EMBO/soda-nel huggingface dataset:
Nuclear Dishevelled targets gene regulatory regions and promotes tumor growth (2021)
Figure 8

Figure 8. Nuclear DVL3 influences gene expression and spheroid formation

(A) Immunofluorescence of HA-tagged DVL3 localization in MDA-MB-468 cells. The cells were probed with an HA antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of actin (green) and nuclear staining (blue) is shown as ACTIN/DAPI, merge of DVL3 (red) and nuclear staining (blue) is shown as HA/DAPI, merge of DVL3 (red) and actin (green) is shown as HA/ACTIN, and, merge of DVL3 (red), nuclear staining (blue) and actin (green) is shown as MERGE for empty vector (EV), HA-DVL3-WT and HA-DVL3-NESm transient transfected cells. Scale bar: 10µm.

(B) RT-qPCR-based analysis of expression changes of DVL3 in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells.

(C) RT-qPCR-based analysis of expression changes of KMT2D in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells.

(D) RT-qPCR-based analysis of expression changes of ARAP1, RFX5, GBAT2, WDR74, SNORD3D, HAUS5, TAP1, HLA-A, PDG1, and, MR1 in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells.

(E) Images and quantification of spheroid areas in stable MDA-MB-468 cell line (EV, HA-DVL3-WT and HA-DVL3-NESm). The data are presented as the mean ± SD, n = 8 showing statistical difference after 32h for HA-DVL3-WT respect to EV and48h for HA-DVL3-NESm respect to EV *p < 0.05. Independent t tests were performed per time point for statistical comparison. Scale bar: 300µm.

(F) Proposed model of the role of DVL3, colocalizing with transcription factors such as ZBTB7B and chromatin modifying enzymes as KMT2D in transcriptional regulatory region and regulate transcription.

Data information: For B-D panels transcript levels were normalized to actin transcript levels and an independent t test was used for statistical comparison. * p < 0.05; data show means ± SD, n=3.

Expanded View Figure legends
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles (2016)
Figure 2

Figure 2. SPOP localizes to nuclear speckles or other nuclear bodies. Confocal microscopy images of fixed NIH 3T3 cells transiently expressing (A) full length HA-SPOP, (B) GFP-Gli31455, and (C) HA-SPOP+GFP-Gli31-455 are shown. DAPI was used to stain the nucleus, SPOP localization was identified using a anti-HA antibody, nuclear speckle localization was identified using a anti-SC-35 antibody, and Gli31-455 localization was identified via GFP fluorescence. SPOP co-localizes with a marker for nuclear speckles or with the substrate Gli31455. The areas with overlapping HA- and GFP signal contain 75% of the punctate HA signal, and 100% of the punctate GFP signal.
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles (2016)
Figure 3

Figure 3. SPOP nuclear bodies have liquid-like character. (A-B) NIH 3T3 cells were (A) co-transfected with constructs expressing full length SPOP and GFP-Gli31455 or (B) transfected with a construct expressing SC-35-GFP, and GFP fluorescence was monitored in live cells. (A) Snapshots were taken from individual timepoints as noted in the Figure to show photobleaching (at 0 s), recovery, and nuclear body fusion events. The arrow in each panel points to the body that was photobleached and subsequently fuses with another body. These data were not used to calculate the average recovery time as the fusion event precludes accurate measurement of fluorescence recovery. (B) Snapshots at indicated timepoints show a nuclear speckle fusion event. Additional images are shown in Fig EV1. (C) Individual nuclear speckles were photobleached and FRAP was monitored for 90 sec. Data from 45 individual cells and FRAP events were corrected for background, normalized to the minimum and maximum intensity, and the mean is shown with error bars representing the standard error of the mean. The mean characteristic recovery time is indicated ± SEM. (D-E) Nuclear bodies are close to spherical. (D) Aspect ratios for speckles observed in NIH 3T3 cells transfected with constructs expressing HA-SPOP (151 cells) (D), or GFP-Gli31-455 and HA-SPOP (155 cells) (E) were calculated as described previously (Brangwynne et al, 2011). Representative individual images of cells are shown as insets in each panel.
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles (2016)
Figure 4

Figure 4. Self-association defective SPOP mutants do not localize to nuclear speckles. (A) SEC chromatograms of given loading concentrations of SPOP28-359 are shown. The concentrations were normalized to the monomer molecular weight, i.e., identical concentrations of dimeric SPOP ΔBACK (see Fig EV2) and oligomeric SPOP28-359 contain identical numbers of protomers. (B) SEC chromatograms for SPOP constructs defective in self-association in one or both oligomerization domains are shown. Proteins were injected at the same loading concentration (533 µM) and the elution volume of globular molecular weight standards is noted above the graph. (C) SEC chromatograms of SPOP28-359 (200 µM), SPOP mutBACK (718 µM), and mixtures of the two (ratios given in the Figure, SPOP at 200 µM) are shown. The elution volume of globular molecular weight standards is noted above the graph. (D) Constructs for expressing full-length HA-SPOP or HA-SPOP mutants capable of oligomerization through only one (or neither) of the self-association domains were transfected into NIH 3T3 cells. DAPI was used to stain the nucleus and SPOP localization was identified using an anti-HA antibody. Experiments were performed at least twice on four biological samples. Multiple cells were examined and representative cells are shown. For additional images see Appendix Fig S3. Scale bar represents 5 µm.
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles (2016)
Figure 5

Figure 5. SPOP forms higher-order oligomeric species in cells. (A) Substrate binding was assayed by anisotropy using a fluorescently labeled substrate peptide. All SPOP proteins bound substrate with an affinity similar to that of SPOP28-359 (4-8 µM, see Table 2). Experimental data are shown as circles; the solid lines are fits to eq. 2 (Roehrl et al, 2004). (B) In vitro crosslinking assays were performed for SPOP28-359 and each mutant at 30 μM protein with the amide-specific BS3 crosslinker. Crosslinking for SPOP ΔBACK and MATH domain are shown to demonstrate that crosslinking conditions do not lead to non-specific crosslinking of protein species. (C) Crosslinking reactions were performed on whole cell lysates from cells expressing wild type SPOP, SPOP mutBACK, SPOP mutBTB, or SPOP mutBTB-BACK. SPOP1, SPOP2 and SPOPn identify SPOP monomers, dimers and larger species, respectively. For loading levels see Appendix Fig S4.
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles (2016)
Figure 6

Figure 6. The SPOP oligomerization domains dimerize with different affinities. (A-C) The BTB domain dimerizes with nanomolar affinity. (A) SV-AUC data for 0.2 nM AF488-SPOP ΔBACK. Fluorescence scans were collected for 12 h and are plotted against distance from the axis of rotation (circles). The data were subjected to standard c(s) analysis in SEDFIT (Schuck, 2000) and the results of this fit are shown (solid lines, rainbow color scheme). (B) Sedimentation coefficient distributions for a selection of values from the dilution series are shown. (C) The isotherm of weighted-average s-values vs. concentration (black circles), was fit to a dimer self-association model and reveals a KD of 1.11 nM, [95% confidence interval 0.88, 1.40 nM] (solid line). (D) The BACK domain dimerizes with micromolar affinity. Experimental weight-average molar mass (Mw) from CG-MALS for SPOP mutBTB (black circles) was fitted to a self-association model (see text for details) for BACK-BACK dimerization (black line) yielding a KD of 36 ± 3 μM (average and standard deviation from 3 independent experiments). Fits to the monomer/dimer equilibrium model and to alternative monomer/trimer, tetramer and pentamer equilibrium models are depicted as black solid line and as dashed and grey lines, respectively. Residuals for all models are depicted in the lower panel.
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles (2016)
Figure 7

Figure 7. Indefinite SPOP self-association fits to an isodesmic model. (A) Cartoon schematic depicting the proposed isodesmic self-association model. KD1 and KD2 represent the domain-mediated dimerization affinities for the BTB domain and BACK domains, respectively. KD2 is identical for all association steps independent of oligomer size. At large oligomer sizes, KD2 may increase due to entropic penalties, resulting in KD2*. The N- and C-termini contain neither defined domains nor low-complexity sequences but may add additional self-association behavior as evidenced by aggregation; these interactions were not dissected due to the poor reversibility of aggregation (Fig EV2B). (B) Experimental weight-average molar mass (Mw) from CG-MALS for SPOP28-359 (orange circles) was fitted to an isodesmic self-association model in which SPOP dimers are the self-associating unit (orange line). The largest SPOP oligomer taken into account was an undecamer of SPOP dimers ((SPOP2)11). The fits from three independent experiments yielded a KD2 of 2.4 ± 0.4 µM. Lines for fits of the data to self-association models that assume formation of individual oligomeric species instead of isodesmic self-association are shown for reference (gray lines). (C) Experimental weight-average molar mass (Mw) from CG-MALS data (circles) and fits (solid lines) are depicted for each SPOP construct assayed, showing that a progressive increase in SPOP oligomer size is observed only when both self-association domains are interaction-competent. This Figure is comprised of data shown in Figures 6D, 7B, and Appendix Fig S5A for direct comparison. (D) Ribbon diagram illustrating structural model of an octamer SPOP assembly. In a 27 µM SPOP solution three percent of the oligomeric assemblies would be equal in size or larger. The domains are colored as in Figures 1 and 7A (MATH, green; BTB, red; BACK, blue), with alternating monomers shown in gray for clarity. The model was built using two available crystal structures (PDB ID 3HQI (Zhuang et al, 2009) and 4HS2 (van Geersdaele et al, 2013)), and no further assumptions.
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles (2016)
Figure 8

Figure 8. SPOP forms dynamic higher-order oligomers that can be analyzed quantitatively. (A) Native MS spectrum of 30 µM SPOP28-359 confirms that SPOP self-associates through addition of dimers. The increase in mass indicates that the dimer is the stable building block within the assemblies. (B) Graphical representation of the SPOP concentration of each oligomeric species within a 27 µM SPOP28-359 sample. The cartoons’ sizes are scaled based upon the fraction present within the total sample. (C) Concentration-dependence of the SPOP oligomer size distribution (mole fraction) from isodesmic modeling of CG-MALS data.
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles (2016)
Figure 9

Figure 9. The self-association deficient SPOP mutant mutBTB has a substrate degradation defect in vivo. UAS transgenes were expressed under the control of an epithelial driver C765-Gal4 in Drosophila melanogaster. (A) UAS vector served as a control and yielded a normal wing. Longitudinal veins are denoted by numbers 1-5. Expression of (B) SPOP WT resulted in a modest Hh loss-of function phenotype with fusion of LV3 and LV4 (arrow). (C) mutBTB acts in a dominant-negative manner, as evidenced by a modest Hh gain-of function phenotype with expansion of the LV3-LV4 intervein space. Expression of (D) mutBACK and (E) mutBTB-BACK does not induce a wing patterning defect. ~50 animals each were analyzed from two independent crosses.
The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1 (2019)
Figure 1

Figure 1. Effect of Nanog on erythropoietic development.

A. Dox-induced prolongation of Nanog expression in Nanogtg embryos up to E9.5 results in lack of blood (left) and downregulation of erythropoietic gene expression. The center and right panels show whole-mount in situ hybridization for Hbb-bh1 (in embryos with intact yolk sacs) and for the long non-coding RNA Redrum. Asterisks mark the aorta-gonad-mesonephros region (AGM) and arrows the tailbud. Embryos of the same genotype but not treated with dox were used as controls (-dox). Scale bars, 500 µm.

B. Endomucin staining of vessels in control (-dox) or treated (+dox) E9.5 Nanogtg embryos. On the right, higher magnifications of the boxed areas. Scale bar, 500 µm.

C. Representative FACS plot of the distribution of the CD71 and Ter119 populations in dissected yolk sacs from untreated and dox-treated E9.5 Nanogtg embryos.

D. Quantification of the CD71+ Ter119+ population in controls (-dox, black dots; n=8) and Nanog expressing (+dox, red dots; n=7) E9.5 yolk sacs. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 Nanogtg embryos. ***P. < 0.0005; Student's t-test. Horizontal line represents mean values and error bars standard deviation (SD).

E. Representative FACS plots showing the distribution of cKit and CD41 populations in yolk sacs from untreated controls (-dox) and Nanog expressing (+dox) E9.5 Nanogtg embryos.

F. Quantification of different progenitor populations in yolk sacs from control (-dox, black dots; n=8) and Nanog expressing (+dox, red dots; n=7) E9.5 embryos. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 Nanogtg embryos. Horizontal line represents mean values and error bars SD.

G. Differences in the expression levels of Nanog and selected hematopoietic genes in the CD71+ Ter119+ population of control (-dox; n=7) and Nanog-expressing (+dox; n=4) E9.5 embryos. **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.
The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1 (2019)
Figure 2

Figure 2. Wild type ES cells rescue erythroid maturation in chimeric embryos.

A. Experimental design for chimera generation, and contribution of GFP cells to chimeric embryo (right hand side panels).

B. Freshly dissected dox-treated Nanogtg E10.5 embryos without (control) and with (chimera) contribution of wt-ESGFP cells (left, brightfield; right, GFP). Arrows mark the presence of blood in chimeric embryos that is absent from controls. Scale bar, 500 µm.

C. Representative FACS plots showing of red blood cell maturation as determined by CD71/Ter119 staining in single dox-treated E10.5 control (left) and chimeric (right) embryos.

D-F. Quantification of the CD71+ Ter119+ population in single dox-treated E10.5 embryos (D; control, n=18; chimera, n=10), untreated embryos (E; control, n=7; chimera, n=12), and in GFP- cells (not derived from wild type ES cells) from dox-treated embryos (F; control, n=18; chimera, n=10). ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.
The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1 (2019)
Figure 3

Figure 3. Nanog-knockout ES cells show increased potential to generate erythroid precursors.

A. Quantification of colony-forming units generated by wild type (wt) and knockout (Nanog-/-) ES cells after culture of EBs for 5 (D5), 6 (D6), or 7 (D7) days and plating disaggregated cells in different hemogenic-promoting conditions). Panels on the left show representative images of mouse hematopoietic colonies obtained after 12 days of culture in specific media. CFU-GEMM, progenitors giving rise to granulocytes, erythrocytes, monocytes, and megakaryocytes; BFU-E, burst forming units-erythroid; Ery-P, colony forming primitive erythroid; CFU-GM, granulocyte-monocyte precursors; CFU-M, monocyte precursors; CFU-G, granulocyte precursors. No CFU-GEMM are detected at D5 and no BFU-E at D7. For both wt and knockout cells, n=3 each with 3 technical replicates. *P < 0.05, **P < 0.005, ***P < 0.00005; Student's t-test. Horizontal line represents mean values and error bars SD.

B. RT-qPCR determination of the relative expression of Brachyury and selected hematopoietic genes in control (wt, right) and knockout (Nanog-/-, left) ES cells (n=3) during 10 days of EB differentiation in hematopoietic-cytokine-enriched medium. Black arrowheads indicate the peak of Brachyury expression and white arrowheads the time of maximum hematopoietic-gene expression.
The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1 (2019)
Figure 4

Figure 4. Induced Nanog expression blocks erythroid maturation in adult mice.

A. Experimental design for the treatment of adult Nanogtg mice.

B. Representative FACS plots showing the distribution of different populations distinguished by CD71/Ter119 staining in whole bone marrow from untreated (-dox) or treated (+dox) adult mice. S0 (double negative cell), S1 (proerythroblast), S2 (basophylic erythroblast), S3 (polychromatic erythroblast), and S4 (orthochromatic erythroblast) are different stages of blood maturation.

C. Quantification of the S1-S4 erythroid populations (-dox, n=4; +dox, n=5). *P < 0.05, **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.

D. Representative FACS plots showing the distribution of CD16/32 and CD34 hematopoietic precursors sorted from the cKit+Sca1-LIN- bone marrow of untreated (-dox) or treated (+dox) adult Nanogtg mice.

E. Quantification of precursor populations based on CD16/32 and CD34 sorting, as total number of cells per individual femur (-dox, n=5; +dox, n=6). *P < 0.05, **P < 0.005; Student's t-test. Horizontal line represents mean values and error bars SD.

F. RT-qPCR quantification of the relative expression of hematopoietic genes in megakaryocyte-erythroid progenitors (MEP) (-dox, n=8; +dox, n=5). *P < 0.05, ****P < 0.00005; Student's t-test. Horizontal line represents mean values and error bars SD.

G. Experimental design for the transplant of Nanogtg bone marrow to wild type recipients and treatment of chimeric mice.

H. Contribution of Nanogtg transplanted bone marrow cells to peripheral blood before (left) and after (right) dox treatment. Percentage of host derived cells (CD45.1+) are shown in black, and of donor derived cells (CD45.1/CD45.2 double +) in red. Individual mice are indicated on the x-axis (n=7).

I. Contribution of Nanogtg transplanted cells to LSK, CMP, GMP and MEP populations purified from bone marrow. Percentage of host derived cells (CD45.1+) are show in black, and of donor derived cells (CD45.1/CD45.2 double +) in blue. Individual mice are indicated on the x-axis (n=7).
The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1 (2019)
Figure 5

Figure 5. Direct transcriptional regulation of Tal1 expression by Nanog

A. Expected and observed number of mesodermal (Flk1+) cells of the E7.0 mouse embryo expressing Nanog and selected mesodermal or hematopoietic gene expression, based on single cell RNA-seq data (Scialdone et al., 2016). Statistical significance was calculated with a hypergeometric test.

B. PCA showing the distribution of Flk1+ E7.0 mesoderm cells expressing Nanog (green) or Tal1 (red). The single cell expressing both genes is shown in yellow and indicated by an arrow.

C. E6.5 Nanogtg embryos after 8 hours ex-utero culture in the presence (+dox) or absence (-dox) of doxycycline. Scale bar, 100 µm.

D. RT-qPCR quantification of the relative expression of Nanog, Tal1, and Klf1 in individual untreated embryos (-dox) or treated embryos (+dox) (n=5). **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.

E. Whole mount in situ hybridization of Tal1 in E7.5 untreated (-dox) or in utero treated (+dox) Nanogtg embryos. Scale bar, 100 µm.

F. UCSC browser view of the Tal1/Stil1 region (mm9; chr4:114,705,753-114,756,741), indicating the presence of the NANOG binding peak, determined by ChIP-seq, in EpiLCs (2 replicates are shown) but not in ES cells (Murakami et al., 2016); the binding peak was deleted by CRISPR/Cas9 genome editing (scissors).

G. RT-qPCR determination of relative expression in wild type and CRISPR-deleted embryos (n=5) of Tal1 (wt, n=19; deleted, n=13), Klf1 (wt, n=3; deleted, n=6), Gfi1b (wt, n=10; deleted, n=8), Runx1 (wt, n=13; deleted, n=5) and Stil (wt, n=19; deleted, n=13). **P < 0.005, Student's t-test. Horizontal line represents mean values and error bars SD.

H. Experimental design for ES to EpiL cell differentiation of Nanogtg cells and two independent clones (Nanogtg;dTal1del#1 and Nanogtg;dTal1del#2) where the binding site for NANOG distal to Tal1 has been deleted (left). On the right, relative expression of Tal1 determined by RT-qPCR for each ES cell line (ESC; n=9 for all three lines) and EpiL cells without (EpiLC; Nanogtg and Nanogtg;dTal1del#1, n=8; Nanogtg;dTal1del#2, n=6) or with dox treatment (EpiLC +dox; n=9 for all three lines). The genotype of the cell lines is indicated below. Values were normalized to Nanogtg ESC. *P < 0.05, **P < 0.01, ns = not significant; ANOVA with Fisher post-test. Horizontal line represents mean values and error bars standard error of the mean (SEM).

EXPANDED VIEW FIGURE LEGENDS
Sonic hedgehog is a regulator of extracellular glutamate levels and epilepsy (2016)
Figure 1

Figure 1. Shh release and its pathway activation in response to epileptic stimulations.

Representative western-blots of the cortical (Ctx) or hippocampal (Hip) extracts from mice at the indicated time after seizure activity in pilocarpine (A,B) or kindling (D,E) models. For D and E, samples obtained from mice evoked with a single kindling stimulation to induce seizure activity as evidenced in EEG. (C,F) Statistics of Gli1 or Shh expression levels shown in A,B or D,E respectively. n=8-14 mice in C and n=8-23 mice in F. (G) Shh levels assayed by ELISA from mouse cortex and hippocampus at the indicated time after the initiation of status epilepticus (SE) induced by pilocarpine (n=7-10). (H,I) Shh levels assayed by ELISA in the medium of slices (H, n=9) or hippocampal neurons (I, n=6) incubated with picrotoxin (Pic) or Mg2+-free (0Mg) for the indicated times. (J) Representative western-blots and statistics of Gli1 expression levels from hippocampal neurons incubated with 0Mg for the indicated times (n=13-19). α-tubulin (α-Tub), a loading control. Data were mean + SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Control (Ctrl) with student’s t test.