EMBO/soda-nel huggingface dataset:
Phase-separated foci of EML4-ALK facilitate signalling and depend upon an active kinase conformation (2021)
Figure 1

Figure 1. EML4-ALK V1 and V3 activation induces the formation of cytoplasmic foci

A. H3122 cells expressing endogenous EML4-ALK V1 and HEK293 transfected with YFP-EML4-ALK V1 WT and KD were stained for either anti-ALK or anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.

B. Violin plots showing the number of EML4-ALK V1 cytoplasmic foci per cell. Data represents 30-50 counts from three biological replicates. ***P<0.001, ****P<0.0001 in comparison to HEK293 YFP-EML4-ALK-V1 KD by one-way ANOVA analysis.

C. H2228 cells expressing endogenous EML4-ALK V3 and HEK293 transfected with YFP-EML4-ALK V3 WT and KD were stained for either anti-ALK or anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.

D. Violin plots showing the number of EML4-ALK V3 cytoplasmic foci per cell. Data represents 30-50 counts from four biological replicates. ***P<0.001, ****P<0.0001 in comparison to HEK293 YFP-EML4-ALK-V3 KD by one-way ANOVA analysis.

E. Time-lapse imaging of transfected HEK293 YFP-EML4-ALK V1 WT to observe the movement of cytoplasmic foci. Representative still images are shown of an area of cytoplasm containing YFP-EML4-ALK V1 WT and V3 WT foci at the times indicated. Scale bar, 1 μm.

F. Time-lapse imaging of transfected HEK293 YFP-EML4-ALK V3 WT to observe the movement of cytoplasmic foci. Representative still images are shown of an area of cytoplasm containing YFP-EML4-ALK V1 WT and V3 WT foci at the times indicated. Scale bar, 1 μm.

G. Kymographs showing the movement of YFP-EML4-ALK V1 WT, YFP-EML4-ALK V3 WT and YFP-EML4-ALK V3 KD over the duration of the observation period. Scale bars; 1 μm horizontal; seconds vertical.

H. Whisker plot showing the calculated velocity of single events in the kymograph. Data represent 20 counts from 10 kymographs. n=2. Error bar represents SD of two biological replicates. *P< 0.05 in comparison to YFP EML4-ALK V3 KD by unpaired t test.
Phase-separated foci of EML4-ALK facilitate signalling and depend upon an active kinase conformation (2021)
Figure 2

Figure 2. Cytoplasmic EML4-ALK V1 and V3 foci contain downstream signalling proteins

A, B. H3122 and H2228 cells were stained for anti-ALK (green), anti-GRB2 (red), anti-SOS1 (red), anti-pC-KITY721 (red), anti-PI3K p85β or anti-pPLCγ2Y759 and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.

C. HEK293 cells transfected with YFP-EM4L-ALK V1 WT or V3 WT for 48 hrs and treated with 5% or 10% 1,6-hexanediol. Representative still images after 5 min (taken from time-lapse movies) are shown of cells treated with 10% 1,6-Hexanediol or DMSO. Scale bars, 10 μm; the arrowheads indicate cytoplasmic foci.

D. Box plot showing the number of cytoplasmic foci in DMSO and 5% or 10% Hexanediol (5 min). Data represent 5-10 counts from 3 independent experiments (n=3). Error bar represents SD of three biological replicates. ****P< 0.0001 in comparison to YFP-EML4-ALK V3 WT DMSO by one-way ANOVA analysis.

E. Representative western blots of HEK293 transfected EML4-ALK V1 WT and V3 WT for 48 hrs. Cells were treated with either 5% or 10% 1,6-Hexanediol for 5 minutes. DMSO was used a control. Lysates were analysed for the phosphorylation and expression of the indicated antibodies. GAPDH was used as a loading control. Data representative of n=2 experiments.
Phase-separated foci of EML4-ALK facilitate signalling and depend upon an active kinase conformation (2021)
Figure 3

Figure 3. Different ALK inhibitors dissolve or maintain cytoplasmic EML4-ALK V1 and V3 foci and redirect V3 to microtubules

A, B, C, D. HEK293 cells were transfected with EML4-ALK V1 WT or V3 WT or Kinase Dead (KD) for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.

E, F. Violin plots show the number of cytoplasmic foci per cell from A and B, respectively. Data represent counts from >30 cells, n= 4. *P< 0.05, ***P< 0.001, ****P< 0.0001 in comparison to DMSO by one-way ANOVA.

G, H. Violin plots show the number of cytoplasmic foci per cell from C and D, respectively. Data represent counts from >30 cells, n= 4. ****P< 0.001 in comparison to DMSO by one-way ANOVA.

I. HEK293 cells were transfected with YFP-EML4-ALK V1 WT or V3 WT for 48 hours and treated for 4 hours with ALK inhibitors, ceritinib (500 nM), alectinib (100 nM) and lorlatinib (100 nM) or an equivalent volume of DMSO. Western blots of pALKY1604 and α-tubulin were used to assess relative abundance in transfected HEK293 cells.

J. After photobleaching, the fluorescence intensity of an area of a HEK293 cell transfected with YFP-EML4-ALK V1 WT, V3 WT in the presence or absence of ALK inhibitors, or YFP-EML4-ALK V1 or V3 KD, was plotted as a function of time. Each curve is the average of data of 40-50 individual cells. Error bar represents SD of three biological replicates.

K. Still images from FRAP analysis of YFP-EML4-ALK V1, V3 WT, KD or ALK inhibitors. Magnified views of a selected area are shown. Time is shown in seconds.
Phase-separated foci of EML4-ALK facilitate signalling and depend upon an active kinase conformation (2021)
Figure 4

Figure 4. The deletion of 12N blade of EML4-ALK V1 is redirected to microtubules upon ALK inhibition

A. Linear representation of the structure of EML4-ALK V1 and V3 variants and the deletion 12N blade mutant including residue numbering indicating domain boundaries.

B. HEK293 cells were transfected with EML4-ALK V1 deletion 12N blade for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.

C. Violin plot shows the number of cytoplasmic foci per cell from B. Data represent counts from >30 cells, n= 3. ****P< 0.0001 in comparison to DMSO by one-way ANOVA.

D. Models summarizing the domain structures of EML4 and EML4-ALK proteins consisting of a coiled-coil region (dark green) followed by basic region (black), 12N blade (orange), blades 1-4 (purple) and blade 5 (lilac). The ALK JM domain is coloured light blue and the kinase domain is coloured light green.
Phase-separated foci of EML4-ALK facilitate signalling and depend upon an active kinase conformation (2021)
Figure 5

Figure 5. Structure and dynamics of the ALK kinase domain in the presence of TKIs

A. View of ALK (grey) bound to alectinib (magenta) centred on the active site highlighting the αC-helix (cyan), αAL-helix (orange) and Lys-Glu salt bridge (blue-red), based on the crystal structure (PDB:3AOX).

B. Magnified views of ALK, in the vicinity of the Lys-Glu salt-bridge, bound to inhibitors: alectinib (ALE, PDB:3AOX), ceritinib (CER, PDB:4MKC) and lorlatinib (LOR, PDB:4CLI). The cyan wiremesh is a 2Fo-Fc electron density map. The black line marks the interatomic distance between terminal atoms in the Lys-Glu salt-bridge.

C. Schematic illustration of the regulatory features of the ALK kinase domain and the effects of mutations. The side chains of key residues are shown as sticks. The αC-helix of inactive ALK interacts with the JM domain through hydrophobic contacts and the αAL-helix through polar contacts. The interactions between αC-helix, the JM domain and the A-loop are destabilised by mutations such as F1174L, R1275Q that drive the activation of ALK. The Lys-Glu salt-bridge can be disrupted by an inactivating mutation (K1150M).

D. HEK293 cells were transfected with EML4-ALK V3 ∆JM 1058-1100 for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.

E. Violin plot representing the number of cytoplasmic foci counted per cell from D. Data represent counts from at least 30 cells, n=3. ****P< 0.0001 in comparison to DMSO by one-way ANOVA.
Phase-separated foci of EML4-ALK facilitate signalling and depend upon an active kinase conformation (2021)
Figure 6

Figure 6. Point mutations in ALK kinase domain alter EML4-ALK V3 localisation

A-F HEK293 cells were transfected with A. YFP-EML4-ALK V3 K1150M, C. R1275Q or E. F1174L constructs and treated with ALK inhibitors or DMSO for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. B, D, F. Violin plots representing the number of cytoplasmic foci counted per cell from A, C and E. Data represent counts from at least 30 cells, n=3 or 4. ****P< 0.0001 in comparison to DMSO by one-way ANOVA.
Phase-separated foci of EML4-ALK facilitate signalling and depend upon an active kinase conformation (2021)
Figure 7

Figure 7. Stoichiometric mismatch between EML4 and ALK proteins is required for foci formation

A. Linear representation of the structure of EML4-ALK V3 and EML4-GyrB fusion proteins including residue numbering indicating domain boundaries.

B. HEK293 cells transfected with YFP-EML4-GyrB and either untreated (DMSO) or treated with Coumermycin A1 to induce dimerization of GyrB before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.

C. Violin plot showing the number of cytoplasmic foci per HEK293 cell transfected with YFP-EML4-GyrB. Data represent counts from >30 cells, n= 4. ****P< 0.0001 in comparison to DMSO by unpaired t test.

D. Schematic model showing the mechanism of EML4-GyrB foci formation. EML4-GyrB foci are formed by dynamic cross-linking interactions between the EML4 trimerization domain (TD) and dimerization of GyrB domain in the presence of coumermycin A1.
The E3 ubiquitin ligase RNF114 and TAB1 degradation are required for maternal-to-zygotic transition (2016)
Figure 1

Figure 1. Expression of Rnf114 during mouse early embryonic development and effect of Rnf114 knockdown on early embryonic development.

(A) Quantitative analysis of Rnf114 mRNA by realtime-PCR in various mouse tissues and organs shows that Rnf114 was predominately expressed in oocytes.

(B) Immunohistochemistry analysis of RNF114 in mouse ovary shows that RNF114 was mainly expressed in the oocytes (Scale bar = 100um).

(C) Knockdown efficiency of Rnf114 in the 2-cell stage embryos derived from Rnf114 siRNA #1 or siRNA #2 injected zygotes was verified by realtime-PCR and western blotting. NC indicates negative control injected with non-silencing siRNA. Three independent experiment replicates were performed for realtime-PCR, error bars represent s.e.m.; **:P<0.01, ***:P<0.01 in unpaired two-tailed t-test.

(D) RNF114 depletion impaired the early embryonic development. Rnf114 siRNA #1 or siRNA #2 injected zygotes were cultured and quantified (siRNA1#, n = 120; siRNA2#, n = 128; three independent experiments); NC indicates negative control (n = 193; six independent experiments). The error bars represent s.d., *:P<0.05, **:P<0.01, ***:P<0.001 in unpaired two-tailed t-test.

(E) Representative images to show early embryonic development defects. The embryos arrested at two-cell stage are indicated by arrows (Scale bar = 100um).
The E3 ubiquitin ligase RNF114 and TAB1 degradation are required for maternal-to-zygotic transition (2016)
Figure 2

Figure 2. RNF114 substrate screening by human protein microarrays.

(A) In vitro ubiquitination assay. Purified His-RNF114 was subjected to ubiquitination assays with the indicated components and the samples were analyzed by immunoblotting with anti-FLAG (Ub) antibody. Lanes 1–4 indicate negative controls. The results show that recombinant RNF114 efficiently catalyzed the formation of polyubiquitin chains.

(B, C) Purified wild-type and RING mutated His-RNF114 were stained by Coomassie Blue (B). Purified wild-type and RING mutated His-RNF114 were subjected to ubiquitination assays together with E1, Ubc4, FLAG-Ub, and ATP. The results show that mutant RNF114 lacked ubiquitination activity (C).

(D) Screening for proteins ubiquitinated by RNF114 on a proteome array. Putative substrates of RNF114 were identified on Ubiquitin Ligase Substrate Identification on ProtoArray® Human Protein Microarrays v5.0. Ubiquitination reactions were performed on quintuplicate arrays using the indicated components, the five sub-array are respectively negative controls, E1+E2 (no E3), WT RNF114 (two arrays, duplicate), and mutated RNF114.

(E) A heat map comparing hits between E1+E2, E1+E2+WT RNF114, and E1+E2+mutated RNF114 reactions. Candidate substrates of RNF114 selected for further characterization are indicated in red font; HIP1, indicated by blue font, was considered to be a false positive because it was also detected in the mutated RNF114 reaction. Black font indicates proteins with ubiquitin-binding domains such as UIM, UBA or CUE or proteins without clearly reported functions, which were excluded from further investigation.
The E3 ubiquitin ligase RNF114 and TAB1 degradation are required for maternal-to-zygotic transition (2016)
Figure 3

Figure 3. Verification of RNF114 substrates.

(A) RNF114 substrate validation. HEK293 cells were co-transfected with constructs expressing FLAG-tagged substrates, wild-type or mutated His-RNF114 and EGFP, and western blot was performed with anti-FLAG and anti-His antibodies. The results show that the protein levels of CD74, TAB1, OPTN, TNIP1, APPL1, PSAT1, and RALGPS1 were reduced in the presence of wild-type RNF114 compared to co-transfecting with mutant RNF114.

(B) RNF114-dependent in vitro ubiquitination of substrates. Substrate ubiquitination assays were performed with purified RNF114, the reaction products were immunoblotted using anti-His antibody to detect the ubiquitin (upper panel) or anti-FLAG antibody detecting the substrate protein (middle panel), and the lower panel showed the merged signals. The results show that clear polyubiquitin chains were formed in the systems containing recombinant CD74, TAB1, TNIP1, PSAT1 or RALGPS1.

The relative intensity of western blot were quantified and normalized, the data was plotted at the bottom of corresponding lanes.
The E3 ubiquitin ligase RNF114 and TAB1 degradation are required for maternal-to-zygotic transition (2016)
Figure 4

Figure 4. RNF114 mainly targets TAB1 for degradation during MZT.

(A) Mouse zygotes were microinjected with sterile ddH2O (control group) or Ralgps1, Cd74, Tnip1, Psat1, or Tab1 mRNA. During 96–100 h culturing, the percentages of embryos at various stages were counted under microscopy. The results show that overexpression of Tab1 impaired the early embryonic development. Three independent experiment replicates were performed, error bars represent s.d.; *:P<0.05, ***:P<0.001 in unpaired two-tailed t-test.

(B) TAB1 reduced with mouse early embryonic development, while abnormally accumulated at 2-cell stage once treated with Rnf114 siRNAs. TAB1 was detected by western blotting with anti-TAB1 antibody. The relative level of TAB1 protein after Rnf114 siRNAs treatment were presented as mean±s.e.m. (*:P<0.05 in unpaired two-tailed t-test, four independent experiment replicates).

(C) Detection of endogenous TAB1 ubiquitination at 1-cell or 2-cell stages. Endogenous TAB1 proteins were immunoprecipated by anti-TAB1 antibody at either 1-cell or 2-cell stages, the immunoprecipates were detected by either anti-TAB1 antibody or anti-ubiquitin antibody. The results show that more Ub chains on TAB1 were detected in the two cell stage embryos than in the zygotes, when the TAB1 proteins were at comparable level.

(D) RNF114-dependent TAB1 degradation during early embryo development. (a-c) Representive time lapse embryo images were shown for each treatment. Scale bar = 20um. (a’-c’) Quantification of the GFP fluorescent signals for respective treatments in (a-c). n=14, 9 or 12 for the NC, Rnf114 siRNA1# or Rnf114 siRNA2# treatment group respectively. Gray traces: Tab1-GFP fluorescent signals were normalized to the first time point; Black trace: average signal of all embryos for each treatment.

(E) Tab1 knockdown partly rescued the early embryo development defect observed with Rnf114 knockdown. Both Rnf114 and Tab1 siRNAs were injected into the GV oocytes, then the oocytes matured, and were fertilized by IVF, and the fertilized eggs were transferred into KSOM media, cultured for 48–50 h. The percentages of embryos arrested at 2-cell stage were counted. Three independent experiment replicates were performed, error bars represent s.d.; **:P<0.01 in unpaired two-tailed t-test.
The E3 ubiquitin ligase RNF114 and TAB1 degradation are required for maternal-to-zygotic transition (2016)
Figure 5

Figure 5. Developmental defect caused by TAB1 accumulation might be due to failure of NF-κB pathway activation.

(A) NF-κB activation during MZT. Immunofluorescence staining of p65 shows nuclear translocation of p65 at the two-cell stage and thereafter. Scale bar = 25um.

(B) The protein levels of IκBα and its phosphorylated form (p-IκBα) during mouse early embryonic development were detected by western blotting, representative western blotting of IκBα and p-IκBα was shown on the left and the quantitation analysis (right panel) indicated that the ratio of p-IκBα/total IκBα was gradually increased from MII oocytes to 4-cell stage embryos (three independent experiment replicates were performed, error bars represent s.e.m.).

(C) NF-κB pathway inhibition using the specific inhibitor Bay11-7082 resulted in two cell stage arrest. Three independent experiment replicates were performed, error bars represent s.d.; ***:P<0.001 in unpaired two-tailed t-test.

(D, E) Representive immunofluorescence staining of p65 after knockdown of Rnf114 by Rnf114 siRNA #1 or siRNA #2 or overexpression of Tab1 by Tab1 mRNA injection (shown in D. Scale bar = 25um.), the percentages of two-cell stage embryos with p65 nuclear translocation (shown in E.) were significantly decreased after Rnf114 knockdown or Tab1 overexpression (chi-square test was performed for statistical analysis).

(F) Western blot analysis at 2-cell stage showed the levels of p-IκBα were downregulated in the Rnf114 siRNA #1, siRNA #2 injected groups.

(G) Western blot analysis at 2-cell stage showed the levels of p-IκBα were downregulated in the exogenous Tab1 mRNA injected groups, ddH2O was injected as control. Dotted line indicates a cropped lane.
Chimeric Pneumoviridae fusion proteins as immunogens to induce cross-neutralizing antibody responses (2017)
Figure 1

Figure 1. Antigenic characterization of postfusion hMPV F chimeras with amino acids from hRSV F antigenic site II. A) Partial amino acid sequences of the hMPV F (blue) and hRSV F(red) antigenic site II (Lopez et al., 1998;McLellan et al., 2011b;Toiron et al., 1996). Partial sequences of the F-415 and F-416 chimeras are color coded to indicate the origin of their antigenic site II residues. Amino acids shared by hMPV F and hRSV F are colored purple. The remaining sequences of F-415 and F-416 are derived from hMPV F. B) Surface representation of the hMPV F structure in the postfusion conformation (Mas et al., 2016). One of the protomers is shown as a blue ribbon. Antigenic site II is magnified and colored as in (A) for the F-415 and F-416 chimeras. C) ELISA binding results of mAbs specific for hRSV F (left panels) or hMPV F (right panels) with the proteins indicated at left. mAbs specific of hRSV F bind epitopes of antigenic site II (Mota and 47F) or antigenic site IV (101F). mAs specific of hMPV F bind epitopes of antigenic site II (MF9, MF12, MF14 and MF15) or antigenic site IV (MF16).
Chimeric Pneumoviridae fusion proteins as immunogens to induce cross-neutralizing antibody responses (2017)
Figure 3

Figure 3. Antibody responses of mice inoculated twice with either wild-type proteins or chimeric proteins with a postfusion hMPV F backbone. Groups of BALB/c female mice (n = 5) were inoculated twice (four weeks apart) i.m. with 10 µg/dose of the proteins indicated at the top of each panel, folded in their respective postfusion conformations. One week after the last dose, mice were sacrificed and their blood collected. A) Each mouse serum (identified by a number) was tested in ELISA for antibody binding to the postfusion F proteins indicated at the bottom. The results represented in the y axis for each individual mouse serum correspond to the inverse dilution that gave 50% of maximal binding, expressed in log10 units (EC50 (log10)). Each number represents an individual mouse. Short horizontal bars indicate mean values for each group. P values were calculated as indicated under Statistical Analysis. Only relevant P values are shown. *P = 0.00045. Differences were considered significant when P < 0.05. B) Mouse sera were also tested in microneutralization assays with hRSV and hMPV, as indicated at the bottom. The results represented in the y axis correspond to the inverse dilution that inhibited 50% of the viral infectivity (IC50 (log10)). P values: *P = 0.01001 and **P = 0.00271. Long horizontal lines in A and B indicate detection limits.
Chimeric Pneumoviridae fusion proteins as immunogens to induce cross-neutralizing antibody responses (2017)
Figure 4

Figure 4. Protection against hRSV challenge with hMPV F backbone chimeras. Groups of BALB/c mice (n = 5) were inoculated three times, four weeks apart, with 20 µg of the postfusion F proteins indicated above each panel. One group (n = 5) was inoculated only with the CpG adjuvant, as indicated. Two other groups of mice (n = 8) were similarly inoculated with either F-415 or F-416, as indicated at top of the panels. A) Mouse sera, collected just before challenging with hRSV (A2 strain), were used in microneutralization assays against the viruses indicated at bottom. Statistical results are shown as in Fig. 3B. *P = 0.00085 and **P = 0.01986. B) Five days after virus inoculation, mice were sacrificed and the amount of virus in lung extracts was measured in a plaque assay. Mean virus titer for each group is indicated by short horizontal bar.
Chimeric Pneumoviridae fusion proteins as immunogens to induce cross-neutralizing antibody responses (2017)
Figure 5

Figure 5. Antigenic characterization of prefusion hRSV F chimeras with amino acids from hMPV F antigenic site IV. A) Partial amino acid sequences of the hRSV F (red) and hMPV F (blue) antigenic site IV (Lopez et al., 1998;McLellan et al., 2010;Wu et al., 2007). Partial sequences of the F-410 and F-411 chimeras, color coded to indicate the origin of their antigenic site IV residues. Amino acids shared by hMPV F and hRSV F are shown in purple. The remaining sequences of F-410 and F-411 are derived from prefusion hRSV F. B) Surface representation of the hRSV F structure stabilized in the prefusion conformation, as reported (McLellan et al., 2013a). One of the protomers is shown as a red ribbon. Antigenic site IV is magnified and colored as in (A) for the F-410 and F-411 chimeras. C) ELISA binding results of mAbs specific for hRSV F (left panels) or hMPV F (right panels) with the proteins indicated at left. mAbs specific of hRSV F bind to epitopes of antigenic site Ø (D25), antigenic site II (Mz and 47F) or antigenic site IV (101F and 107F). mAs specific of hMPV F bind to epitopes of antigenic site II (MF14), antigenic site IV (MF11, MF16 and MF29) or an epitope shared by hRSV F and hMPV F (MPE8). .