EMBO/soda-nel huggingface dataset:
NEDD4L-mediated Merlin ubiquitination facilitates Hippo pathway activation (2020)
Figure 5

Figure 5. Lysine 396 is the Ubiquitin conjugation residue on Merlin.

(A) LN229 cells stably transduced with Flag-tagged wild-type Merlin or the S518D mutant were treated with thapsigargin and subjected to immunoprecipitation with a Flag antibody and followed by SDS-PAGE. Outlined gel pieces were analyzed by mass spectrometry.

(B) Numbers of unique peptides of Merlin or Ubiquitin from each outlined gel pieces in (A) are shown.

(C) The percentage of ubiquitinated forms for each peptide from each immunoprecipitated product are shown.

(D) and (E) Mass spectrometry spectrums of the indicated peptides from each immunoprecipitated product are shown.

(F) Merlin-depleted LN229 cells expressing His-Ubiquitin were stably transduced with the indicated HA-tagged Merlin forms or an empty vector. These cells treated with DMSO or thapsigargin were subjected to nickel affinity purification in a denaturing step followed by western blotting for HA. Arrows indicate mono-ubiquitinated Merlin. The red asterisk indicates native Merlin and black asterisks indicate non-specific signals. The ratio of mono-ubiquitinated to native Merlin in each lane of the input blot was quantified by Image J and is shown under the blot.

(G) Merlin-depleted LN229 cells were stably transduced with an empty vector, Flag-tagged wild-type (WT) Merlin or its K396R mutant. These cells were detached (S, suspension) by trypsinization using the procedure described in the Methods section and reseeded (A, attached) for two hours. Cell lysates were subjected to immunoprecipitation with a Flag antibody followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.

(H) Merlin-depleted LN229 cells stably transduced with wild-type (WT) Merlin or its K396R mutant were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.

(I) Merlin-depleted LN229 cells stably transduced with HA-tagged wild-type (WT) Merlin or its K396R mutant were treated as described in (G) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.
NEDD4L-mediated Merlin ubiquitination facilitates Hippo pathway activation (2020)
Figure 6

Figure 6. Merlin ubiquitination is required for activating Lats1.

(A) LN229 cells were treated with DMSO or thapsigargin (TG) for the indicated time and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.

(B) and (C) Merlin-depleted LN229 cells were reconstituted with wild type (WT) Merlin or its mutants, treated with DMSO or thapsigargin (TG), and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.

(D) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting.

(E) Merlin-depleted LN229 cells were stably transduced with empty vector, Flag-tagged wild-type (WT) Merlin, or its K396R mutant. These cells were detached (S, suspension) by trypsinization using the procedure described in Methods and reseeded (A, attached) for two hours. Cell lysates were subjected to western blotting.

(F) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L (+) or a scrambled siRNA (-) were detached (Susp., suspension) by trypsinization using the procedure described in Methods and reseeded (Attach, attached) for two hours. Total lysates from these cells were subjected to western blotting.

(G) LN229 cells transfected with indicated siRNAs were seeded for 3 hours. CTGF or CYR61 mRNA in these cells was quantified by q-RT-PCR. *P<0.05, **P<0.01, ****P<0.0001. Mean±s.e.m, N=4 biological repeats, unpaired t-test.
NEDD4L-mediated Merlin ubiquitination facilitates Hippo pathway activation (2020)
Figure 7

Figure 7. Merlin ubiquitination is important for its interaction with Lats1.

(A) LN229 cells stably transduced withHA-Lats1 (two leftmost panels) or vector only (right panel) were treated with DMSO or thapsigargin (TG) and subjected to PLA using HA and Merlin antibodies. Scale bar = 10 μm.

(B) PLA signals (dots) in each cell from the results in (A) were quantified. Mean±s.e.m, two-way ANOVA. *P<0.05, ***P<0.001, ****P<0.0001. Each data point represents an image field containing an average of 10 cells. N=4-9 images for each condition as indicated. All images were collected from one experiment. Two independent experiments were performed and gave similar results.

(C) Merlin-depleted LN229 cells were stably transduced with HA-tagged wild-type (WT) Merlin or its mutants. These cells were treated with thapsigargin (TG) for 15 minutes and subjected to PLA using HA and Lats1 antibodies. Scale bar = 20 μm.

(D) PLA signals (dots) in each cell from the results in (C) were quantified. Mean±s.e.m, Ordinary one-way ANOVA. ****P<0.0001. Each data point represents an image field containing an average of 10 cells. NVector=8, NWT=16, NK396R=18, NK159R=17 images. All images were collected from one experiment. Two independent experiments were performed and gave similar results.

(E) Merlin-depleted LN229 cells stably transduced with empty vector, Flag-tagged wild-type (WT) Merlin, or its K396R mutant were subjected to immunoprecipitation with a Flag antibody followed by western blotting.

(F) HEK293T cells were transfected with indicated proteins and subjected to immunoprecipitation with a Flag antibody. The lysates and immunoprecipitated products were subjected to western blotting.

(G) Merlin-depleted LN229 cells were stably transduced by HA-tagged Merlin. These cells were then transduced with a pool of four siRNAs against NEDD4L or a scrambled control siRNA and treated with DMSO or thapsigargin (TG). PLA was performed in these cells using HA and Lats1 antibodies, and the PLA signals (dots, showed in Figure EV5E) in each cell were quantified. Mean±s.e.m, two-way ANOVA. ****P<0.0001. Each data point represents an image field containing averagely 10 cells. N=12 images in all conditions, except for Nsi-Control:TG=13 images. All images were collected from one experiment. Two independent experiments were performed and showed similar results.

(H) LN229 cells treated with DMSO or thapsigargin (TG) were subjected to cytosolic (C)/membrane (M) fractionation followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.
NEDD4L-mediated Merlin ubiquitination facilitates Hippo pathway activation (2020)
Figure 8

Figure 8. Merlin ubiquitination contributes to growth control and tumor suppression.

(A) Met5-A cells stably transduced with the indicated shRNAs targeting Merlin or a scrambled shRNA were subjected to the BrdU incorporation assay. Scale bar = 40 μm.

(B) The percentage of BrdU positive cells from (A) was quantified. Mean±s.e.m, N=3 biological repeats, paired t-test. **P<0.01, ***P<0.001.

(C) Met5-A cells stably transduced with the indicated shRNAs targeting NEDD4L or a scrambled shRNA were subjected to the BrdU incorporation assay. Scale bar = 40 μm.

(D) The percentage of BrdU positive cells from (C) was quantified. Mean±s.e.m, N=3 biological repeats, paired t-test. **P<0.01, ***P<0.001.

(E) Meso-33 cells transduced with vector or the indicated Merlin forms were subjected to the BrdU incorporation assay. Scale bar = 40 μm.

(F) The percentage of BrdU positive cells from (E) was quantified. Mean±s.e.m, N=3 biological repeats, ordinary one-way ANOVA. *P<0.05, **P<0.01.

(G) Meso-33 cells stably transduced with the indicated shRNAs targeting NEDD4L or a scrambled shRNA were then transduced with vector or Merlin prior to the BrdU incorporation assay. The percentage of BrdU positive cells was quantified. Mean±s.e.m, N=3 biological repeats, two-way ANOVA.

(H) FC-1801 cells stably transduced with vector or the indicated Merlin forms were subjected to the sphere formation assay. Scale bar = 500 μm.

(I) The number of spheres in each well from (H) was quantified. Mean±s.e.m, N=3 biological repeats, paired t-test. **P<0.05.

(J) FC-1801 cells stably transduced with vector or the indicated Merlin forms were subcutaneously injected into nude mice. Tumor sizes in each group were quantified and plotted. Mean±s.e.m, NVector=8 mice, NWT=10 mice, NK396R=10 mice, NS518D=8 mice, two-way ANOVA. P values of the last measurements (day 37 after injection) are shown. *P<0.05, **P<0.01.

(K) Tumors were collected when the vector group in (J) reached the end point (day 37 after injection).

(L) A proposed model showing how Merlin is activated to induce Lats1 phosphorylation on the plasma membrane.

Expanded View Figure Legends
Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1 production (2008)
figf1

a, Schematic structure of wild-type or truncated (&Dgr;) Atg16L1 protein. CCD, Coiled-coil domain; WD, WD40 domain. Here and below, suffix &agr; or &bgr; indicates isoform &agr; or &bgr;. b–d, MEFs were cultured in nutrient-rich medium (fed) or Hanks' buffer (starved) for 1 h. Total cell lysates were prepared, and blotted with the indicated antibodies (b). &agr;, &bgr;, indicate Atg16L1 isoform &agr;, &bgr;; &Dgr;CCD indicates Atg16L1&Dgr;CCD; asterisk indicates a non-specific band. Atg16L1&Dgr;/&Dgr; indicates mice expressing &Dgr;Atg16L1. The number of endogenous LC3 or GFP-Atg5 dots was counted (c, d). The results shown are mean + s.d. (n > 20). e, Electron micrograph of starved MEFs. f, Degradation of long-lived proteins in MEFs. Wortmannin is an autophagy inhibitor. The results shown are mean + s.d. Statistical significance (P value) was determined by the Student's t-test.
Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1 production (2008)
figf2

a, Cytokine production from macrophages stimulated with LPS (100 ng ml-1) for 24 h. Statistical significance was determined by the Student's t-test. *P  0.01. b, IL-1&bgr; production from macrophages stimulated with indicated ligands. c, IL-1&bgr; production from LPS-primed macrophages infected with S. typhimurium (multiplicity of infection, m.o.i., 1), or stimulated with ATP or MSU for 1 h. d, Expression levels of caspase-1 and IL-1&bgr; in macrophages. Lys., cell lysates; Sup., culture supernatants. e, LPS-induced production of IL-1&bgr; from macrophages with the indicated phenotype. f, Expression levels of caspase-1 and IL-1&bgr; in macrophages treated as in e. g, Effect of the ROS scavenger FeTPPS (25 µM), N-acetyl-l-cysteine (NAC; 25 mM) or P2X7 receptor antagonist oxidized ATP (250 µM) on IL-1&bgr; production. h, ROS in LPS-stimulated macrophages were detected by CM-H2DCFDA staining.
Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1 production (2008)
figf3

a, Macrophages were stimulated with LPS (100 ng ml-1) for the indicated time period. The number of endogenous LC3 dots within each cell was counted. The results shown are mean + s.d. (n > 100). b, Expression levels of p62 and LC3 in macrophages. c, IL-1&bgr; and IL-6 production by wild-type or Atg7-deficient macrophages stimulated with LPS. Statistical significance was determined by the Students t-test. *P  0.01. d, Macrophages were pre-treated with or without 10 mM 3MA and then stimulated with the indicated ligands.
Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1 production (2008)
figf4

a, b, Fetal liver chimaeric mice were given 5% DSS in drinking water for 7 days. The survival (a) and weight loss (b) of each mouse genotype were plotted. The results shown are mean ± s.d. Statistical significance was determined by the Students t-test. c, Typical distal colon appearance 6 days after the initiation of DSS administration. The results shown are mean ± s.d. (n = 3, each group). d, Expression levels of IL-1&bgr; and IL-18 in serum (n = 5, each group). e, f, Atg16L1-deficient chimaeric mice given 5% DSS in drinking water were intraperitoneally injected with both anti-IL-1&bgr; and anti-IL-18 neutralizing antibodies (squares; n = 5) or isotype control IgG (circles; n = 5) at days 1, 3, 5 and 7. The survival (e) and weight loss (f) of each mouse genotype were plotted.
Positional proteomics reveals differences in N-terminal proteoform stability (2016)
Figure 6

Figure 6 - N-terminal proteoforms may display different stabilities. A. Degradation profiles of AIMP2, MARE2 and AN32E dbTIS and aTIS proteoforms as representative examples of N-terminal proteoforms that differ in stability. B. MS spectra of the N-termini of two proteoforms originating from the aminoacyl tRNA synthase complex-interacting multifunctional protein 2 (AIMP2): a dbTIS indicative N-terminus with processed iMet and starting at the second amino acid Pro, an aTIS indicative peptide starting at position 3 and retaining its iMet. As evident from the MS spectra shown, these N-terminal proteoforms displayed largely different turnover rates. C. Differences in turnover times were calculated for 100 dbTIS and aTIS proteoform pairs and presented in ascending order. The extent of N-terminal truncation in each pair was colour-coded to visualize the absence of correlation between the number of lacking amino acids and the direction of turnover shift. D. An increase in disorder content between aTIS and dbTIS proteoforms can be observed for a multitude of dbTIS/aTIS pairs.
Positional proteomics reveals differences in N-terminal proteoform stability (2016)
Figure 8

Figure 8 - Comparison of protein turnover measured by pSILAC and CHX-chase. A. Jurkat cells were treated with 100 µg/ml CHX for 0, 0.5, 1.5, 4, 8, 12 or 24 hours. Protein degradation was monitored by Western blotting and stabilities of several short-lived endogenous proteins were confirmed using antibodies (including lamin B, securin, β-catenin, GCIP interacting protein p29) and compared to stable proteins (such as GAPDH and actin). Proteoform-specific bands were used to calculate protein half-lives which were in a good agreement with turnover times obtained from pSILAC B-C. Validation of the differential turnover time of the dbTIS and aTIS derived proteoforms of MARE2 and AN32E. Selective C-terminal V5-tagged proteoforms were overexpressed in HCT116 cells for 24 hours and CHX pulse-chase experiments were performed as described for Jurkat cells. Degradation of overexpressed proteoforms was monitored by an anti-V5 antibody and compared to the turnover of stable proteins such as actin and tubulin.
ECM dimensionality tunes actin tension to modulate endoplasmic reticulum function and spheroid phenotypes of mammary epithelial cells (2022)
Figure 3

Figure 3. Ligation of rBM in 3D modulates filamin to alter ER function.

(A) Representative immunofluorescence microscopy images of MCF10A MECs ligated with either a rBM in 2D or 3D for 18h and stained with antibody targeting filamin (green). F-actin was counterstained with phalloidin (red). Images show maximum intensity z-projections of xy confocal stacks (left) and middle focal plane of confocal stacks from MECs (right). Yellow arrows indicate filamin aggregates in individual MECs ligated to rBM in 2D. Yellow blunt-end lines highlight the extent of filamin distribution away from the cell edge. Scale bar, 10 μm.

(B) The expression level of filamin in MCF10A MECs ligated to rBM in 2D and 3D for 18h was assessed via immunoblot and quantified relative to actin loading control (mean ± SEM; n=5 independent biological replicates). ***P = 0.0008 (Student's t test).

(C) Representative fluorescence microscopy images of MCF10A MECs co-expressing the ER-plasma membrane contact site reporter GFP-MAPPER and shRNA targeting filamin (FLMN) or luciferase (Luc) ligated to rBM in 2D for 18h. Scale bar (whole cell), 10 μm; Scale bar (magnified), 2 μm.

(D) Quantification of the levels of GFP-MAPPER at the plasma membrane versus total cellular fluorescence in MCF10A MECs stably expressing an shRNA targeting filamin (FLMN) or luciferase (Luc) ligated to rBM in 2D (mean ± SEM; 2D + shLuc, n=38; 2D + shFLMN , n=39 cells from three independent experiments). **P < 0.0021 (Student's t test).

(E) Representative immunoblots of filamin, phosphorylated EIF2a (pEIF2a), total EIF2a and alpha-tubulin in cell lysate from MCF10A MECs that express shRNA targeting filamin (shFLMN) or luciferase (shLuc) and ligated to a rBM in 2D or 3D for 18h and corresponding quantification data (mean ± SEM; n=4 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. 2D-shLuc versus 3D-shLuc, ***P = 0.0003. 2D-shLuc versus 2D-shFLMN, ***P = 0.0004.

(F) Bar graph of the relative levels of ATF3 mRNA as measured by qPCR in MCF10A MECs expressing shRNA targeting filamin (shFLMN) or luciferase (shLuc) and ligated with rBM in 2D or 3D for 18h (mean ± SEM; n=4 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. 2D-shLuc versus 3D-shLuc, ***P = 0.0005. 2D-shLuc versus 2D-shFLMN, *P = 0.0359.

(G) Representative immunoblots of filamin, phosphorylated EIF2a (pEIF2a), total EIF2a and alpha-tubulin in cell lysate from MCF10A MECs ligated to rBM in 3D and treated with ethanol (mock) or doxycycline (FLMN) for 18h to induce filamin expression and corresponding quantification data (mean ± SEM; n=5 independent biological replicates). **P = 0.0018 (Student's t test).

(H) Model of how ECM dimensionality affects ER function via filamin.
Mannose receptor-derived peptides neutralize pore-forming toxins and reduce inflammation and development of pneumococcal disease (2020)
Figure 1

Figure 1. MRC-1 co-localizes with the bacterial CDCs, PLY, LLO and SLO, in human dendritic cells. (A-C) Human DCs were incubated with a non-cytolytic dose (0.2 μg/ml) of purified (A) PLY, (B) LLO or (C) SLO for 45 min. Immunofluorescence staining shows that PLY, LLO and SLO (green) co-localize with MRC-1 (red) in DCs and EEA-1 (purple, early endosomes). All scale bars, 5 μm. Images are representative of three independent experiments.
Mannose receptor-derived peptides neutralize pore-forming toxins and reduce inflammation and development of pneumococcal disease (2020)
Figure 3

Figure 3. MRC-1 peptides bind to bacterial CDCs and inhibit their induction of hemolysis and cytolysis of macrophages. (A) ELISA showing the dose-dependent binding of plate-bound MRC-1 peptides P2, P3, and the control peptides CP1 and CP2 to PLY (0-0.5 μM). BSA was used as negative control to show the binding specificity. Data are mean ± s.e.m. of two independent experiments, each containing three replicates per condition.

(B) Hemolysis assay (n=4) of 1 μg/ml purified PLY in the presence of increasing concentrations of MRC-1 peptides, P2, scrambled P2, P3 and control peptide CP2(1-1000 μM). Data represent mean ± s.e.m. * denotes P < 0.05 by one-way ANOVA with Dunnett's post hoc test for multiple comparisons. Exact P values are shown in Appendix Table S4.

(C) LDH cytotoxicity assay in human THP-1 macrophages stimulated with purified PLY, LLO or SLO (0.5 μg/ml) in the presence or absence of 100 μM peptides P2, scrambled P2, P3 or control peptide CP2 for 18 h. Cholesterol (100 μM) was used as positive control to inhibit hemolysis. Data are mean ± s.e.m from 4 independent experiments. **** denotes P< 0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.

(D) Binding of FITC-labelled peptides P2 and CP2 to wild-type pneumococci, TIGR4 (T4) and isogenic PLY mutant (T4Δply) was visualized by fluorescence microscopy. Scale bars, 10 μm. In magnified images, scale bars, 1 μm. Images are representative of three independent experiments.

(E) The hemolytic activity of wild-type pneumococci, TIGR4 (T4) and PLY mutant, T4Δply in the presence of 100 μM peptide P2 and CP2. Data are the mean ± s.e.m. of three independent experiments. *** denotes P < 0.001 by one-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.
Mannose receptor-derived peptides neutralize pore-forming toxins and reduce inflammation and development of pneumococcal disease (2020)
Figure 5

Figure 5. Treatment with peptide P2 reduces development of pneumococcal disease in vivo. (A) Survival percentage of 3-4 dpf zebrafish embryos (n ≥156) upon infection with S. pneumoniae T4 alone or its isogenic PLY mutant, T4Δply. Injection with E3 growth medium served as mock control. *** denotes P < 0.0005 and **** denotes P < 0.0001 by Mantel Cox test. Exact P values are shown in Appendix Table S4.

(B) Zebrafish survival percentage upon infection with T4 alone or together with peptide P2 or CP2 or P2-conjugated CaP NPs (P2-NPs). *** denotes P < 0.0005 and **** denotes P < 0.0001 by Mantel Cox test. Exact P values are shown in Appendix Table S4.

(C) Survival of mice (n=10) upon intranasal infection with 2x106 CFU of S. pneumoniae T4 together with peptide P2 or CP2 or P2-NPs over 3 days post infection. Infected mice were checked twice daily in the morning at 9 am (early check) and in evening at 7 pm (late check) for clinical symptoms. Unloaded NPs and the isogenic PLY mutant strain (T4Δply) served as negative controls. * denotes P < 0.05 and ** denotes P < 0.005 by Mantel Cox test. Exact P values are shown in Appendix Table S4.

(D-F) Bacterial load (D) and levels of pro-inflammatory cytokines (E) TNF-α and (F) IL-12 in the lungs of infected mice (n=10) were measured post sacrifice. Data represent mean ± s.e.m. * denotes P < 0.05, ** denotes P < 0.01 and **** denotes P <0.0001 by one-way ANOVA with Bonferroni's post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.

Legends to Expanded View Figures
Molecular Basis of Ion Permeability in a Voltage-Gated Sodium Channel (2016)
Figure 1

Figure 1: Patch clamp measurements of the ion selectivity of wildtype and E178 mutant NavMs channels.

A) Left: Representative current traces from wildtype NavMs channels showing the first 0.1 s of 0.5 s activations from -180 mV holding potential recorded with the indicated extracellular cations. Right: Resulting current-voltage profiles profiles, where current magnitudes were normalized to the maximum inward current measured in the sodium condition.

B) Left: Representative current traces from mutant channel E178D (as in A). Right: Resulting current-voltage profiles (error ± SEM; n = 6-9 for NavMs; n = 4-8 for E178D).
Molecular Basis of Ion Permeability in a Voltage-Gated Sodium Channel (2016)
Figure 4

Figure 4: Comparison of ion binding in sodium and potassium channels.

Left: Sodium ions (cyan) in the selectivity filter of the NavMs sodium channel. Only two monomers (in white and pink) are shown for clarity. Coordinates are from the AB tetramer of PDBID 5BZB.

Right: Potassium ions (blue) in the selectivity filter of the KcsA potassium channel. As for the NavMs structure, only two monomers (in grey and maroon) are shown for clarity. Coordinates are from PDBID 1K4C (Zhou et al., 2004).

The two crystal structures were first superimposed using SSM (Krissinel and Henrick, 2004), and are drawn using the same scale, and with the correct ratio between the diameters of the ions used, so that the differences in the internal dimensions of their selectivity filters can be seen, thus making it clear how much more space there is available in the NavMs SF for intervening water molecules. In the KcsA channel, the ions are directly coordinated to the polypeptide backbone atoms, whereas in the NavMs SF, the closest atoms (side chains) are obviously at longer distances than direct bonding to the ions, enabling interactions via water molecules.