (A and B) show mean ± SD, and data are pooled from three independent experiments performed in triplicates. Images in (B and C) are representative of three independent experiments.
A unique bacterial tactic to circumvent cell-death crosstalk induced by blockade of caspase-8. (2020)
Figure 2

Figure 2. Shigella OspD3 inhibits necroptosis

(A) HT29 cells were infected with Shigella WT, S325, or ospD deletion mutants, and incubated for 8 h. Cell lysates were subjected to immunoblotting.

(B,C) HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor, RIPK3 inhibitor, or caspase inhibitor (z-VAD), and then incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (B) and cytotoxicity assay (C), respectively. *P<0.05, n.s., not significant (one-way ANOVA).

(D,E) HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ospD3. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (top) and cytotoxicity assays (bottom), respectively. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting (inset). n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).

Data information: Graphs in (C-E) show mean ± SD, and data are pooled from three independent experiments performed in triplicates. Images in (A), (B), (D), and (E) are representative of three independent experiments. Molecular weights in immunoblots are in kDa.
A unique bacterial tactic to circumvent cell-death crosstalk induced by blockade of caspase-8. (2020)
Figure 3

Figure 3. OspD3 inhibits necroptosis by degradation of RIPK1 and RIPK3

(A) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h, and then cell lysates were subjected to immunoblotting.

(B) 293T cells were transfected with Myc-tagged ospD expression plasmids. After 24 h, cells were harvested and subjected to immunoblotting. Arrows indicate cleaved RIPK1.

(C) Multiple sequence alignment of the OspD family: Shigella OspD3, EPEC EspL, and EHEC EspL2. Conserved amino acids are indicated by asterisks. Typical protease catalytic sites are colored in red.

(D) 293T cells were transfected with a series of plasmids expressing ospD3 point mutants. After 24 h, cells were harvested and subjected to immunoblotting. Arrows indicate cleaved RIPK1.

(E,F) HT29 cells were infected with Shigella WT, S325, ∆ospD3, ∆ospD3/D3 (∆ospD3 complemented with wild-type ospD3), or ∆ospD3/D3CS (∆ospD3 complemented with a protease activity-deficient mutant, in which the cysteine residue at position 64 was replaced by serine) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (E) and cytotoxicity assays (F), respectively. *P<0.05 (one-way ANOVA).

Data information: Graph in (F) shows mean ± SD, and data are pooled from three independent experiments performed in triplicates. Images in (A), (B), (D), and (E) are representative of three independent experiments. Molecular weights in immunoblots are in kDa.
A unique bacterial tactic to circumvent cell-death crosstalk induced by blockade of caspase-8. (2020)
Figure 4

Figure 4. Shigella OspC1 effector prevents caspase-8 activation

(A) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cells were harvested and subjected to measurement of caspase-8 activation. Caspase-8 activity is reported as relative light units (RLU) of infected samples, minus the value in uninfected samples. *P<0.05 (one-way ANOVA).

(B) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cell lysates were subjected to immunoblotting. Arrows indicate cleaved forms of caspase-8, PARP, GSDMD, and IL-18, respectively.

(C) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Aliquots of cellular supernatants were subjected to cytotoxicity assay. *P<0.05; n.s., not significant (one-way ANOVA).

(D) HT29 cells were infected with Shigella WT or ∆ospC1 mutant and incubated for 8 h. Infected cells were then fixed and stained with cleaved caspase-8 (green), rhodamine-phalloidin (red), and DAPI (blue). Percentages of positive cells are shown in the graph at right (*P<0.05; unpaired two-tailed Student's t-test). Scale bar: 100 μm.

(E) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cells were harvested and subjected to measurement of caspase-3/7 activation. Caspase-3/7 activity is reported as relative light units (RLU) of infected samples minus uninfected samples. *P<0.05 (one-way ANOVA).

(F) HT29 cells were infected with Shigella WT, S325, or ∆ospC1 strains, and incubated for up to 8 h. After incubation for the indicated times, cells were subjected to real-time Annexin V apoptosis assay. Annexin V binding is reported as relative light units (RLU) in infected samples minus the value in uninfected samples. *P<0.05 (one-way ANOVA).

Data information: Graphs in (A) and (C)-(F) show mean ± SD, and data are pooled from three independent experiments performed in triplicates. Images in (B) and (D) are representative of three independent experiments. Molecular weights in immunoblots are in kDa.
A unique bacterial tactic to circumvent cell-death crosstalk induced by blockade of caspase-8. (2020)
Figure 5

Figure 5. Shigella T3SS effector OspC1-mediated caspase-8 inhibition triggers necroptosis

(A,B) HT29 cells were infected with Shigella WT, ∆ospC1, ∆ospD3, ∆ospC1∆ospD3, or ∆ospC1∆ospD3/ospC1 (∆ospC1∆ospD3 complemented with ospC1) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (A) and cytotoxicity assay (B), respectively. *P<0.05; n.s., not significant (one-way ANOVA).

(C) HT29 cells were infected with Shigella WT, ∆ospD3, or ∆ospC1. Cell lysates obtained at the indicated time points were subjected to immunoblotting.

(D,E) HT29 cells treated with DMSO or caspase-8 inhibitor were infected with Shigella WT, ∆ospC1, ∆ospD3, or ∆ospC1∆ospD3 strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (D) and cytotoxicity assay (E), respectively. *P<0.05; n.s., not significant (unpaired two-tailed Student's t-test).

(F) HT29 cells treated with the control or caspase-8 siRNAs were infected with the indicated Shigella strains and incubated for 8 h. Cell lysates were subjected to immunoblotting. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting.

Data information: Graphs in (B) and (E) show mean ± SD, and data are pooled from three independent experiments performed in triplicates. Images in (A), (C), (D), and (F) are representative of three independent experiments. Molecular weights in immunoblots are in kDa.
Insert L1 is a central hub for allosteric regulation of USP1 activity (2021)
Figure 1

Fig.1) UAF1 activates USP1 allosterically by relieving the insert 1 and 3 mediated auto-inhibition of USP1 catalytic activity.

A) Schematic diagram of the USP1 subfamily and the USP1 deletion mutants tested in this study.

B) Single point activity assays of USP1WT and deletion mutants on Ub-Rho show signifi­cantly increased activity in mutants where insert L1 and L3 are both deleted.

C) Single point activity assays of USP1WT and deletion mutants (± UAF1) on Ub-Rho show the loss in hyper-activation of USP1ΔL1L3 upon addition of UAF1.

D) Michaelis-Menten analysis of USP1 deletion mutants against ubiquitin-rhodamine (Ub-Rho) shows that USP1ΔL1L3 has significantly higher activity compared to WT and other mutants (n=2, biological replicates).

E) Michaelis-Menten analysis of USP1 deletion mutants (+UAF1) against Ub-Rho shows that all deletions mutants have similar catalytic activity (n=2, biological replicates).

F) Comparing activity of USP1WT and USP1ΔL1L3 on a peptide substrate i.e. LRGG-AMC (100µM). USP1ΔL1L3 cleaves the peptide substrate more efficiently compared to USP1WT (n=2, technical replicates).

G) Comparing activity of USP1WT and USP1ΔL1L3 (+UAF1) on a peptide substrate i.e. LRGG-AMC (100µM). Addition of UAF1 allows cleavage of LRGG-AMC by both USP1WT and USP1ΔL1L3 (n=2, technical replicates).
Insert L1 is a central hub for allosteric regulation of USP1 activity (2021)
Figure 2

Fig.2) Insert L1 of USP1 contains a PIP motif which is essential for activity of USP1 on PCNA-Ub.

A) Quantification of gel-based activity assays where the activity of USP1WT and deletion mutants (+UAF1) on PCNA-UbTAMRA is compared. The USP1ΔL1 mutant has reduced activity on PCNA-UbTAMRA compared to the other USP1 variants tested (n=2, biological replicates).

B) Multiple sequence alignment of USP1 insert L1 which highlights the conservation of the PIP and APIM motif in USP1 across species, the residues mutated in this region are indicated with an asterisk.

C) Quantification of gel-based activity assays where the activity of USP1 PIP mutants (+UAF1) on PCNA-UbTAMRA is compared. Both the PIP mutants have reduced activity on PCNA-Ub compared to USP1WT (n=2, biological replicates).

D) FP based binding assays of USP1 mutants (+ UAF1) with PCNA-UbTAMRA show reduced binding of USP1ΔL1 and USP1PIP1 compared to USP1WT (n=2, technical replicates).

E) Quantification of gel-based activity assays where the activity of USP1WT+UAF1 on PCNA-UbWT is compared with PCNA-UbPIM1 shows that PCNA-UbWT is cleaved much faster compared to PCNA-UbPIM1 (n=2, technical replicates (example shown here from 1 of 2 biological replicates))

F) Comparing activity of USP1WT and USP1PIP1 (+UAF1) on PCNA-UbWT and PCNA-UbPIM1 in gel-based assays shows that the activity of USP1WT is severely reduced by the PIM1 mutation in PCNA, whereas USP1PIP1 activity is not further affected indicating that PIP1 and PIM mutation affect the same interaction (n=2, technical replicates (example shown here from 1 of 2 biological replicates)).