EMBO/sd-character-level-ner huggingface dataset:
Nup50 and MEL28 do not depend on each other for chromatin binding (A, B) Demembranated sperm chromatin was preincubated in Xenopus egg extract, depleted for MEL28/ELYS or Nup50 (B) or control treated (Mock, A).Figure 3(A, B) Demembranated sperm chromatin was preincubated in Xenopus egg extract, depleted for MEL28/ELYS or Nup50 (B) or control treated (Mock, A). After 10 min, membranes were added to the reaction. Reactions were stopped at the indicated time points by fixation and analyzed by confocal microscopy after immunostaining with α-MEL28/ELYS (A), α-Nup50 (B), and mAb414 (A, B). Chromatin was stained with DAPI (blue in the overlay). Scale bar: 10 μm.
Figure 6(A) Distribution of ZFP207 binding sites across common transcript features. (B) Scatterplot depicting ranked distribution of ZFP207 binding sites. The top 5 strongest binding sites are highlighted. (C) Venn diagram of in vitro RIP-seq and AS upon Zfp207 knockdown. (D) Bar- and dot plots show significantly enriched GO terms for biological processes for ZFP207 bound genes. Top 30 GO terms are shown. (E) FISH of poly(A)+ RNA distribution in shScr, sh1 and sh2 stained with 5´Cys3-oligo dT(50). Nuclei were counterstained with DAPI. Scale bar, 20 µM. Quantification of (F) nuclear, (G) cytoplasmic, and (H) of nuclear/cytoplasmic poly(A)+ FISH signals. (I) Relative nuclear and cytosolic total RNA levels in sh1 and sh2 compared to shScr. Data are presented as mean ± SEM of n ≥ 3 independent biological experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001 (shScr vs. sh1 or sh2); unpaired Student's t-test. J. ZFP207 modulates mouse ESCs pluripotency and neuroectodermal differentiation by integrating co- and post-transcriptional mechanisms.
Profiling of the breast cancer cell-line panel according to ATRA sensitivity.Figure 1A panel of 42 breast cancer cell lines was challenged with increasing concentrations of ATRA (11 nM-10 μM) for 3, 6, and 9 days, and cell growth was determined.The graphs show the growth-inhibitory effect exerted by the indicated concentrations of ATRA in SKBR3, HCC-1954, and MDA-MB436 cells which are representative of lines characterized by a high, intermediate and low ATRA score, respectively. Each point is the mean ± SD of three replicate cultures. **Significantly lower than the corresponding vehicle-treated group (P-value < 0.01, Student's t-test). Cell lines are ranked in ascending order according to the ATRA score. The plot distinguishes four separate groups of cell lines (D-A) with increasing ATRA scores, as indicated by the colored scale on the right. The cell lines are also grouped in tertiles, T1-T3, according to an ascending ATRA score. Each calculated value is representative of at least two independent experiments.
Loss of gba Impairs Both Autophagic and Proteasomal Degradation Machinery.Figure 1(A) Autophagic markers from mixed midbrain gba+/+, gba+/−, and gba−/− cultures were analyzed via western blotting using antibodies as indicated. (B) Autophagic flux was analyzed in gba+/+ and gba−/− midbrain neurons. Neurons were treated with 100 nM bafilomycin A1 and 1 μM rapamycin and flux assayed by comparing LC3I and LC3II levels via western blotting. β-actin was used as a loading control.(C) Densitometry analyzes of autophagy of (B) expressed as a ratio of LC3II/LC3I. Error bars, ± SEM. *p < 0.05. (D) Accumulation of p62/SQSTM1 was analyzed via immunoblotting. β-actin was used as a loading control. (E) Midbrain neurons were treated with either 10 μM MG132 or vehicle control DMSO. Proteins were extracted and analyzed via western blotting and stained with anti-ubiquitin antibodies. (F) Ubiquitin linkage of isolated midbrain analyzed using western blotting with antibodies as indicated. β-actin was used as a loading control. (G) Proteasomal activity (chymotrypsin activity) was measured using the Proteasome Glo assay where activity is proportional to the released luciferase. RFU, relative luciferase units. Data represent the mean ± SEM (n = 3, each experiment contained triplicates for each genotype). *p < 0.05.
Figure 3A. Representative time-lapse spinning disk confocal images of n = 3 live COS7 cells stably expressing mCherry-KDEL (green) and transiently expressing GFP-RAB7 (orange). Zoom insets shows a region of interest containing a mobile RAB7-positive endosome as denoted by the white arrows. First row represents the ER (mCherry-KDEL) (green), the second row shows MDA-generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and GFP-RAB7 (orange). Scale bar: 10µm, zoom insets 3µm. Images related to movie EV11. Images were acquired for 90 frames at 1Hz. B. Kymograph of panel (A). C. MDA quantification of panel (A) and figures EV2A-G normalized to 1 showing average ER movement surrounding static endosomes and mobile endosomes. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=15 cells per condition from 3 independent experiments). All images acquired for 90 frames at 1Hz. Significance two-tailed student t-test. *P<0.05, ** P<0.01, **** P<0.0001 ns = not significant. D. Violin plots of fractional distance analysis showing the average distribution of endosomes in cells from panel (E) and fig. EV3A. Graph represents mean ±s.d. of n=15 cells per condition from 3 independent experiments. Dashed lines represent median and solid lines represent interquartile range. Cartoons representing the overexpression and control LE/Ly phenotype. Significance two-tailed student t-test. **** P<0.0001 E. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP-TMD (green) and in lower panels transiently expressing mScarlet-RILP (magenta) and SiR-lysosome-stained LE/Lys (orange). Zoom insets shows region of interest containing ER (GFP-TMD). First row represents ER (GFP-TMD) (green), the second row shows MDA-generated ER movement (magenta), third row shows a merge of the two preceding channels. Scale bar: 10µm, zoom insets 3µm. Images related to movie EV19 and movie EV20. Images were acquired for 90 frames at 1Hz. F. MDA quantification of panel (E) and figure EV3A normalized to 1 showing total average ER movement. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=15 cells per condition from 3 independent experiments). All images acquired 90 frames at 1Hz. Significance two-tailed student t-test. **** P<0.0001 ns = not significant.
hnRNPA2 interactions and neurodegeneration are altered by tyrosine phosphorylation.Figure 7hnRNPA2 interacts with transport granule components hnRNPF and ch-TOG in the phase separated state. Disease mutations (X) D290V and P298L induce aggregation of hnRNPA2 in vitro, while D290V induces tdp-1-dependent neurodegeneration in a C. elegans model. hnRNPA2 LC tyrosine phosphorylation alters hnRNPA2 LC LLPS in vitro, prevents interaction with hnRNPF and ch-TOG, reduces aggregation in vitro, and expression of an activated tyrosine kinase reduces D290V-associated neurodegeneration in the C. elegans model.
Fig.Figure 6a, iPS-derived 14-day old organoid displaying complex morphological structures of brain-like tissues, parenthesis. Scale bar 500 μm. The ventricular zone (VZ) with radial glial cells stained with nestin (magenta) and neurons stained with TUJ1 is shown at higher magnification. Scale bar 20 μm. b, Reduced Nestin/Pax-6-positive progenitors and increased TUJ1-positive neurons in Seckel organoids. In ventricular zones of WT organoids, there are significantly more Pax6 positive cells than TUJ1 positive cells, which is not the case in Seckel organoids (*p<0.05). TUJ1/Pax6 ratio in graph on the right shows significantly more TUJ1 positive cells vs. Pax6 cells in Seckel organoid ventricular zones (* p<0.05). Error bars are s. e. m. Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 10 WT organoids (2520 cells). Seckeln = 10 organoids (2024 cells). Scale bar 20 μm. c, (i) Seckel organoid tissues contain increased numbers of cilia with an obvious observation of enlarged lateral ventricular lumen (* p<0.05). Graph at far right shows an increased lumen area as well as thinner ambient VZ in Seckel organoids. (*p<0.05). Error bars are s. e. m. t-test. n=6 for WT organoid and 6 for Seckel organoid. Scale bar 20 μm. (ii) Seckel organoid cells display longer cilia. Error bars are s. e. m. *** p<0.001, t-test. n=3 for WT organoids and 4 for Seckel organoids. Scale bar 1 μm. d, Seckel organoids electroporated with constructs expressing GFP alone (i), GFP with CC5-GFP and (ii) GFP with CPAP-GFP (iii). Middle panel shows staining with anti-GFP antibodies (red) to show the transfected area. Note the presence of increased Pax6-positive (magenta) NPCs in CC5 and CPAP electroporated tissues compared to GFP control. Likewise, there is a larger neural epithelium with multiple RGs (arrowheads). Bar diagrams at right shows rescue effects of CPAP and CC5 electroporation compared with GFP alone (iv) lower TUJ1/Pax6 ratio (**p<0.01 and ***p<0.001 respectively) (v) smaller lumen diameter (**p<0.01) and (vi) shorter cilia length (**p<0.01 and ***p<0.001 respectively). Error bars are s. e. m. One-way ANOVA followed by Dunnett's multiple comparisons test. n = 3 organoids with 225 cells for GFP, 537 cells for CPAP and 569 for CC5. At least 10 ventricles from 3 independent organoids were used to measure the CPAP diameter. t test. Error bars are s.e.m. Scale bar 20 μm.e, Rescue effect of CC5 and CPAPelectroporation on cleavage planes of phospho-vimentin-positive RGs in Seckel organoids. Compared to GFP control, electroporation of CC5 or GFP increases the proportion of horizontally orientated SeckelRGs (for CC5 ***p<0.001) and decreases the portion of obliquely (for CC5 *p<0.05) or vertically oriented SeckelGFP (for CPAP and CC5 **p<0.01). Error bars are s.e.m. Two-way ANOVA followed by Tukey's multiple comparisons test. n = 4 organoids. 100 cells for GFP control, 112 cells for CPAP and 99 cells for CC5.
Figure 1A The family pedigree, where circles denote female members, squares male members, solid symbols affected members, and white symbols asymptomatic members with normal physical exam; the dots indicate heterozygous carriers and double line denotes a consanguineous marriage. The pictures show scapular winging which is a consistent clinical sign in affected individuals. B Hematoxylin and eosin staining (H&amp;amp;E) of skeletal muscle from patient II.1 shows histological features of moderate to severe dystrophic pattern. (Scale bar, 50 μm). C T1-weighted MRI axial images at thigh and calf levels show that the fatty degeneration is more prominent in thigh muscles, equally affecting posterior and anterior compartment, with relative sparing of the rectus femoris, sartorius and gracilismuscles until late stages (4, 10 and 11, respectively). Strikingly, the fatty tissue is located in the internal parts of almost all the affected muscles in thigh (1, 2, 3, 5-9), while the external regions are spared. At calf level, only the gastrocnemius medialis muscle (12) shows this pattern, while the soleus (13) is diffusely involved. Patient II.2 (PII.2) shows late-stage thigh muscles with an unusual involvement of the tibialis posteriormuscle (14) in the lower leg.
The yeast growth phenotype is a readout for the kinase phosphotransferase activity.Figure 2A. Statistical comparison of the number of yeast growth phenotypes between kinase group and control protein group. In each strain, across all conditions, the percentage of kinases that cause a phenotype upon exogenous expression is significantly higher than the percentage of control proteins, than cause a growth phenotype upon overexpression. Data are from a single large-scale growth measurement. The boxes extend from 1st to 3rd quartile with the central band representing the median. The whiskers extend to the furthest point up to 1.5 times the interquartile range away from the nearest quartile, with any further points marked as outliers. P-values are calculated using the Mann-Whitney test. B. Growth comparisons of selected kinases with the kinase‑dead mutant match. 24h growth curves (OD595) from liquid culture of yeast strains (W303) expressing human kinase in the presence or absence of 3.2% (v/v) DMSO. Upper panel: BUB1: BUB1 wild type, BUB1-KD: BUB1(K821M); upper middle panel: NEK6: NEK6 wild type, NEK6-KD: NEK6(K74M&K75M); lower middle panel: PKCα-ac: PKCα(A25E), PKCα-KD: PKCα(K368R); lower panel: Control with empty plasmid. Error bars represent SD from three biological replicates. C. Human protein kinases phosphorylate large sets of yeast proteins. Upper panel: In total, 27 human protein kinases showed activity towards yeast proteins visible on western blots through enhanced reactivity with phospho-motif antibodies on yeast proteins in comparison to other human kinases, kinase‑dead mutant versions or vector control samples. Commercially available pS/T-antibodies with relatively low sequence specificities were chosen to enable detection of phosphorylation of the yeast proteome by human kinases. Lower panel: Examples of western blots with equal amount of whole yeast cell lysate from strains expressing human kinases loaded. When developed with the indicated phospho-motif recognizing antibodies, immunoreactive bands indicated phosphorylation of yeast proteins.
Adipocyte EV modify melanoma mitochondrial dynamics, a process that promotes melanoma aggressiveness and is exacerbated by obesity A) 1205Lu cells exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1, were stained with a MitoTracker probe, fixed and observed by confocal microscopy.Figure 51205Lu cells exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1, were stained with a MitoTracker probe, fixed and observed by confocal microscopy. Arrows indicate the presence of mitochondria in membrane protrusions. On the images to the right, the outline of cells is shown in dotted lines. Scale bars: 20µm. Right panel, quantification of the distance between mitochondria and nuclei (n=15). Western blot analysis of the indicated mitochondrial fission proteins in melanoma cells treated, or not, with EV from 3T3-F442A adipocytes (3T3-EV). Tubulin (TUB) is used as a loading control. 1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1. Cell migration was then evaluated in Boyden chamber assays (left panel; n=6). Right panel, 3T3-F442A EV were added to 1205Lu cells, which were immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group) (right panel). 1205Lu cells were transfected, or not, with 2 different siRNA targeted against DRP1 (siDRP1#1 or 2) or an untargeted siRNA (siUT). Top, 48h after transfection, protein extracts were prepared and DRP1 expression was evaluated by western blot. Bottom, 36h post transfection, cells were exposed to 3T3-F442A EV (3T3-EV) and cell motility was tracked by video microscopy (n = 40 cells per group). 1205Lu cells were exposed to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) and immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group).
Transport of Cvt complex in SEY6210 (wild-type) harboring 2μ plasmid encoding API under growing and nitrogen starvation conditions.Figure 4Transport of Cvt complex in SEY6210 (wild-type) harboring 2μ plasmid encoding API under growing and nitrogen starvation conditions. Vegetative cells grown in SD medium were observed (A-E). For starvation, logarithmically growing cells in SD were transferred to SD(-N) for 25 min (F-H) or 1 h (I-K) in the presence of 1 mM PMSF. Samples were prepared for electron microscopy as described in Fig. 1. (A) Freeze-substitution fixation image of Cvt vesicle (marked with an arrow). (B) Immunostaining of a Cvt vesicle (marked with an arrow). (C and D) Higher magnification image of Cvt vesicle. Arrowhead and double arrowheads show the inner and outer membrane of a Cvt vesicle, respectively. (E) Freeze-substitution fixation image of a membrane sac enclosing small portion of a large Cvt complex (marked with an arrow). (F) Freeze-substitution image of the wrapping of a Cvt complex by the isolation membrane (marked with an arrow). (G) Freeze-substitution image of a Cvt vesicle (marked with an arrow) fusing to a vacuole. (H) Freeze-substitution image of autophagic body containing a Cvt complex. (I) Immunostaining image depicting the wrapping of a Cvt complex. The arrow marks the enwrapping membrane. (J) Immunostaining of an autophagosome containing a Cvt complex. (K) Immunostaining of an autophagic body containing a Cvt complex in the vacuole. V, vacuole. Transport of Cvt complex in SEY6210 (wild-type) harboring 2μ plasmid encoding API under growing and nitrogen starvation conditions. Vegetative cells grown in SD medium were observed (A-E). For starvation, logarithmically growing cells in SD were transferred to SD(-N) for 25 min (F-H) or 1 h (I-K) in the presence of 1 mM PMSF. Samples were prepared for electron microscopy as described in Fig. 1. (A) Freeze-substitution fixation image of Cvt vesicle (marked with an arrow). (B) Immunostaining of a Cvt vesicle (marked with an arrow). (C and D) Higher magnification image of Cvt vesicle. Arrowhead and double arrowheads show the inner and outer membrane of a Cvt vesicle, respectively. (E) Freeze-substitution fixation image of a membrane sac enclosing small portion of a large Cvt complex (marked with an arrow). (F) Freeze-substitution image of the wrapping of a Cvt complex by the isolation membrane (marked with an arrow). (G) Freeze-substitution image of a Cvt vesicle (marked with an arrow) fusing to a vacuole. (H) Freeze-substitution image of autophagic body containing a Cvt complex. (I) Immunostaining image depicting the wrapping of a Cvt complex. The arrow marks the enwrapping membrane. (J) Immunostaining of an autophagosome containing a Cvt complex. (K) Immunostaining of an autophagic body containing a Cvt complex in the vacuole. V, vacuole.
Mitotic GSCs and CBs exhibit non-overlapping old versus new histone H3 patterns, but overall symmetric old and new segregation patterns.Figure 2(A-C) Telophase GSCs marked by H3S10p (gray) expressing old (green) and new (red) histone H3 show non-overlapping patterns (A). Telophase GSCs expressing old (green) and new (red) histone H4 show moderate levels of overlap (B). GSCs expressing old (green) and new (red) histone H2A show high levels of overlap (C). (D) A control where the old and new fluorescent tags have been switched shows the non-overlapping patterns of old (red) versus new (green) H3Rev histone in mitotic telophase GSCs marked by H3S10p (gray). (E) Quantification of log2 ratios of total old and new histone inherited by each future daughter cell of the GSC division, where a value of 0 is equal inheritance at exactly a 1:1 ratio. Data gathered over 9 separate experiments for H3Rev GSCs, H2A GSCs, and H4 GSCs. Data gathered over 16 separate experiments for H3 GSCs. Values are mean + 95% CI. (F-H) Telophase CBs marked by H3S10p (gray) expressing old (green) and new (red) histone H3 also show non-overlapping patterns. (F) Telophase CBs expressing old (green) and new (red) histone H4 (G) and H2A (H), which depict more overlap like GSCs. (I) A control with switched old and new fluorescent tags shows the non-overlapping old old (red) versus new (green) H3Rev histone patterns in mitotic telophase CBs marked by H3S10p (gray). (J) Quantification of log2 ratios of total old and new histone inherited by each future daughter cell of the CB division, where a value of 0 is equal inheritance at exactly a 1:1 ratio. Data gathered over 9 separate experiments for H3Rev CBs and H2A GSCs. Data gathered over 12 separate experiments for H4 CBs and H3 CBs. Values are mean + 95% CI.
Figure 8A. Schematic diagram of the I-PpoI system for inducing DNA breaks in the nuclear human genome. Addition of 4-OHT to U-2-OS-pEP15 cells stably expressing the I-PpoI endonuclease fused to the estrogen receptor (ER) induces nuclear translocation of the fusion protein and cleavage cleavage of FokI recognition sites in nuclear DNA resulting, leading to DSB induction. B, C. Quantitative PCR with reverse transcription (qRT-PCR) analysis of SLCO5a1 (B) and RYR2 expression levels (C) (left panels) U-2-OS HA-ER-I-PpoI cells depleted of CDKL5 transiently transfected with FLAG-tagged CDKL5 wild type (WT) or a K42R-mutated kinase dead (KD) mutant, or empty vector, at the times indicated after inducing I-PpoI. The mean ± SD from two qPCR replicates of two independent experiments is shown. Statistical significance for all the data was assessed by two-way-ANOVA-test *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001; ns - not significant. I-PpoI-mediated cutting efficiency in the relevant gene is shown in the right-hand panel D. Schematic diagram depicting CDKL5 functions in nucleus and cytosol.
Tyrosine phosphorylation of hnRNPA2 LC alters LLPS and prevents partitioning of hnRNPF PLD or TOG D1.Figure 3A) Concentration of protein remaining in the supernatant after centrifugation, which is an inverse measure of phase separation, changes with salt concentration. hnRNPA2 LC shows low phase separation (high protein remaining in the supernatant) at low salt conditions and near complete phase separation (no protein remaining in the supernatant) at high salt concentrations. In contrast, tyrosine phosphorylated hnRNPA2 LC shows higher phase separation (less protein remaining in the supernatant) at low salt concentrations than at high salt concentrations. Conditions: 20 µM protein, 20 mM MES pH 5.5 150 mM urea, salt concentration as indicated, 25˚C. Error bars are standard deviation of three replicates. B) While hnRNPA2 LC shows no droplets in low salt conditions and droplets in high salt conditions, tyrosine phosphorylated hnRNPA2 LC shows droplets in all salt concentrations tested, although more droplets are present at low salt conditions. Conditions: 20 µM proteins, 20 mM MES pH 5.5 150 mM urea with 0 mM, 50 mM, or 500 mM NaCl as indicated. Scale bar: 10 µm. C) Fluorescence micrographs showing that although TOG D1 and hnRNPF PLD partition into hnRNPA2 LC droplets (rows 1 and 3), they are unable to partition into tyrosine phosphorylated hnRNPA2 LC droplets (rows 2 and 4). Conditions: 20 µM proteins (~1% fluorescently labeled), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. D) Phosphomimic variants containing 5 or 12 serine to glutamate substitutions (hnRNPA2 LC5E and hnRNPA2 LC12E, respectively) both allow partitioning of TOG D1 and hnRNPF PLD, even though hnRNPA2 LC12E barely phase separates at these conditions, indicating that increased negative charge is insufficient to prevent partitioning of TOG D1 and hnRNPF PLD. See Appendix Figure S7B for quantification of LLPS of hnRNPA2 LC5E and hnRNPA2 LC12E. Conditions: 20 µM proteins (~1% fluorescently labeled), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm.