EMBO/soda-nel huggingface dataset:
The Super Elongation Complex mediates phase transition of SPT5 during transcriptional pause release (2022)
Figure 1

Figure 1. NELF and DSIF form stable clusters at the paused Pol II genes.

A, B (A) Confocal imaging of FOS DNA FISH with concurrent SPT5 and Pol II-S5P IF showing that SPT5 and Pol II-S5P occupied the FOS loci after serum starvation. Zoomed-in views of the merged regions are indicated by the white arrow. (B) Same as Fig 1A. but for FOS DNA FISH with concurrent SPT5 and NELFE IF.

C Percentage of FISH foci overlap with IF puncta in Fig 1A and B. Results are plotted with four technical repeats from two biological replicates. Error bars represent standard deviations.

D Violin plot showing the number of cells containing nuclear puncta in Fig EV1F. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. For Pol II-S5P, **p = 0.0034. For Pol II-S2P, ***p = 0.0004. For SPT5, p = 0.8631. For NELFE, **p = 0.0015. For NELFA, ***p < 0.0001.

E Confocal images showing that SPT5, NELFA, Pol II-S5P and AFF4 formed nuclear puncta in HCT 116 cells before and after treated with 1% 1,6-hexanediol (abbreviation as 1,6-hex) for 30 minutes.

F Violin plot showing the number of cells containing nuclear puncta in Fig 1E. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. For SPT5, p = 0.1301. For NELFA, p = 0.2756. For Pol II-S5P, p = 0.8179. For AFF4, ***p < 0.0001.

G-I ChIP-qPCR showing that the occupancies of Pol II (G), SPT5 (H), and NELFE (I) at the promoters and gene bodies of HSP70, GAPDH, and MYC in HeLa cells. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent standard error of means. Results are plotted with four technical repeats from two biological replicates. Two-tailed, unpaired Student's t test was performed. For Pol II ChIP, HSP70_promoter, **p = 0.0014; GAPDH_promoter, p = 0.8385; GAPDH_gene body, ***p < 0.0001; MYC_promoter, p = 0.1765; MYC_gene body, ***p = 0.0002. For SPT5 ChIP, HSP70_promoter, **p = 0.0033; GAPDH_promoter, p = 0.9603; GAPDH_gene body, **p = 0.0012; MYC_promoter, p = 0.9898; MYC_gene body, ***p < 0.0001. For NELFE ChIP, HSP70_promoter, p = 0.3913; GAPDH_promoter, p = 0.9347; MYC_promoter, p = 0.8558.
The Super Elongation Complex mediates phase transition of SPT5 during transcriptional pause release (2022)
Figure 2

Figure 2. SPT5 carries an intrinsic property to form clusters.

A NCPR (Net Charge Per Residue) analyses (top) of SPT5. The scores are assigned between -1 and 1. The score above 0 indicates positive charge (blue), while the score blew 0 indicates negative charge (red). The intrinsic disorder calculated by PONDR (Predictor of Natural Disordered Regions) VSL2 algorithm (bottom). The intrinsic scores are shown on the y-axis, and amino acid positions are shown on the x-axis. The scores are assigned between 0 and 1, and the score above 0.5 indicates disorder. The grey regions represent the indicated IDRs under investigation in this study.

B Fluorescence microscopy images showing that FLAG affinity purified NELFE and SPT5 formed core-shell like clusters in 100 mM NaCl containing buffer.

C Normalized FRAP curves for eGFP-SPT5. The bleaching events occurred at 0 second. Confocal images were embedded within the plot showing FRAP of the in vitro eGFP-SPT5 clusters. Results shown are from three biological replicates. The arrowhead represents the bleached region.

D Domain organization of SPT5. NTR, N-terminal IDR; NGN, NusG N-terminal domain; KOW, Kyprides, Ouzounis, Woese domains; CTR, C-terminal IDR. NLS, nuclear localization signal. The residuals phosphorylated by P-TEFb and the Pol II interacting regions in the SPT5 sequence are indicated.

E Live cell imaging of HEK-293T cells expressing eGFP-SPT5 together with mCherry-SPT5, mCherry-SPT5ΔNTR, or mCherry-SPT5ΔCTR close to endogenous level.

F Violin plot showing the clusters area (top) and circularity (bottom) of SPT5 with SPT5, SPT5ΔNTR, or SPT5ΔCTR in Fig 2E. Each n > 20. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. ***p < 0.001.

G Normalized FRAP curves for eGFP-SPT5 (green) and mCherry-SPT5 (purple) in the SPT5 homotypic clusters, while eGFP-SPT5 (yellow) and mCherry-SPT5ΔNTR (blue) in the SPT5/SPT5ΔNTR heterotypic aggregates. The bleaching events occurred at 0 second. Results shown are from three biological replicates.

H Confocal images showing FRAP analysis of the NELFA-IDR-eGFP and SPT5-CTR-mCherry clusters in vitro. The arrowhead represents the bleached and recovered cluster.

I Normalized FRAP curves for NELFA-IDR-eGFP and SPT5-CTR-mCherry clusters. The bleaching events occurred at 0 second. Results shown are from two biological replicates.
The Super Elongation Complex mediates phase transition of SPT5 during transcriptional pause release (2022)
Figure 4

Figure 4. SPT5 translocates from the pausing clusters to the liquid-like elongation droplets.

A Fluorescence microscopy images showing the phase-separated droplets formed in 50 mM NaCl containing buffer with 2 μM NELFA-IDR-eGFP and 10 μM SPT5-CTR-mCherry and 2 μM BFP or AFF4-IDR-BFP in the presence of 10% PEG-8000.

B Fluorescence microscopy images showing the phase-separated AFF4-IDR-eGFP and SPT5-CTR-mCherry droplets formed in 50 mM NaCl containing buffer in the presence of 10% PEG-8000. The concentration ratios are indicated as CAFF4-IDR/CSPT5-CTR.

C Dot plot showing the droplet area in Fig 4B. Fields per condition n = 5.

D Live cell confocal images showing FRAP of the heterotypic eGFP-AFF4 + ENL and mCherry-SPT5 droplets. The arrowhead represents the bleached and recovered droplet.

E Normalized FRAP curves for eGFP-AFF4 + ENL (green) and mCherry-SPT5 (purple) in the AFF4/ENL/SPT5 heterotypic droplets. The bleaching events occurred at 0 second. Results shown are from three biological replicates.

F Time-lapse fluorescence images showing that the heterotypic AFF4-IDR-eGFP and SPT5-CTR-mCherry droplets rapidly fused into one spherical droplet upon contact. The images were subjected to illuminate every 2 second. The droplet formation buffer contained 10% PEG-8000 and 50 mM NaCl.

G Time-lapse fluorescence images of the nucleus of the HeLa cell expressing eGFP-AFF4, eGFP-ENL and mCherry-SPT5 subjected to illuminate every 1 minute for the times indicated. The SEC and SPT5 heterotypic droplets underwent spontaneous fusion as indicated by arrows.
The Super Elongation Complex mediates phase transition of SPT5 during transcriptional pause release (2022)
Figure 5

Figure 5. Disruption of the SEC clusters increases the SPT5 containing pausing clusters.

A Confocal imaging of FOS RNA FISH with concurrent SPT5 and AFF4 IF showing that SPT5 and AFF4 co-occupied the FOS loci after serum treatment. Zoomed-in views of the merged regions are indicated by the white arrow.

B The three columns showing average FOS FISH signal and average SPT5 and AFF4 immunofluorescence signal in Fig 5A. centered on the FISH foci (see Methods). Data shown was analyzed from at least four biological replicates.

C Confocal imaging of FOS RNA FISH with concurrent SPT5 IF in wild type (WT) and ENL KO HCT116 cells after serum treatment. Zoomed-in views of the merged regions are indicated by the white arrow.

D The two columns showing average FOS FISH signal and average SPT5 immunofluorescence signal in Fig 5C. centered on the FISH foci (see Methods). Data shown was analyzed from at least four biological replicates.

E Confocal imaging of nascent RNA with EU staining in WT and ENL KO HCT116 cells.

F Violin plot showing the intensity of cells in Fig 5E. Results are representative of two biological replicates, each n > 70. Two-tailed, unpaired Student's t test was performed. ***p < 0.001.

G Western blot analysis of AFF4, SPT5, and ENL in HCT 116 cells after KL-1 treatment. α-Tubulin was used as a loading control.

H Confocal images showing SPT5 in nuclear puncta in WT or ENL KO cells treatment with KL-1.

I Violin plot showing the number of cells containing nuclear puncta in Fig 5H. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. ***p < 0.001.

J-M (J) Confocal images showing co-localization of Pol II-S5P with SPT5 in nuclear puncta in KL-1 treated HCT 116 cells. Results shown are representative images from three independent experiments. (K) Boxplots showing the mean values of the Pearson correlation coefficient of co-localization ratios of SPT5 and Pol II-S5P in Fig 5J. Central band is median; boxes represent 1st and 3rd quartile (25th and 75th percentile, respectively) and whiskers 1.5× interquartile range. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. KL-1 vs. DMSO, **p = 0.0050. (L) Same as Fig 5J. but for SPT5 and NELFA. (M) Same as Fig 5K. but for SPT5 and NELFA. Two-tailed, unpaired Student's t test was performed. KL-1 vs. DMSO, *p = 0.0100.
The Super Elongation Complex mediates phase transition of SPT5 during transcriptional pause release (2022)
Figure 6

Figure 6. SPT5 phosphorylation promotes the incorporation of SPT5 into the elongation droplets.

A The recombinant SPT5-CTR-eGFP proteins were purified, and analyzed by SDS-PAGE followed by Coomassie blue staining. The arrowhead represents the purified protein.

B Fluorescence microscopy images showing that 2 μM SPT5-CTR-eGFP formed phase-separated droplets in the NaCl containing buffer.

C Droplet number (top) and droplet area (bottom) analyses are shown. Central band is median; boxes represent 1st and 3rd quartile (25th and 75th percentile, respectively) and whiskers 1.5× interquartile range. Results are representative of two biological replicates, each n > 6. Two-tailed, unpaired Student's t test was performed. For droplet number, SPT5-CTR + P-TEFb (- ATP) vs. SPT5-CTR, p = **0.0015. SPT5-CTR + P-TEFb (+ vs.- ATP), **p = 0.0032. For droplet area, SPT5-CTR + P-TEFb (+ vs. - ATP), ***p < 0.001.

D The recombinant SPT5-CTR-mCherry and SPT5-CTR-TE-mCherry proteins were purified, and analyzed by SDS-PAGE followed by Coomassie blue staining. The arrowhead represents the purified protein.

E Fluorescence microscopy images showing the phase-separated droplets formed in 50 mM NaCl containing buffer with 10 μM AFF4-IDR-eGFP and 10 μM SPT5-CTR-mCherry or SPT5-CTR-TE-mCherry in the presence of 10% PEG-8000.

F Droplet number (top) and droplet area (bottom) analyses are shown. Central band is median; boxes represent 1st and 3rd quartile (25th and 75th percentile, respectively) and whiskers 1.5× interquartile range. Results are representative of two biological replicates, each n > 6. Two-tailed, unpaired Student's t test was performed. AFF4-IDR + SPT5-CTR-TE vs. AFF4-IDR + SPT5-CTR, **p = 0.0029.

G Fluorescence microscopy images showing the phase-separated droplets formed in 50 mM NaCl containing buffer with 2 μM NELFA-IDR-eGFP and 10 μM SPT5-CTR-mCherry or SPT5-CTR-TE-mCherry in the presence of 10% PEG-8000.
Chromosome misalignments induce spindle-positioning defects (2016)
Figure 1

Figure 1. Chromosome misalignments cause spindle misorientation

A. Histograms of spindle angle of GFP-H2B U2OS cells transfected with either GAPDH or Spindly siRNA and filmed on rectangular micropatterns. Cells were filmed 48 hours post-transfection. B. Mitotic timing of control-depleted U2OS cells or CLIP-170-depleted cells. CLIP-170-depeleted cells are split in two categories; ‘aligned’ and ‘misaligned’, relating to the observed chromosome misalignment phenotype. C. The histograms of spindle angles for the two different categories of CLIP-170-depleted U2OS cells (Left graph with alignment defect, right graph without alignment defect). D and E. Mitotic timing and spindle angle histograms of control (GAPDH)-depleted U2OS cells and CENP-E-depleted cells.
Chromosome misalignments induce spindle-positioning defects (2016)
Figure 2

Figure 2. Plk1 restricts cortical LGN localization near misaligned chromosomes

A. HeLa cell stably expressing GFP-LGN and transiently expressing RFP-H2B was treated with noscapine to induce chromosome misalignments. Cells were imaged every 5 minutes. The time is relative to NEB. The red arrows indicate cortical regions where the chromosomes are in close proximity to the cell boundaries, accompanied by the displacement of LGN from the cortex. B. Similar assay as described in supplementary figure 2D was carried out in the presence of a small molecule inhibitor against Aurora-B (ZM447439) in combination with the proteasome inhibitor MG-132 to keep cells arrested in mitosis and in B. with an inhibitor against Plk1 (BI2536). Drugs were added at the same timing as Hoechst, 30 minutes prior to imaging.
Chromosome misalignments induce spindle-positioning defects (2016)
Figure 3

Figure 3. Loss of KT-localized Plk1 rescues cortical LGN enrichment in the proximity of misaligned chromosomes and spindle misorientation

A. Immunofluorescence images and quantifications of KT-localized Plk1 in U2OS cells after PBIP1 depletion. Cells were either Mock transfected or with PBIP1 siRNA for 72 hours. Cells were treated with nocodazole for 1 hour and fixed. Cells were stained with the centromere marker ACA, Plk1 and DAPI. B. Representative images and histograms showing the effect of chromosome position on cortical LGN enrichment after 72 hours of Mock and PBIP1 depletion. C. Live cell images and histograms of GFP-H2B U2OS cells Mock transfected or with siPBIP1 filmed on rectangular micropatterns in the presence of a CENP-E inhibitor. The spindle angles for both conditions were scored at 32 minutes after NEB. D. The total spindle angle displacement within the first hour of mitosis was determined by measuring the spindle angle at each time point and calculating the sum of the Δ spindle angle between each time points.
An in vivo avian model of human melanoma to perform rapid and robust preclinical studies (2022)
Figure 1

Figure 1- Set up and characterization of melanoma cell lines and patient samples engrafted in the avian embryo.

A. Schematic diagram of the engrafting procedure of melanoma cell lines or melanoma biopsies in the chick embryo.

B. 3D views (light-sheet imaging) of HH25 chick embryos engrafted with the A375P::GFP stable melanoma cell labeled with an anti-GFP antibody (in red) and an anti-HNK1 antibody (in white, migrating and early post-migrating NCCs).

C,D. Immunolabeling of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody (in green in C, in brown in D, A375P:GFP cells), an anti-HNK1 antibody (in white, migrating and early post-migrating NCCs), an anti-Ki67 antibody (in red in C, cycling cells), an anti-SOX10 antibody (in red in D, also stains chick endogenous NCCs) and an anti-MITF antibody (in yellow in D). Nuclei were stained with Hoechst (in blue). In C, the upper left photo shows a grafted embryo prior to cryosection. In D, right panels are enlargements of the lower left panel. Scale bar: 200 µm.

E. Tumor take rate of 13 melanoma patient samples engrafted in a series of chick embryos, ranked according to their assigned Stage (I to IV).

F. 3D views (light-sheet imaging) of HH25 chick embryos engrafted with different melanoma patient samples, showing different patterns of tumor cell localization 50 hours post-engraftment.

G. Immunofluorescent labeling of HH25 chick embryo sections 50 hours post-engraftment of OF-MEL-020 patient sample, labeled with CFSE (in green) prior to the graft. An anti-SOX10 antibody (in red) and an anti-MITF antibody (in yellow) were used. Nuclei were stained with Hoechst (in blue). Right panels are enlargements of the dotted area in the left panel.

Data information: So: Somite; NT: Neural Tube; No: Notochord; DRG: Dorsal Root Ganglia; SG: Sympathetic Ganglia; Coe: Coelom
An in vivo avian model of human melanoma to perform rapid and robust preclinical studies (2022)
Figure 2

Figure 2 - Melanoma cells retain their phenotypic traits and associated response to targeted therapies in the AVI-PDXTM model.

A,B. Immunofluorescent labeling (A) of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody, an anti-MITF antibody (pink) and an anti-SOX10 (white) antibody. Nuclei were stained with Hoechst. Right panels are enlargements of the dotted area in left panel. In B, the mean fraction of MITF-negative (MITFneg), -low (MITFlow) and -positive (MITFpos) A375P::GFP cells in the tumor mass and in migrating cells was quantified (n = 16 slices from 3 embryos). Error bars show SEM. Mann-Whitney test comparing tumors vs migrating cells for each class of MITF expression was performed, p-values are indicated on the graph.

C,D. Immunofluorescent labeling (C) of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody and an anti-Ki67 antibody. Nuclei were stained with Hoechst. Right panels are enlargements of the dotted area in the left panel. In D, the mean fraction of Ki67-positive (Ki67pos) A375P::GFP cells in the tumor mass and in migrating cells was quantified (n = 25 slices from 3 embryos). Arrows point at cells having a mesenchymal morphology. Error bars show SEM. Student t-test, **p = 0.0070.

E. RNASeq analysis of GLO and GLO-R cells; gene signatures published in (Verfaillie et al, 2015), associated with either a proliferative or an invasive melanoma phenotype were scored in both cell lines. P-values are indicated in the graphical representation. F,G. 3D views (light-sheet imaging, F) of HH25 chick embryos engrafted with GLO or GLO-R cells labeled with CFSE (in green) prior to the graft. The total volume occupied by tumor cells and the number of segmented CFSE-positive objects was quantified in G to calculate a mean compaction index (see details in the Materials and Methods section) of tumors for each cell line. Each dot represents a tumor analyzed in a different embryo. Bars and error bars indicate mean + SEM. Mann-Whitney test, p < 0.0001.

H-K. 3D views (H, J) and quantification of tumor volumes (I, K) of HH25 chick embryos engrafted with GLO (H, I) or GLO-R (J, K) cells and treated with a combination of Vemurafenib and Cobimetinib, or Vorinostat alone, or a combination of the three molecules. The number of embryos analyzed for each experimental condition are indicated on the graphs. Bars and error bars indicate mean + SEM. Student t-tests, **: p < 0.01, ns: not significant. Exact p-values are indicated on the graphs.
An in vivo avian model of human melanoma to perform rapid and robust preclinical studies (2022)
Figure 3

Figure 3 - The AVI-PDXTM paradigm efficiently models patient clinical response to targeted therapies.

A,B. 3D views (A) and quantification of variations in the mean tumor volume (B) of HH25 chick embryos engrafted with BRAFwt (OF-MEL-028) or BRAFV600E (OF-MEL-020, OF-MEL-027) patient samples and treated with excipient or a combination of Vemurafenib and Cobimetinib. The number of embryos analyzed for each patient sample is indicated on the graphs. Student t-test for OF-MEL-020; Mann-Whintey test for OF-MEL-028, *: p < 0.05, ns: not significant. Exact p-values are indicated on the graphs.

C,D. Survival rate (left axis) and mean body surface area (BSA, right axis) of chick embryos injected with increasing doses of Dabrafenib (C) and Trametinib (D). The maximum tolerated dose (MTD, in red on the X-axis) was defined as the higher dose of drug associated with a survival rate higher than 75% and a mean BSA similar (i.e., non-statistically different) from embryos treated with excipient (17.1 and 0.0228 mg/kg respectively). N=10 replicates per experimental group. Error bars indicate SEM. ns, non-significant using Student's t-test compared with excipient.

E-H. 3D views (E) and quantification of tumor volumes (F-H) of HH25 chick embryos engrafted with 3 different patient samples (OF-MEL-033 (F), OF-MEL-034 (G), OF-MEL-035 (H)) and treated with excipient or a combination of Dabrafenib and Trametinib. The clinical response of each patient after a 3 months treatment with Dabrafenib/Trametinib is indicated above the graphs. Error bars indicate SEM. Mann-Whitney tests, exact p-values are indicated on the graphs.
Miz1 is required to maintain autophagic flux (2013)
f1

(a) Miz1ΔPOZNes mice move slowly and lack motor coordination. The panel shows the average speed of movement and time on a rotarod (maximum: 300 s) maintained at constant speed of control and age-matched Miz1ΔPOZNes mice. The average age of mice tested was 16 months. P-values were calculated using an unpaired two-tailed Student’s t-test (the number of animals tested is shown below each graph). (b) Defect of cerebellar growth in Miz1ΔPOZNes mice. The panels show sagittal sections of brains of control and Miz1ΔPOZNes mice at the indicated age. Dotted lines show the circumference of the cerebellum. For each mouse, the cerebellar surface area was determined; numbers show the ratio between control and Miz1ΔPOZNes mice. The number of mice analysed for each genotype is indicated on the right. Scale bar, 1 mm. (c,d) Histology of the cerebellum. Sections of the cerebellum of 11-month-old control (c) and Miz1ΔPOZNes (d, please note different scale) mice were stained with antibodies directed against calbindin, a marker for PCs, and glial fibrillary acid protein (GFAP), a marker for Bergman glial cells. Boxes indicate the area of a detail picture in the overviews. GFAP staining is shown for adjacent section (arrowheads: regions of missing PCs). Scale bar, 500 μm (upper panels) and 100 μm (lower panels). (e) Enhanced apoptosis in the granular layer of cerebella in Miz1ΔPOZNes mice. The upper panel shows representative TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) staining of cerebella of age-matched control and Miz1ΔPOZNes mice. The lower panel shows a quantification of staining observed in four control and five Miz1ΔPOZNes mice. Error bars show s.e.m. P-value was calculated using an unpaired two-tailed Student’s t-test.
Miz1 is required to maintain autophagic flux (2013)
f2

(a) ISH documenting the expression of Miz1 mRNA in PCs of the cerebellum in wild-type mice. Left panel shows ISH, right panel shows haematoxylin and eosin staining. Scale bar, 50 μm. (b) IHC documenting the presence of Miz1 in the nuclei of PCs. The panel on the left shows staining with α Miz1 (H190) antibody, staining on the right is a control staining lacking primary antibody. Scale bar, 50 μm.