EMBO/sd-character-level-ner huggingface dataset:
Figure 5A. HEK293 cells were co-transfected with CDKL5NLS (wild type "WT" or kinase-dead "KD" K42R mutant) and either FLAG-EP400 (left), FLAG-ELOA (middle) or FLAG-TTDN1 (right). 24 h later cells were incubated with H2O2 (500 µM) for 15 min before being harvested and lysed. Protein extracts were subjected to immunoprecipitation with anti-FLAG-agarose beads. Precipitates were subjected to SDS-PAGE and blotting with antibodies shown (bottom panels) or staining with Coomassie Brilliant Blue (top panels). The bands corresponding to the FLAG-tagged proteins were excised from the gels in A. and processed for mass spectrometric detection of relevant phospho-peptides. Three independent co-transfection experiments were done for every condition. B. Boxplots showing VSN-normalised intensity of phospho-peptides corresponding to EP400 pSer729, ELOA pSer311and TTDN1 pSer40 from the experiment in A. The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. In all cases the data was derived from 3 biological replicates. C. Boxplots of the VSN-adjusted TMT reporter ion intensities for all peptides for each TMT label in the case of FLAG-EP400, FLAG-ELOA and FLAG-TTDN1 from the experiment in A.The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. Datapoints further removed than the whiskers are plotted individually. The experiment was conducted using three biological replicates within each respective group, each TMT channel represents a single biological replicate. D. Left: Anti-FLAG precipitates from HEK293 cells transiently expressing FLAG-tagged CDKL5 (wild type "WT" or a K42R kinase-dead "KD" mutant) were incubated with the synthetic peptides indicated, in the presence of [γ-32P]-labelled ATP-Mg2+ and peptide phosphorylation was measured by Cerenkov counting. Data are represented as mean ± SEM from three independent experiments. Right: Same but anti-FLAG precipitates were subjected to SDS-PAGE and autoradiography to detect CDKL5 autophosphorylation, or western blotting with CDKL5-pTyr171 antibody specific for the CDKL5-Tyr171 autophosphorylation site
Bcl-2 promotes SC-driven tumour initiation.Figure 7A Schematic of tumour experiment timeline in K15ΔNLef1 genetic background. B,C Tumour incidence (B) and frequency (C) in Ctr, Bcl-2EOE, K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice (n=10 biological replicates; mean ± standard error of mean (SEM)). D Histology of sebaceous tumours of K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice. Scale bar, 100µm.
Figure 6Mice from cohorts 1-3 were tested in the MWM at the indicated ages. (A-D, M, N) Learning curves of mice in cohorts 1 (A-D) and 3 (M, N). Data at day 0 are from the first trial on day 1. Data from cohort 2 are shown in EV5B-G. Compared with NTG controls, only hTau-A152T(L1) mice showed significant learning impairments at 4-6, 17-19 (cohort 1, P < 0.001) and 17-21 (cohort 3, P < 0.01) months of age by Cox proportional hazards model analysis with mixed effects and Holm adjustment for the following comparisons for each age range: NTG versus CaMKII-tTA, NTG versus TRE-hTau-A152T(L1), and NTG versus hTau-A152T(L1) for cohort 1, and NTG versus CaMKII-tTA, NTG versus hTau-WT(L32), NTG versus hTau-A152T(L1), and hTau-WT(L32) versus hTau-A152T(L1) for cohort 3. (E-L, O-V) Performance of mice from cohorts 1 (E-L), 3 (O-R) and 1-3 combined (S-V) in a probe trial (platform removed) 24 h after the last training trial. (E-H, O, P, S, T) Latency to reach original platform location. (I-L, Q, R, U, V) Number of times mice crossed the original platform location (black bars) or the average of their crossings of corresponding locations in the three other quadrants (white bars). Cohort 1: n = 12 (A-L) NTG; 13 (A, B, E, F, I, J), 11 (C, G, K), or 9 (D, H, L) TRE-hTau-A152T(L1); 11 (A, B, E, F, I, J), 10 (C, G, K), or 9 (D, H, L) CaMKII-tTA; and 14 (A, B, E, F, I, J), 13 (C, G, K), or 12 (D, H, L) hTau-A152T(L1) mice. Cohort 3: n = 17 (M-R) NTG; 12 (M, O, Q) or 11 (N, P, R) CaMKII-tTA; 13 (M, O, Q) or 12 (N, P, R) hTau-WT(L32); and 10 (M-R) hTau-A152T(L1) mice. Cohorts 1+2+3: n = 47 (S, U), or 39 (T, V) NTG; 11 (S, U) or 9 (T, V) TRE-hTau-A152T(L1); 34 (S, U) or 31 (T, V) CaMKII-tTA; 13 (S, U) or 12 (T, V) hTau-WT(L32); and 43 (S, U) or 36 (T, V) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by Cox proportional hazards/mixed effects model analysis (A-D, M, N), one-way ANOVA with post-hoc Tukey (E-H, O, P) or Dunnett's (S, T) test, or one-tailed paired t test with Holm adjustment (I-L, Q, R, U, V). Values are means ± SEM.
Nup50 is a chromatin binding protein 3 µM recombinant EGFP, EGFP-tagged MEL28/ELYS (aa 2290-2408) or EGFP-tagged Nup50 were incubated with empty or DNA-coated magnetic beads, which were chromatinized with Xenopus egg extracts (right panel) or unchromatinized DNA-beads (left panel).Figure 43 µM recombinant EGFP, EGFP-tagged MEL28/ELYS (aa 2290-2408) or EGFP-tagged Nup50 were incubated with empty or DNA-coated magnetic beads, which were chromatinized with Xenopus egg extracts (right panel) or unchromatinized DNA-beads (left panel). After 3h the beads were re-isolated, washed, co-stained with DAPI and analyzed by confocal microscopy. Scale bar: 10 µm.
Figure 4A. Immunofluorescence microscopy was performed using the indicated antibodies in hTERT-RPE-1 cells treated with control, PCM1 or Plk4 siRNA. Regions around the basal body (insets) are enlarged on the right hand side. Scale bars, 5 μm (left) and 1 μm (right).B. Quantification. At least 150 cells were counted (n=3). Statistical analysis was performed using two-tailed unpaired student's t-tests. ***p<0.001, ****p<0.0001. C. hTERT-RPE-1 cells treated with PCM1 siRNA were transfected by an empty vector (EV) or plasmids producing PCM1-WT*, -S327A* or -S372E*, and immunofluorescence microscopy performed. Enlarged images of the regions around the basal body are shown in insets. Scale bars, 5 μm and 1 μm (inset). D. Quantification. At least 200 cells were counted (n=3). Statistical analysis was performed using two-tailed unpaired student's t-tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. not significant. E. Model. Plk4 plays a decisive role in not only centriole duplication but also centriolar satellite integrity and primary cilium formation. The latter two roles, at least in part, are executed via PCM1 phosphorylation. Plk4 directly or indirectly phosphorylates PCM1 at S372 (I), which promotes its own dimerisation/oligomerisation (II) and interaction with other components of centriolar satellites (III). These processes ensure PCM1 acts as a structural platform for centriolar satellites. Processes II and III are not necessarily sequential steps. It is shown that centriolar satellites and PCM1 play a critical role in centriole duplication and assembly [25, 49]. Under conditions of serum starvation or developmental cues, centriolar satellites deliver various factors from the cytoplasm to the basal body, thereby helping formation of cilia [26, 30, 37, 50, 51].
Figure 1(A) Heatmap showing the altered metabolic pathways in PKM2 KO MCF7 and MDA-MB-231 cells relative to the parental cells (n=4). Fatty acid metabolism (Acyl Carnitine) and long chain fatty acid pathways were significantly changed in both cell lines. (B) Heatmap showing the differential fatty acid β-oxidation pathway metabolite levels between PKM2 KO MCF7 and MDA-MB- 231 cells, and their respective parental counterparts (n=4). (C) Decreased levels of free carnitine and modified carnitine species in PKM2 KO cell lines as compared with their parental counterparts (n=4). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. n.s means no significance. (D) Heatmap showing the decreased levels of long chain fatty acids in PKM2 KO cell lines relative to their parental counterparts (n=4). (E) Increased acetyl-CoA in PKM2 KO MCF7 and MDA-MB 231cells as compared with their parental counterparts (n=4). (F) Decreased total cholesterol in PKM2 KO MCF7 and MDA-MB 231cells as compared with their parental counterparts (n=3). ( Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. n.s means no significance. (G) Confocal imaging of neutral lipid droplets stained with BODIPY493/503 or Nile red dye in PKM2 KO cells relative to the parental cells. Data represent one of three independent experiments with similar results. Scale bar, 25μm.
Figure 4(A - C) Fluorescence images (upper) and quantifications (lower) of GFP-tagged CST-1 (A  B) or GFP-labeled F-actin (C) and mCherry tagged plasma membrane and histone in WT and mig-2 null mutant animals. The membrane protrusions in the white boxes are enlarged 3 times at the bottom. Yellow dashed lines indicate the cell periphery. White lines indicate the line to measure the fluorescence intensity. Green lines denote the CST-1 fluorescence intensity; red lines denote the plasma membrane fluorescence intensity; double-headed arrows denote the distance between the GFP signal and cell periphery. Scale bars, 5 μm. (D) Distance between GFP::CST-1 or GFP::Moesin ABD and the plasma membrane at the leading edge in WT and mig-2 null mutants. Error bars indicate SD. N = 9 - 14. ***, P  0.001 by Student's t test. (E - F) Fluorescence images (left) and quantifications (right) of GFP-tagged CST-1 and mCherry tagged plasma membrane and histone in WT and mig-2 null mutant animals. Blue lines, sites of measurements; dotted lines, cell periphery; Green lines; CST-1 fluorescence intensity; red lines, mCherry fluorescence intensity. Scale bars, 5 μm. (G) Quantification of the fluorescence intensity ratio of GFP::CST-1 and mCherry-membrane between the leading edge and the lagging edge of AQR in WT and mig-2 null mutant animals. Error bars indicate SD. N = 20. ***, P < 0.001 by Student's t test. (H) Proposed model of Hippo-RhoG signaling negative feedback loop in the prevention of secondary fronts during directional cell migration.
Figure 2(A) Representative images of WT and Mfn2 KO MEFs co-transfected with plasmids encoding mitochondria-targeted RFP and ER-targeted GFP. Scale bar: 5 μm. (B) After 48 h, the cells were fixed and the degree of colocalization between ER and mitochondria based on red and green fluorescent pixel overlap was analyzed using Mander's coefficient (n= 30 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM. *p<0.05, two-tailed Student's t-test. (C) Representative EM micrographs of WT and Mfn2 KO cells. Scale bar: 250 nm. (D) Average of ER-mitochondria distance (n= 111-168 mitochondria). *p<0.05, Mann-Whitney test. Data are presented as mean ± SEM. (E) Quantification of the percentage of mitochondria in contact with the ER distributed at the indicated distance range (n= 3 independent experiments in which were analyzed 627-779 mitochondria from 68-98 cells). Data are presented as mean ± SEM. *p<0.05, two-tailed Student's t-test. Data information: Note that experiments were performed at the same time, so the values of WT and Mfn2 KO are the same but the figures have been split in two for the sake of linearity. F) WT and Mfn2 KO MEFs were co-transfected with GFP and ERMITO-RLuc plasmids. After 24 h, the transfected cells were sorted and plated for another 24 h, after which RLuc was analyzed, the activity of which requires the proper apposition of the ER and mitochondria to reconstitute the split RLuc (n= 3 independent experiments). Data are presented as mean ± SEM. *p<0.05, two-tailed Student's t-test. G) Determination of mitochondrial Ca2+. Fluorescence was recorded every second in WT and Mfn2 KO MEFs that were loaded with Rhod2. Mitochondrial Ca2+ was released with the addition of the uncoupler CCCP (10 μM) as indicated (n= 40 cells from 4 independent experiments).Data are presented as mean ± SEM. H) Determination of ER Ca2+. WT and Mfn2 KO MEFs were transfected with plasmid encoding the ER-located Ca2+ sensor LAR-GECO1. After 48 h, fluorescence was recorded every 3 seconds. ER Ca2+ release was induced with caffeine (20 mM) as indicated (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM. I-K) Mitochondrial Ca2+ was determined in WT and Mfn2 KO MEFs loaded with Rhod2. The effect of (I) caffeine-induced (20 mM), (J) IP3R activation with ATP-induced (100 μM) and (K) histamine-induced (100 μM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM. (L, M) (L) MMP potential was measured in WT and Mfn2 KO cells treated with vehicle or methyl-pyruvate (5 mM) for 24 h (n= 3 independent experiments). *p<0.05, one-way ANOVA followed by Tukey's post hoc test. (M) Effect of methyl-pyruvate in mitochondrial Ca2+ uptake after caffeine stimulation (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM.
ATRA-dependent perturbations of the transcriptome in primary tumors.Figure 12Tissue slices corresponding to the indicated patients were treated with vehicle (DMSO) or ATRA (0.1 μM) for 48 h. Whole-genome gene expression studies were performed on the extracted total RNA using a microarray platform.The heat-map shows the genes significantly up- or down-regulated by ATRA (P < 0.005, paired t-test) in either Luminal-A and -B (Lum) or triple-negative (TN) tumors, and the results are expressed as the log2 ratio observed between the ATRA and vehicle-treated samples. A Venn diagram of the genes up- or down-regulated by ATRA in TN and Lum tumors is shown. The number of genes commonly or selectively regulated in TN and Lum tumors is indicated. The heat-map shows the regulation patterns of the retinoid-dependent genes significantly modulated by ATRA (P < 0.001) in either TN or Lum tumors. The symbols highlighted in red represent the five genes differentially and significantly regulated by ATRA in Lum versus TN tumors. The panel shows the estrogen-receptor (ESR1) pathway, which is significantly enriched for genes regulated by ATRA in Lum tumors. The green arrows indicate up-regulatory or stimulating interactions, while the red arrows indicate down-regulatory or inhibitory interactions. The gray arrows indicate unknown types of interactions. The red and blue dots above the protein symbols indicate the effect of ATRA in Lum tumors (red = up-regulation; blue = down-regulation).
Figure 2A,B Representative bright-filed (A) and AP staining (B) images of the WT and Mettl5 KO mESCs cultured in 2i/Lif medium. Scale bars represent 100 μm and 50 μm, respectively. C RT-qPCR analysis of the expression of pluripotency genes in WT and Mettl5 KO mESCs. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; ***, P<0.001. D Western blot analysis of the expression of pluripotency markers in WT and Mettl5 KO mESCs. α-TUBULIN was used as a loading control. E Immunostaining analysis of the expression of pluripotency markers (POU5F1, SOX2 and NANOG) in WT and Mettl5 KO mESCs, respectively. DAPI (blue) was used as a nuclear counterstain. Scale bars represent 10 μm.
Figure 1(A) EVD flGAG-POL and spliced shGAG mRNAs are distinguishable using specific PCR primer sets (arrows) for quantification and northern analysis. (35S) Cauliflower Mosaic Virus 35S promoter, (Pr) protease, (IN) integrase, (RT-RNase) reverse-transcriptase-RNase; red squares: stop codons. (B) sRNA-seq reads profile of EVD expressed from 35S:EVDWT in WT (black) or rdr6 (red). (RPM) reads per million. Positions are indicated in nucleotides (nt) from the start of the 35S sequence. Dashed vertical lines: shGAG and GAG-POL 3' ends. (C) Northern analysis of EVD RNA isoforms using a probe for the GAG region or for ACTIN2 (ACT2) as a loading control. (D) qPCR quantification of shGAG and flGAG-POL normalized to ACT2 and to GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE C SUBUNIT (GAPC) levels. qPCR was performed on n=3 biological replicates; bars: standard error. (**) = p-value < 0.01 (two-sided t-test between indicated values). (E) Western analysis of GAG and RDR6 with Coomassie (coom.) staining as a loading control. Arrow indicates cognate RDR6 protein band. (F-G) sRNA-seq profiles from EVD de-repressed in the ddm1 (F) or ddm1 dcl1 (G) backgrounds. Different siRNA size categories are stacked. Nomenclature as in (B).
Figure 8(A-B) Lipofuscin accumulation in the retina: the number of auto fluorescent puncta per section was quantified. ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer. n=3-6. Scale bar = 50 µm. TMEM106B and PGRN deletion results in increased autofluorescence in the spinal cord of 5-month-old mice. n=3. Scale bar = 10 µm. TMEM106B and PGRN deletion results in increased autofluorescence in the thalamus of 5-month-old mice. n=3. Scale bar = 10 µm. (G, H) Western blot analysis of lysosomal proteins in the spinal cord lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Asterisk indicates non-specific bands. n=3.
Figure 1(A) Schematic of SILAC-mass spectrometry. Wild-type (WT) and Ews-/- mBA cells were labeled by heavy or light amino acid, respectively. Differently labelled proteins were analyzed by mass spectrometry. (B) Network analysis of mass spectrometry results. (C) NAD+/NADH ratio was measured in mBA cell lines and 17.5 days embryo liver. Data represented as mean ±SEMs, and obtained from three different cell line and embryo liver. Significance determined by Student's t test, two-tailed, *P < 0.05, **P < 0.01. (D) Immunostaining of 17.5 days embryo tissues (mid-brain, heart, liver, and skin) with anti-PAR antibody. Insets show higher magnification. Scale bar indicates 100 μm (insert). (E) Wild-type (WT) and Ews-/- embryos mid-brain at E17.5 days were subjected to TUNEL assay. Scale bar indicates 100 μm (lnsert).
Figure 5A). LIF enhanced laser-induced CNV. Intravitreal administration of LIF (10 ng and 100 ng, Sigma) and OSM (10 ng) after laser-induced CNV. PBS was used as vehicle control. OCT-A imaging and immunostaining of choroid flatmounts was carried out 10 days after laser induction. Quantification of CNV area is shown. Dashed line circles indicate the CNV in OCT-A images. n = 5. Dot plot shows all data points from three independent experiments. Scale bar = 100 µm. B) Sodium Iodate was administered to induce retinal and CC damage in mice. After sodium iodate injection, the indicated amounts of LIF, CT-1 or OSM were injected in the eyes. Choroid capillaries were imaged by OCT-A system, n=5. The avascular area in CC is indicated in yellow. C.) H & E staining of eyes treated with different IL-6 proteins following sodium iodate injection (n = 5). Representative images are shown. Scale bar = 100 µm. D). Avascular area in choroid in sodium iodate model was determined and quantified using Image J. E). Thickness of retina in H&E staining was quantified and bar graph was shown using ImageJ. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.
Figure 6Mathematical modelling of death rate changes as bacteria adapt during entry to stationary phase, see Methods for details. The key assumption is that changes in maintenance rate β are due to changes of the proteome. To understand adaption, we thus need to understand how much bacteria can remodel their proteome when nutrients run out (A - left). We combine our key assumption with the following quantitative laws: 1. Death rate is set by maintenance rate; 2. Proteome sectors depend linearly on growth rate (Hui et al., 2015), 3. adapt at the same time-scale during nutrient shifts (Erickson et al., 2017). Combined, these relations allow us to compute how death rate depends on the proteome adaptation (A - right). We use the Flux Controlled Regulation (FCR) model (Erickson et al), to calculate the relative adaptation $\frac{\left( \phi_{X} - \phi_{X,0} \right)}{\phi_{X}^{'}}$ in various scenarios. Adaptation at the end of growth. When the nutrient concentration decreases, the nutrient uptake rate slows down with a Michaelis-Menten type kinetics (top). Depending on the uptake affinity, bacteria will experience either a sharp or smooth decrease of growth rate (bottom left, bright orange: 10 µM, dark orange: 1 mM). During this slow down, the proteome adapts (bottom center), with lower affinities (dark orange) showing a slight improvement of proteome adaptation, which results in a slightly lower death rate (bottom right). Adaptation in complex media. A medium supplemented with multiple nutrients (one - red, two - green, ten - blue) leads to a step-wise decrease of growth rate during nutrient depletion (bottom left). Due to the extended periods of slow growth after exhaustion of primary nutrients, bacteria can adapt their proteome substantially (bottom center), and decrease their death rate (bottom right).
Figure 2Immunoprecipitation of SPATC1L in the testis using an anti-SPATC1L antibody. IgG was used as a control. In, input; IgG, normal rabbit serum. Proteins associated with SPTAC1L, identified by LC-MS/MS analysis. Proteins immunoprecipitated by an anti-SPATC1L antibody were analyzed by LC-MS/MS. Spec., spectrum count; Seq., sequence count (number of non-redundant peptides identified for each protein); Seq. cov., sequence coverage calculated based on the amino acid count; MW, molecular weight calculated using TurboSEQUEST software. Interaction between SPATC1L and PKA subunits. Samples precipitated with an anti-SPATC1L antibody were analyzed by immunoblotting with anti-RIα and anti-Cα antibodies. Immunoprecipitation of SPATC1L was confirmed by immunoblotting with anti-SPATC1L antibody. IgG was used as a control. IP, immunoprecipitation; In, input; IgG, immunoglobulin G from normal rabbit serum. Expression of PKA subunits during spermatogenesis, examined by immunoblotting for RIα and Cα in testicular cells (TC), testicular sperm (TS), and epididymal sperm (S). Anti-ADAM2 and anti-α-tubulin antibodies were used as controls. Localization of SPATC1L and RIα during spermatogenesis. Isolated spermatogenic cells were immunostained for SPATC1L (red) and RIα (green), and counterstained with the nuclear dye Hoechst 33342 (blue). Overlay with bright field image is shown at right in each cell type. TC, testicular cell (spermatocyte); RS, round spermatid; ES, elongating spermatid; TS, testicular sperm. Scale bar = 5 μm.
Munc13 catalyzes opening of the Syx1 linker region via binding to the Munc18-1 domain 3a.Figure 4A Schematic depiction of smFRET assay. Positions of FRET label pair (S95C/S171C) in Syx1 are indicated as yellow pentagrams. 6×His-tagged Syx1 bound to Munc18-1 was immobilized on coverslip coated with PEG/PEG-NTA-Co2+. Syx1 linker region is colored by black. B Example histograms of smFRET efficiency of Syx1 in complex with Munc18-1 WT in the absence and presence of the MUN domain, and within the SNARE complex. C−E Example histograms of smFRET efficiency of Syx1 bound to Munc18-1 QKAA (C), KKEE (D), and L348R (E) in the absence or presence of the MUN domain. The QKAA mutation strongly impaired the shift of smFRET efficiency in the presence of the MUN domain. F Quantitative results of the Gaussian peak center positions of the FRET efficiency distributions in C−E. G Effects of QKAA, KKEE, and L348R mutations on the interaction between the Munc18-1/Syx1 complex and the MUN domain by GST pull-down assay combined with immunoblotting. Results were shown with 6×His-tag antibody and GST-tag antibody.