EMBO/soda-nel huggingface dataset:
Pathogenic ACVR1R206H activation by Activin A-induced receptor clustering and autophosphorylation (2021)
Figure 2

Fig. 2. ACVR1R206H and SMAD2/3 signaling influence the kinetics of SMAD1/5 phosphorylation.

A, B. Parental HEK293T, HET and HOM1 cells were treated with Activin A or Activin B (A) or BMP2 (B) for the indicated times. Quantifications are shown on the right.

C. Parental HEK293T, S2/3 dKO1, S6/7 dKO1 and S6/7 dKO2 cells were treated with Activin A for the indicated times.

D. Parental HEK293T, S2/3 dKO1 and HOM1 cells were treated with Activin A for the indicated times.

Data information: In A-C quantifications are the average normalized intensities ± SEM of between 3 and 10 (A and C) or 2 (B) independent experiments, and in the case of Activin A and Activin B, include a second ACVR1R206H homozygous clone (HOM2, see Fig. EV2A). In A, quantifications are the levels of pSMAD1/5 normalized to Actin, and in B and C, the quantifications are expressed as fold changes relative to 1 h of ligand stimulation. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. The p-values are from two-way ANOVA with Dunnett's post-hoc test. In D quantifications are from a representative experiment and are the intensities of pSMAD1/5 normalized to Actin, expressed as fold change relative to untreated cells for each clone. In all panels, Western blots of whole cell lysates were probed with the antibodies indicated. S2/3 dKO1, SMAD2/3 double knockout clone 1; S6/7 dKO1/2, SMAD6/7 double knockout clones 1 and 2.
Pathogenic ACVR1R206H activation by Activin A-induced receptor clustering and autophosphorylation (2021)
Figure 3

Fig. 3. Distinct type I receptor requirements for Activin A-mediated SMAD1/5 phosphorylation by ACVR1WT and ACVR1R206H.

A. Parental HEK293T, HET and HOM1 cells were treated with high (20 ng/mL) or low (1.25 ng/mL) concentrations of Activin A for 1 h, in the presence or absence of either 1 µM LDN-193189 or 10 µM SB-431542. Quantifications are the levels of pSMAD1/5 normalized to the loading control (Actin), expressed as fold change relative to untreated for each clone. Quantifications are the average normalized intensities ± SEM of 3 independent experiments.

B. Parental HEK293T, HET and HOM1 cells were treated with either Activin A or BMP4/7 (both at 20 ng/mL) for 1 h, in the presence or absence of either 1 µM LDN-193189 or 10 µM SB-431542. Quantifications are the levels of pSMAD1/5 normalized to the loading control (Actin or Tubulin), expressed as fold change relative to untreated for each clone. Quantifications are the average normalized intensities ± SEM of 3-5 independent experiments. Note that the Activin A quantifications also includes data generated with high dose Activin A in Fig. 3A as they are independent replicates.

C. ICR-B169 and HSJD-DIPG-007 cells were treated with either Activin A or BMP4/7 (both at 20 ng/mL) for 1 h, in the presence or absence of either 1 µM LDN-193189 or 10 µM SB-431542. Quantifications are the levels of pSMAD1/5 normalized to the loading control (Actin) expressed as fold change relative to BMP4/7 induction in each clone and are the average normalized intensities ± SEM of 3 independent experiments.

D. Parental HEK293T, ACVR1B/C dKO, HOM1 and HOM1 ACVR1B/C dKO cells were treated with 20 ng/mL Activin or BMP2 for 1 h. Quantifications are the average intensities of pSMAD1/5 normalized to Actin ± SEM of 3 independent experiments.

Data information: In all panels, Western blots of whole cell lysates were probed with the antibodies indicated. hi, high; Act, Activin A; SB, SB-431542; LDN, LDN-193189. The p-values (for A, B and D) are from a t-test with Holm-Sidak correction, for comparison between indicated pairs. ns, not significant; *, p< 0.05; **, p<0.01, ***, p<0.001; ****, p<0.0001. The p-values in C are from two-way ANOVA with Tukey's post hoc correction.
Pathogenic ACVR1R206H activation by Activin A-induced receptor clustering and autophosphorylation (2021)
Figure 4

Fig. 4. The structural, but not enzymatic, function of ACVR2A/B is essential for ACVR1R206H signaling.

A. Parental HEK293T, ACVR2A KO, ACVR2B KO, ACVR2A/B dKO, HOM1, HOM1 ACVR2A KO, HOM1 ACVR2B KO, HOM1 ACVR2A/B dKO cells were treated with Activin A or BMP2 for 1 h.

B. ACVR2A/B dKO cells were transfected with increasing concentrations of either wildtype or kinase-dead ACVR2A (ACVR2AWT or ACVR2AKR respectively) and treated with Activin A for 1 h. Parental HEK293T cells served as a control.

C. HOM1 ACVR2A/B dKO cells were transfected with increasing concentrations of either wildtype or kinase dead ACVR2A (ACVR2AWT or ACVR2AKR) and treated with Activin A for 1 h. HOM1 cells served as a control.

Data information: In all panels, Western blots of whole cell lysates were probed with the antibodies indicated. Representative experiments are shown. Par, parental HEK293T cells.
Pathogenic ACVR1R206H activation by Activin A-induced receptor clustering and autophosphorylation (2021)
Figure 5

Fig. 5. Receptor clustering is sufficient for ACVR1R206H signaling.

A, B. NIH-3T3 cells were either untransfected or transfected with FLAG-SMAD1 together with increasing (doubling) amounts of Opto-ACVR1WT or Opto-ACVR1R206H (5-20 ng). Post transfection, cells were either kept in the dark or exposed to blue light for 1 h. In (B) cells were treated in the presence or absence of 1 µM LDN-193189.

C. NIH-3T3 cells were either untransfected or transfected with either FLAG-SMAD1 or GFP-SMAD3 together with Opto-ACVR1B*. Post transfection, cells were either kept in the dark or exposed to blue light for 1 h, in the presence or absence of 50 µM SB-505124 or 1 µM LDN-193189.

Data information: In all panels, Western blots of whole cell lysates were probed with antibodies against pSMAD1/5 (that detected both endogenous and pFLAG-SMAD1), SMAD1 (which detects both endogenous and FLAG-SMAD1), HA (which detects the Opto-receptors), pSMAD3 (which detects pGFP-SMAD3), SMAD3 (which detects GFP-SMAD3) and either Tubulin or Actin as the loading controls. Lane numbers are given below each blot for clarity.
Pathogenic ACVR1R206H activation by Activin A-induced receptor clustering and autophosphorylation (2021)
Figure 6

Fig. 6. ACVR2A/B are required for Activin clustering.

A-G. His-Activin A-Atto647N was imaged free in solution with no cells (A) or with parental HEK293T cells (B), ACVR2A/B dKO cells (C), parental HEK293T cells pre-incubated with 1.5 μg/mL of rhACVR2A Fc and 15 μg/mL of rhACVR2B Fc (D), HOM1 cells (E), HOM1 ACVR2A/B dKO cells (F) and HOM1 cells pre-incubated with 1.5 μg/mL of rhACVR2A Fc and 15 μg/mL of rhACVR2B Fc (G). Number and brightness (N&B) analysis was performed on raster-scanned images, and a representative experiment is shown. Panels i, Brightness (B) vs intensity 2D plots. Red rectangular box, pixels that contribute to brightness of free, dimeric His-Activin A-Atto647N in solution. Green rectangular box, pixels that contribute to His-Activin A-Atto647N clustered into putative tetramers. Blue and pink rectangular boxes, pixels that contribute to His-Activin A-Atto647N in higher order clusters. Panels ii, Selection map of the image captured reconstructed with the pixels in red, green, blue and pink rectangular boxes shown in the upper panels (i). Panels iii, B histograms, frequency distribution of the number of pixels across various brightness of His-Activin A-Atto647N of the selected image. Bav indicates the average brightness for each image. Panels iv, cumulative B histograms from a representative experiment with 4 independent regions of interest.

H. Average brightness of His-Activin A-Atto647N in conditions (A-G). Means ± SEM are plotted. Each point represents one analyzed image, collected from 2-3 biological replicates.

I. Average brightness of His-Activin A-Atto647N in ICR-B169 and HSJD-DIPG-007 cells alone or with pre-incubation with 1.5 µg/mL of rhACVR2A Fc and 15 µg/mL of rhACVR2B Fc. Source data is in Fig. EV4B-F. Means ± SEM are plotted. Each point represents one analyzed image, collected from 2-4 biological replicates.

Data information: In H and I the p values are from one-way ANOVA with Tukey's post hoc correction. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.
The SiaA/B/C/D signaling network regulates biofilm formation in Pseudomonas aeruginosa (2020)
Figure 1

Fig 1. SiaB and SiaC regulate biofilm formation by modulating intracellular c-di-GMP levels through SiaD.

A Deletion of siaA, siaB, siaC, or siaD regulates biofilm formation. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).

B The intracellular levels of c-di-GMPin PAO1, ΔsiaC, ΔsiaB and the corresponding complemented strains were detected by LC-MS/MS.

C SiaB and SiaC regulated biofilm formation is dependent on SiaD. Biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).

D SiaC is required for the function of SiaD. Biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).

E Bacterial two-hybrid assay reveals an interaction between SiaC and SiaD. The recombinant strains harboring different vectors were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). NA represents the empty vector pBT or pTRG. The strain expressing LGF2 and Gal11P was used as positive controls.

F Pull down assays confirmed the interaction between SiaC and SiaD. Cell lysates of P. aeruginosa containing pMM67EH-siaD-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads.

G SPR measurements of SiaC (left) or SiaCP (right) binding at varying concentrations to SiaD. SiaC specifically interacted with SiaD with a KD of 24.7nM, whereas SiaCP failed to interact with SiaB. Shown are measured binding responses (black) and curve fits to a 1:1 interaction (red). Plots are representative from two experiments with similar results. RU, response units; KD, dissociation constant.

H Production of c-di-GMP by either SiaD or SiaD with SiaC at the indicated time-point was determined by HPLC.

Data information: Data represent the means and SDs of three biological replicates. *P<0.05 and **P<0.01 based on one-way ANOVA test. ns, non-significance. EV represents the empty vector pUCP20 in this and subsequent assays.
The SiaA/B/C/D signaling network regulates biofilm formation in Pseudomonas aeruginosa (2020)
Figure 2

Fig 2. SiaA and SiaB regulate biofilm formation via interaction with SiaC.

A Bacterial two-hybrid assay reveals an interaction between SiaB and SiaC. The recombinant strains harboring different proteins were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). NA represents the empty vector pBT or pTRG. The strain expressing LGF2 and Gal11P were used as positive controls.

B Pull-down assays confirmed the interaction between SiaB and SiaC. Cell lysates of P. aeruginosa containing pMM67EH-siaC-Flag were incubated with GST or GST-SiaB individually, and protein complexes were captured by glutathione beads.

C SPR measurements of SiaC binding at varying concentrations to SiaB. SiaC specifically interacted with SiaB with a KD of 40.1 nM. Shown are measured binding responses (black) and curve fits to a 1:1 interaction (red). Plots are representative from two experiments with similar results. RU, response units; KD, dissociation constant.

D SiaB-mediated regulation of biofilm formation is dependent on SiaC. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).

E Bacterial two-hybrid assayreveals an interaction between SiaC and SiaA PP2C-like domain. NA represents the empty vector pBT or pTRG. The recombinant strains harboring different proteins were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). The strain expressing LGF2 and Gal11P were used as positive controls.

F Pull-down assays confirmed the interaction between SiaC and SiaA. Cell lysates of P. aeruginosa containing pMM67EH-siaA-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads.

G Pull-down assays confirmed the interaction between SiaC and SiaA PP2C-like domain. Cell lysates of P. aeruginosa containing pMMB67EH-siaA386-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads.

H The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).

Data information: Data represent the means and SDs of three biological replicates. **P<0.01 based on one-way ANOVA test. ns, non-significance.
The SiaA/B/C/D signaling network regulates biofilm formation in Pseudomonas aeruginosa (2020)
Figure 3

Fig 3. SiaB phosphorylates SiaC at threonine 68 residue and the phosphorylated SiaC is critical for biofilm formation.

A The relative ATP hydrolysis activity of indicated sample was determined. After ATP hydrolysis reaction, the residual ATP concentration in the reaction buffer was determined by mixing the buffer with luciferase reagent. The emitted light was measured using a microplate luminometer. The amount of ATP hydrolyzed during reaction for each sample represents the ATP hydrolysis activity of each sample. The relative activity of each sample was normalized to that of the sample using SiaB and SiaC. (-) represents no substrate protein was added to reaction mixture.

B MS detection of SiaB peptide L63LYLNTSSIK72 that was covalently modified with one molecule of phosphate. Extracted ion chromatograms of the doubly protonated peptide are shown with peak intensities indicating the relative amount of either the modified or unmodified peptides.

C Determination of modification sites by collision induced dissociation (CID) analysis. The MS/MS spectrum of modified L63LYLNTSSIK72 is shown. The fragment ions b6 and y6 to y9 have a mass increase of 80 corresponding to phosphorylation, suggesting phosphorylation of Thr68.

D SiaB phosphorylates and SiaA dephosphorylates SiaC in vitro. SiaA or SiaAT68A were added to the reactions containing SiaB or SiaA386 and γ32P ATP. Phosphorylated products were separated by SDS-PAGE.

E Over-expression of SiaCT68A restored the ability of ΔsiaA to form biofilm. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).

F Deletion of ΔsiaB in the ΔsiaA background strain restored the ability of ΔsiaA to form biofilm. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).

G Production of c-di-GMP at the indicated time-point by SiaD with SiaC or SiaCT68A was determined by HPLC. To evaluate the effect of SiaB-mediated phosphorylation on SiaD DGC activity, SiaC or SiaCT68A was pretreated by SiaB in the presence of 1 mM ATP before reaction initiation.

Data information: Data represent the means and SDs of three biological replicates. **P<0.01 based on one-way ANOVA test.
The SiaA/B/C/D signaling network regulates biofilm formation in Pseudomonas aeruginosa (2020)
Figure 4

Fig 4. The complex structure of SiaB/SiaC.

A The overall fold of SiaB/SiaC complex. The two SiaB molecules are shown as cartoons in light blue and pink, respectively. The two SiaC molecules are all shown as cartoons in yellow. ADP and TPO68 are shown as spheres in atomic colors (C, cyan; N, blue; O, red; P, orange).

B Detailed interactions between SiaB and SiaC. The interacting residues are showing as sticks. The C-atoms of SiaB and SiaC residues are colored in light blue and yellow, respectively. H-bonds are indicated by black dashed lines.

C SiaCE33A partially restored the biofilm formation of ΔsiaA. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).

D Triple mutation of E61A-Q64A-R67A is important for the function of SiaB. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).

Data information: Data represent the means and SDs of three biological replicates. **P<0.01 based on one-way ANOVA test.
The SiaA/B/C/D signaling network regulates biofilm formation in Pseudomonas aeruginosa (2020)
Figure 5

Fig 5. ADP binding and phosphorylation.

A Fo-Fc omit map of ADP and TPO68. The map was contoured at 2.5 σ level. ADP, TPO68 of SaiC and Asn65 of SiaB are shown as sticks. Na+ ion is shown as black sphere.

B Surface presentation of the ADP binding pocket.

C Detailed interactions between ADP and SiaB. ADP and the interacting residues are shown as sticks.

D Interactions between ADP and residues Leu149 and Gly148. ADP and the interacting residues are shown as sticks.

E Leu110 and Phe174 residues are important for SiaB function. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down). Data represent the means and SDs of three biological replicates. **, P<0.01 based on one-way ANOVA test.

F Conformation of TPO68 and coordination of Na+ ion. Na+ ion and water molecules are shown as spheres in black and red, respectively.

G Comparison of the cofactors bound at the active sites of SiaB/SiaC and BsSpoIIAB/SpoIIAA complexes. For SiaB/SiaC complex, ADP, TPO68 of SaiC and Asn65 of SiaB are all shown as sticks in atomic colors (O, red; N, blue), whereas their C-atoms are colored in cyan, yellow and light blue, respectively. For BsSpoIIAB/SpoIIAA complex, ATP, Ala58 (mutation of Ser58) of SpoIIAA and Asn50 of SpoIIAB are all colored in white. Mg2+ ion and its coordinating water molecules are shown as spheres in green and red, respectively.
Midbody ring disposal by autophagy is a post-abscission event of cytokinesis (2008)
figf1

(a) p62 localizes to the MR during cytokinesis and abscission, and is taken up together with the MR by one daughter cell. U2OS cells were fixed and stained with anti-p62 (red) and anti-MKLP1 (green) antibodies. Scale bars are 10 μm. Colocalization of p62 with MKLP1 of the MR was regularly observed in cells at this stage of cell division. The arrowheads point to MRs, which are shown enlarged in the insets. The quantification is presented in Supplementary Information Fig. S1a. (b) siRNA-mediated depletion of p62 from U2OS cells results in moderate MR accumulation, compared with a control depletion (lamin A/C). For quantification, MRs were counted in control cells and in p62-depleted cells that were negative for p62 staining. Data are means ± s.d. of three independent experiments (n, total number of cells counted; P, probability of a two-tailed paired t-test).
Midbody ring disposal by autophagy is a post-abscission event of cytokinesis (2008)
figf2

(a) Atg8 (LC3) localizes to the MR during abscission. The upper panels show stills from time-lapse microscopy of GFP–Atg8-expressing cells. The arrowhead points to the accumulation of Atg8 at the site of the MR during abscission. The enlarged clipping on the right shows the midbody area at time-point 68 min. Scale bar is 10 μm. (b) Cytosolic MRs are found inside Atg8-positive vesicles. U2OS cells stably expressing GFP–Atg8 were fixed and stained for MKLP1 (red) and α-tubulin (blue). Scale bars are 10 μm and 1 μm for the enlarged boxed areas. Putative different stages of autophagosomes (left) and lysosomes (right) are shown. (c) Juxtanuclear MRs are found in Rab7- and LAMP2-positive lysosomes. U2OS cells stably expressing DsRed-Rab7 were fixed and stained for MKLP1 α-tubulin and DAPI (left panels); wild-type U2OS were stained for MKLP1 and LAMP2 (right panel). Scale bars are 10 μm. The insets are enlargements of the engulfed MRs. Scale bars are 1 μm.
Midbody ring disposal by autophagy is a post-abscission event of cytokinesis (2008)
figf3

(a) Accumulation of MRs in cells treated with the PI(3)K inhibitor 3-MA. Left panels show HeLa cells treated with 3-MA (4 mM) for 3 days. Cells were fixed and stained. Images are colour-inverted for better visualization. The quantification of this experiment is shown on the upper right (mean ± s.d. of three independent experiments; n = total number of cells counted). The lower right panel shows an enlarged area of 3-MA-treated cells in true colour. Scale bars are 20 μm. (b) siRNA-mediated depletion of Atg6 (Beclin-1) and Atg7 from HeLa cells causes MR accumulations. The right panel shows the quantification of MRs per cell (mean ± s.d. of three independent experiments); the left panel the efficiency of Atg6 and Atg7 depletion. (c) Inhibition of lysosomal acidification leads to MR accumulation. Panels on the left show HeLa cells left untreated or treated with chloroquine (20 μM) or ammonium chloride (10 mM) for 3 days. Scale bar are 20 μm. Cells were fixed and stained as indicated. A quantification of this experiment is shown on the left (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).
Midbody ring disposal by autophagy is a post-abscission event of cytokinesis (2008)
figf4

(a) Cell-cycle lengths of different cell lines correlate well with the average amount of MRs. Indicated cell lines were grown on coverslips, fixed and MRs were counted after staining with anti-MKLP1 antibodies. Cell-cycle lengths were assessed as described in the Methods. Data are mean ± s.d. of three independent experiments. (b) The degree of MR persistence depends on the time and not on cell-cycle duration. HeLa cells were either left untreated or were arrested in S-phase with hydroxyurea (HU, 5 mM). Cells were fixed and stained as indicated. Scale bars are 20 μm. The right panel shows quantification of MR counting (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).
SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland (2016)
Figure 1

Figure 1. SHARPIN is expressed in the stromal and luminal epithelial cells of the mammary gland.

A. Immunohistochemical analysis of SHARPIN expression in the human mammary gland. Cross-section of a mammary duct (upper panel) and magnification of the marked area (lower panel). SHARPIN-positive luminal (grey arrow) and stromal cells (red arrow), and the approximate position of the basal lamina (dashed red line) are indicated. Scale bar represents 50 μm. B. Western blot analysis of SHARPIN protein expression in isolated primary mammary epithelial cells (MECs) and mammary stromal fibroblasts (MSFs). CDH1 and vimentin were used as markers of epithelial and stromal cell lineages, respectively. GAPDH served as a control for protein loading. C. FACS-based isolation of mouse mammary gland basal epithelial cells (LinnegCD24intICAM-1hi), mature luminal epithelial cells (LinnegCD24hiICAM-1neg), luminal progenitor cells (LinnegCD24hiICAM-1int), and stromal cells (LinnegCD24neg). D. Quantitative PCR analysis of SHARPIN mRNA expression in cell populations isolated in (C) (mean ± SEM, n = 3 - 5).