EMBO/soda-nel huggingface dataset:
Adherens junction remodelling during mitotic rounding of pseudostratified epithelial cells (2020)
Figure 5

Figure 5. Mutation of Aurora A traps rounded cells in a delayed and extended mitosis, while chemical inhibition of both Aurora A and B prevents mitotic rounding.

(A) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells (pH3, red) in wild-type wing discs, and aurA homozygous mutant wing discs, but not upon inhibition of both AurA and AurB by treatment with VX-680. Note increased numbers of mitotic cells in aurA homozygous mutant wing discs, due to delayed mitotic progression. Scale bars ~20µm (low mag.) and ~1µm (high mag.). n > 15 independent biological replicates.

(B) Live-imaging of MyoII-GFP reveals delayed mitosis in aurA homozygous mutant wing discs, while inhibition of both AurA and AurB by treatment with VX-680 leads to complete loss of mitotic rounding and MyoII-GFP membrane localisation. Asterisks indicate a single mitotic cell. Scale bars ~1µm. Quantification shown on the bottom right (n = 5 independent samples per genotype).

(C) Live-imaging of E-cad-GFP reveals delayed mitosis in aurA homozygous mutant wing discs, cytokinesis failure in UAS.aurB-RNAi expressing wing discs, and complete failure of mitotic rounding and E-cad-GFP downregulation upon inhibition of both AurA and AurB by treatment with VX-680. Asterisks indicate a single mitotic cell. Scale bars ~1µm. Quantification shown on the bottom right (n = 9 independent samples per genotype).
Adherens junction remodelling during mitotic rounding of pseudostratified epithelial cells (2020)
Figure 6

Figure 6. Genetic redundancy between Aurora A and B kinases in vivo.

(A) Striped ptc.Gal4-driven expression of UAS.GFP in a control wing imaginal disc. Scale bars ~20µm. n > 6 independent biological replicates.

(B) Striped ptc.Gal4-driven expression of UAS.GFP in an aurA homozygous mutant wing imaginal disc leads to increased numbers of pH3 positive mitotic cells. n > 7 independent biological replicates.

(C) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.aurB-RNAi in a control wing imaginal disc leads to enlarged cells with large nuclei (marked by DAPI; white arrowhead). n > 6 independent biological replicates.

(D) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.aurB-RNAi in an aurA homozygous mutant wing imaginal disc leads to extrusion and apoptosis of cells (marked by Dcp1 and pyknotic nuclei). n > 8 independent biological replicates.

(E) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.pbl-RNAi in the wing imaginal disc leads to extrusion and apoptosis of cells (marked by Dcp1 and pyknotic nuclei). n > 8 independent biological replicates.

(F) ptc.Gal4-driven expression of UAS.aurB-RNAi in an aurA homozygous mutant endoreplicating salivary gland has no phenotypic consequence. n > 6 independent biological replicates.

(G) MARCM clones (GFP-positive) expressing UAS.aurB-RNAi in an aurA homozygous mutant cells result in single cell clones (i.e.: no cell division), while individual loss of AurA or AurB allows some cell proliferation. Quantification shown on the right (n > 7 independent samples per genotype; Mean +/- 1 S.D. shown). Scale bar ~20µm.
Adherens junction remodelling during mitotic rounding of pseudostratified epithelial cells (2020)
Figure 7

Figure 7. Aurora A and B are continuously required for mitotic rounding

and subsequent cytokinesis.

(A) Live-imaging of MyoII-GFP in a control wing disc epithelium. Asterisk indicates the location of a single mitotic cell about to initiate rounding. Scale bar ~1µm. n > 5 independent biological replicates.

(B) Live-imaging of MyoII-GFP upon treatment with the dual AurA/B inhibitor VX-680 after initiation of mitotic rounding, which rapidly arrests progression of rounding and prevents cytokinesis. Scale bar ~1µm. n > 9 independent biological replicates.

(C) Live-imaging of E-cad-GFP upon treatment with the dual AurA/B inhibitor VX-680 after initiation of mitotic rounding, which rapidly arrests progression of rounding and prevents cytokinesis. Scale bar ~1µm. n > 6 independent biological replicates.

(D) Working model for mitotic rounding and junctional downregulation in pseudostratified Drosophila epithelial cells.
Adherens junction remodelling during mitotic rounding of pseudostratified epithelial cells (2020)
Figure 8

Figure 8. The cuboidal follicle cell epithelium does not downregulate E-cadherin at mitosis.

(A) Early stage Drosophila egg chambers showing giant germ-line nurse cells surrounded by E-cadherin-positive follicle cell epithelium. A follicle cell undergoing mitotic rounding is highlighted with an asterisk (*). Scale bar ~3µm. n > 3 independent biological replicates.

(B) High resolution imaging of the F-actin cortex, E-cadherin, and phospho-MyoII in a mitotically rounded follicle cell. Note maintenance of adherens junctions including both E-cad and the contractile actomyosin ring during mitotic rounding. Asterisks or pH3 staining indicate a single mitotic cell. Scale bar ~3µm. n > 4 independent biological replicates.

(C) Schematic diagram of follicle cell rounding, showing the relatively mild shape change that is insufficient to affect E-cadherin levels during mitotic rounding.

Extended View Figures
Trnp1 organises diverse nuclear membrane-less compartments in neural stem cells (2020)
Figure 1

Figure 1. Characterization of Trnp1 protein.

A, Clustal alignment analysis of Trnp1 orthologs. Highly conserved and similar residues are shown in black and grey, respectively. Asterisks show 100% conserved residues among all orthologs. Predicted disordered and structured regions are depicted with a red line and a black line, respectively. Cylinders represent alpha-helices and arrows represent turns.

B, Analysis of the coiled-coil structure of Trnp1 using the prediction tool COILS.

C, Immunostaining of endogenous Trnp1 and DAPI in P19 cells and E14 cortical cells at 1 day in vitro (div).

D, Co-IP with anti-GFP and WB with anti-Trnp1 antibodies in lysates from P19 cells transfected with plasmids expressing Trnp1-GFP fusion protein and/or Trnp1-IRES-RFP for 24h. Mw (molecular weights): 55 kDa (black line); 35 kDa (blue line); 25 kDa (red line). Input: 0,1%; co-IP: all immunoprecipitated proteins.

E, Representative phase contrast images of in vitro phase separation of the recombinant proteins YFP (upper row) or Trnp1 (lower row) at the indicated concentration in 50 mM Sorenson's buffer (pH 7,6) containing 150 mM salt and 2mM DTT with the crowding agent dextran or RNA when depicted.

Data information: Scale bars: 10 μm (C) and 50 μm (E). In Dextran and RNA condition 10 μm (E).
Trnp1 organises diverse nuclear membrane-less compartments in neural stem cells (2020)
Figure 2

Figure 2. Structure-function analysis of Trnp1.

A, Scheme showing the different Trnp1 truncated/mutant constructs generated here and the region used for generating the Trnp1 antibodies. Note than antibody#1 works for immunofluorescence (IF) and WB while antibody#2 only works for WB.

B, GFP and Trnp1 immunostaining with DAPI labelling in E14 cortical cells transfected for 24 h with plasmids (indicated by arrows) expressing the different truncated/mutant forms of Trnp1 and GFP showing Trnp1 localization.

C, WB of cytoplasmic and nuclear enriched fractions of P19 cells transfected with the different truncated/mutant forms of Trnp1 for 24h. Hist1 and actin serve as loading controls for nuclear and cytoplasmic fractions, respectively. The values under the anti-Trnp1 blots indicate the percentage of the signal relative to the control set to 100% after normalization by α-actin (Image J quantification). The values under the anti-actin and anti-Hist1 blots indicate the percentage of the signal considering that nuclear and cytoplasmic fractions together is 100%.

D, WB showing mutCC Trnp1 and ∆1-16 Trnp1 protein turnover in P19 cells 24 h after transfection in the presence of cycloheximide (CHX) for the time indicated below in hours (h). GFP (half live:24h) was used as a loading control.

E, Co-IP with anti-FLAG magnetic beads and WB with anti-Trnp1 antibodies showing that FLAGTrnp1 interacts with the GFP-fusion and the ∆1-16Trnp1 proteins but not with other truncated Trnp1 proteins indicating IDRs are crucial for Trnp1 homo-oligomerization. Samples were collected 24h after transfection of P19 cells with plasmids expressing the indicated proteins depicted in the panel.

F, Representative phase contrast images of in vitro phase separation of the recombinant ∆1-16Trnp1, ∆1−140Trnp1 and ∆95-223Trnp1 proteins at the indicated concentrations in 50 mM Sorenson's buffer (pH 7,6) containing 150 mM salt and 2mM DTT plus Dextran or RNA when indicated.

G, Violin Dot Plots illustrating quantification of the area of single droplets (N=700) formed by the recombinant Trnp1, ∆1-16Trnp1, ∆1−140Trnp1, ∆95-223Trnp1 proteins at 25 μM concentration in a representative experiment of three repetitions under the conditions shown in F.

H, Histogram depicting the percent of proliferating (Ki67+) cells transfected for 24 h with plasmids expressing control (GFP) or Trnp1-IRES-GFP and its different mutant/truncated forms-IRES-GFP in dissociated E14 cortical cells. Biological replicates: GFP=21; Trnp1=15; FLAGTrnp1=10; mutCC=5; ∆1-16=7; ∆1-1-56, ∆1-97, ∆1-140=3, ∆95-223=5 and ∆140-223=4.

Data information: Each dot in each condition represents an independent experiment. Kruskal Wallis test with Dunn's post hoc test (G-H). **p<0.01, ***p<0.005. Mw: 55 kDa (black line); 35 kDa (blue line); 25 kDa (red line), 15kDa (green line) and 10 kDa (orange line). All experiments were repeated three independent times. Scale bars: 10 μm (B) and 50 μm (F) except for dextran and RNA condition that is 10 μm (F).
Trnp1 organises diverse nuclear membrane-less compartments in neural stem cells (2020)
Figure 3

Figure 3. The N-terminal IDR (first 16aa) of Trnp1 is important for promoting proliferation in vivo.

A-C, Micrographs showing examples of sections of E15 cerebral cortex 2 days after IUE of the plasmids indicated in (A-C) with magnifications showing the indicated immunostainings. Scale bars: 100 µm and 20 µm for magnifications. Filled arrows show electroporated cells expressing Pax6 and Ki67 proteins. Open arrows show electroporated cells negative for Pax6 and Ki67 staining. VZ: ventricular zone; SVZ: subventricular zone; IZ: intermediate zone; CP: cortical plate; V: ventricle.

D, Bar and dot plot showing the distribution as percent of GFP+ cells/bin after IUE and staining as in A-C. A cortical column reaching from the ventricular surface (bottom in A) to the pial surface (top in A) was divided into equal sized bins with bin1 at the ventricular surface and bin 5 at the pial surface.

E-G, Bar and dot plots of the proportion of proliferating (Ki67+) cells (E), the number of NSCs (Pax6+) (F) and the proportion of proliferating (BrdU+) TAPs (Tbr2+)(G) as in A-C showing a significant increase in proliferating NSCs upon Trnp1 IUE compared to control or ∆1-16Trnp1 proteins (E- F) and a significant decrease in proliferating TAPs upon Trnp1 IUE and increase upon ∆1-16Trnp1 IUE compared to control (G) at E15, 2 days after IUE.

Data information: Biological replicates: in D: control=7; Trnp1=6; ∆1-16=8 , in E and F: control=5; Trnp1=6; ∆1-16=7, in G, control=5; Trnp1=5; ∆1-16=4 from 2-4 different litters. Data in dot plots are presented as median±IQR; bars in D represent the mean. Kruskal Wallis test with Dunn's post hoc test (D) and Mann Whitney test (E,F and G). *p< 0.05, **p<0.01, ***p< 0.005
Trnp1 organises diverse nuclear membrane-less compartments in neural stem cells (2020)
Figure 4

Figure 4. Trnp1 interacts with proteins involved in ribosomal biogenesis, splicing, and chromatin remodeling.

A, WB showing co-IP performed with the antibodies indicated on top of the lanes in nuclear lysates treated with the nucleases indicated to the right of the panel from P19 cells transfected with plasmids expressing FLAGTrnp1 for 24h immunoblotted with anti-Trnp1 antibodies. As negative control no antibody was added to the co-IP.

B-C, Volcano plot showing the mean difference of the protein iBAQ between FLAGTrnp1 (B) and FLAG∆1-16Trnp1 (C) interacting proteins versus control plotted against the p-value. Proteins used to experimentally confirm the MS results are indicated with squares and the protein name in (B).

D, STRING analysis of Trnp1 interactors (B). The disconnected nodules are not shown. Proteins cluster have been color coded (green for chromatin association, red for RNA splicing, blue for proteins involved in nucleolus function and light yellow for the rest).

Data information: In panel B and E Black dotted lines indicate the fold change (FC>1.9) and the significance cutoff (t test; p>0.05). Each dot represents a protein. Proteins participating in different functions have been color coded (yellow for chromatin association, green for cell cycle, red for RNA splicing, blue for proteins involved in nucleolus function and black for the rest).
Trnp1 organises diverse nuclear membrane-less compartments in neural stem cells (2020)
Figure 5

Figure 5. Trnp1 changes the architecture of nucleolus, condensed chromatin and nuclear speckles.

A, Superresolution confocal image of endogenous Nucleophosmin (B23), Nucleolin (Ncl) and Trnp1 proteins in E14 cortical cells at 1div. The yellow line indicates the position of the intensity line scan plot (below confocal panels) measuring the grey value of each staining (Trnp1=grey; Ncl=purple and B23=green). Scale bars, 5 µm.

B, Co-IP experiments with lysates from P19 cells expressing FLAGTrnp1 for 24h. IPs and WB with the antibodies indicated show interaction between Trnp1 and Ncl but not with Fbl1 consistent with the MS data.

C-E, Ultrastructural analysis depicting in the nucleolus (arrows for nucleolar interstice) (C), condensed chromatin blocks (arrows) (D) and nuclear speckles or interchromatin granule clusters (arrows for dense cords) (E) in the nucleus of P19 cells transfected with plasmids expressing Trnp1-IRES-GFP or GFP in control cells FACS sorted and fixed 24 h after transfection. Scale bars, 2 µm (D) and 1 µm (C and E). Nu: nucleolus; G: granular component; D: dense fibrillar component; FC: fibrillar center; C: condensed chromatin.

F, WB of lysates from P19 cells transfeted for 24h with plasmids expressing GFP only or Trnp1-IRES-GFP with antibodies indicated.

Data information: All experiments were done at least in duplicate.
Trnp1 organises diverse nuclear membrane-less compartments in neural stem cells (2020)
Figure 6

Figure 6. Trnp1 modulates nucleoli disassembly and M-phase duration in vitro.

A, Representative time-lapse sequence of GFP-Fbl1 in P19 cells expressing the plasmids indicated defining t=0 when the chromatids separate (anaphase).

B-C, Analysis of the number (B) and the total area (C) of the GFP-Fbl1 foci in P19 cells expressing the constructs indicated for a period of 360 minutes. Band width represents SEM and grey areas represent time points with significant differences between the control and the Trnp1 expressing conditions. The ∆1-16Trnp1 expression does not show any statistical difference compared to the control condition.

D, Representative time-lapse sequence including one mitosis of H2B-Venus in P19 cell expressing the indicated constructs.

E, Volcano Plot illustrating the length of M-phase from condensation to de-condensation of chromosomes in minutes. Each dot represents an individual cell from 3 different biological replicates. GFP=61 cells, Trnp1=62 cells and ∆1-16Trnp1: 40 cells.

F, Bar graph showing the percentage of cells expressing Trnp1-IRES-GFP or GFP as control in each phase of the mitosis indicated in different color as indicated at different times after release from synchronization in G2. 100 cells per condition and time were analyzed.

G, Bar and dot plots depicting the proportion of GFP+ NSCs (Pax6+) in mitosis (pHH3+) showing a significant decrease 24h after Trnp1 compared to control IUE. N= 4 brains from 2-3 different litters.

Data information: Imaging started 22 hours after transfection with confocal images taken every 10 minutes for a period of 16 to 72 h. Time 0: chromatid separation. Results are shown as mean±SEM (B and C) or as median±IQR (E, F and G). Mann Whitney test (B, C, E, F) and Kruskal Wallis test with Dunn's post hoc test (G). *p< 0.05; **p< 0.01. ***p< 0.005.
Trnp1 organises diverse nuclear membrane-less compartments in neural stem cells (2020)
Figure 7

Figure 7. Trnp1 regulates size of nucleoli in vivo.

A-C, Micrographs showing GFP (green: electroporated cells (A-C)) and Ncl (grey: nucleoli (A', B' and C')) immunostaining of E15 cerebral cortex 2 days after IUE with plasmids indicated. Position of magnifications shown on the right side are depicted by a white square. V= ventricle; VZ: ventricular zone. Yellow dotted shapes show examples of nucleoli in electroporated cells from the VZ and the non-VZ. Scale bars: 20 µm and 10 µm for magnifications.

D-F Violin plots depicting the nuclear size (D) nucleoli number (E) and nucleoli size (F) from cells electroporated as in A-C with the constructs and the areas indicated on the x-axis. Note that in Trnp1, but not ∆1-16Trnp1, overexpression is sufficient to significantly increase nucleolar size in the non-VZ (where most cells lack endogenous Trnp1). N=10 cells per region from 6 different control and Trnp1-IRES-GFP electroporated embryos and N=15 per region from 3-4 different ∆1-16Trnp1-IRES-GFP electroporated embryos from at least 4 litters.

G-H, Micrographs showing GFP (green: electroporated cells), Pax6 (magenta for NSCs) and Ncl (grey for nucleoli) immunostainings in sections of E15 cerebral cortex 2 days after IUE with plasmids indicated. Scale bar: 20 µm. Yellow dotted shapes show examples of nucleoli in electroporated and yellow non dotted shapes show examples of nucleoli in non-electroporated cells from the VZ and the non-VZ.

I-K Violin plots depicting the nuclear size (D) nucleoli number (E) and nucleoli size (F) from cells electroporated as in G-H with the constructs and the areas indicated on the x-axis. Note that Trnp1 knockdown significantly decreases nucleolar size in the VZ (where most cells express endogenous Trnp1). N=10 cells per region from 7 embryos per condition from at least 4 litters.

Data information: Each dot represents a cell. Boxes in violin plots represent median±IQR. Kruskal Wallis test with Dunn's post hoc test. *p<0.05, **p<0.01, ***p< 0.005.
Trnp1 organises diverse nuclear membrane-less compartments in neural stem cells (2020)
Figure 8

Figure 8. Trnp1 co-regulates the function of several nuclear MLOs.

A-C Micrographs of sections of E15 cerebral cortex 2 days after IUE with plasmids and magnifications indicated. V= ventricle; VZ: ventricular zone. Scale bars: 20 µm and 10 µm for magnifications. Yellow dotted shapes show examples of heterochromatin spots in nuclei of electroporated cells from the VZ and the non-VZ.

D-E, Violin plots depicting the number (D) and size (E) of condensed chromatin spots from cells electroporated as in A-C. Note that Trnp1 overexpression is sufficient to significantly increase the size of condensed chromatin spots, while the ∆1-16Trnp1 acts as a dominant negative reducing the number and size of heterochromatin spots. N=10 cells per region from 5 embryos for GFP and Trnp1 and 8-15 cells from 4 embryos for ∆1-16Trnp1 of at least 4 litters. Each dot represents a cell. Results are shown as median±IQR.

F, Summary of the splicing changes observed in GFP+ cells isolated from E14 cerebral cortex 24 h after IUE with plasmids expressing the shRNA against Trnp1 and its control as determined by RNA-seq analysis. The number of events per category are shown in the bar. SE, skipped exon; RI, intron retention; MXE, mutually exclusive exon; A5SS, alternative donor site; A3SS, alternative acceptor site.

G-H Translation assay using OPP-click reaction in P19 cells transfected for 24 hours with GFP, Trnp1-IRES-GFP or ∆1-16Trnp1-IRES-GFP plasmids and analyzed by immunofluorescence (G) or FACs sorting (H) . Graph in (G) is representative from three different biological replicates. Each dot represents a cell. Control=102; Trnp1=126; ∆1-16Trnp1=97. Graph in (H) shows analysis of 2,000 transfected cells from 1 representative experiment with 2 biological replicates for control and 3 biological replicates for Trnp1 and ∆1-16Trnp1 conditons. The same FACs sorting experiment was repeated two more times. The dotted vertical line shows the mean of the OPP click signal for each condition.

I, Bar and dot plots depicting rRNA transcription for the 18S, 28S, 45S and 47S measured by qPCR in P19 cells transfected for 24h with plasmids expressing Trnp1-IRES-GFP or ∆1-16Trnp1-IRES-GFP compared to control cells expressing GFP . Each dot represents a biological replicate. N=4 for each condition.

Data information: Kruskal Wallis test with Dunn's post hoc test. p-values *p< 0.05; **p< 0.01. ***p< 0.005
Myeloid cells protect intestinal epithelial barrier integrity through the angiogenin/plexin-B2 axis (2020)
Figure 1

Figure 1. ANG is down-regulated in IBD patient samples.

(A) mRNA expression of ANG in colonic tissues from normal controls (n = 25) and UC (n = 27) or CD (n = 46) patients.

(B) Representative western blotting of ANG in colonic tissues from normal controls and UC or CD patients.

(C) Representative images showing ANG immunohistochemical (IHC) staining in normal control and UC or CD sample.

(D) Corresponding statistical analysis of ANG expression score in normal controls (n = 10) and UC (n = 10) or CD (n = 15) patients.

(E) Representative images showing hematoxylin-eosin (HE) staining and ANG IHC staining in mild and severe IBD samples.

(F and G) Statistical analyses of ANG expression scores in mild (n = 18) and severe (n = 19) UC (F) or mild (n = 20) and severe (n = 20) CD (G) patients.

Data information: Scale bar, 100 μm; data are shown as violin plot (A) or mean ± SEM (D, F and G); Kruskal-Wallis with Dunn's multiple comparisons test (A and D) and Mann Whitney test (F and G) are used to determine statistical significance (**P<0.01, ***P<0.001). UC: Ulcerative colitis; CD: Crohn's disease.
Myeloid cells protect intestinal epithelial barrier integrity through the angiogenin/plexin-B2 axis (2020)
Figure 2

Figure 2. Ang deficiency enhances susceptibility to DSS-induced colitis in mice.

(A) Ang mRNA expression in colonic tissue from 2.5% DSS-treated WT mice at indicated time point (n = 4 mice/time point).

(B) Kaplan-Meier curve of 3.5% DSS-treated Ang-deficient mice (Ang-/-, n = 14) or littermate controls (WT, n = 13).

(C, D) (C) Body weight loss and (D) disease activity index (DAI) of WT and Ang-/- mice with (n = 9) or without (n = 6) 2.5% DSS treatment.

(E-H) (E) Colon length, (F) serum FITC-Dextran level, (G) representative HE staining image, and (H) histopathological score of colonic section from the WT and Ang-/- mice on day 9 with (n = 9) or without (n = 6) 2.5% DSS treatment.

(I) Quantitative mRNA expression of cytokine genes in colonic tissue from the WT and Ang-/- mice on day 0 (n = 3) or day 9 (n = 9) during 2.5% DSS treatment.

(J) Soluble cytokine level in supernatant of cultured colonic tissue isolated from WT and Ang-/- mice on day 0 (n = 3) or day 9 (n = 7) during 2.5% DSS treatment.

Data information: Scale bar, 50 μm; data are shown as mean ± SEM; ANOVA with Dunnett's multiple comparisons test (A), Log-rank test for equality of survivor function (B), Mann Whitney test (D, F, H, I and J) and two-tailed unpaired student's t test (C and E) are used to determine statistical significance (*P<0.05, ** P<0.01, ***P<0.001). Ctrl: control; DSS: dextran sulfate sodium; FITC-Dextran: fluorescein isothiocyanate-labeled dextran.