EMBO/sd-character-level-ner huggingface dataset:
Molecular interactions of Okp1, Ame1, and RWD domains in kinetochores A) Scheme of binding sites of Okp1 and Ame1 in budding yeasts' kinetochores that we identified; we also show the binding site for MIND (PDB code: 5T58) in Ame1.Figure 9A) Scheme of binding sites of Okp1 and Ame1 in budding yeasts' kinetochores that we identified; we also show the binding site for MIND (PDB code: 5T58) in Ame1. B) Secondary structure cartoon-diagrams of kinetochore RWD-domain-peptide structures. Ctf19-Mcm21 with Okp1 peptide (our study); S. cerevisiae Spc24-Spc25 with Cnn1 peptide (PDB code: 4GEQ); S. cerevisiae Csm1 with Mam1 peptide (5KTB; we show Csm1 residues 67-181); and human Knl1 with Nsl1 peptide (4NF9). We show structures in orientations superposed on Ctf19, and on the same scale. C) Schematic illustrating binding sites and dynamic regions in COMA-Nkp1-Nkp2. D) Schematic of a partial cross section of a budding yeasts' inner kinetochore around a centromeric nucleosome (blue, viewed from the outer kinetochore). Right side illustrates a dimeric COMA assembly; left side, binding of Chl4-Iml3 to Ctf19-Mcm21 and Mif2. We show identified contacts and a few presumed contacts. Interaction sites with the MIND assembly, which connects the inner kinetochore to the outer kinetochore, are indicated.
Figure 1(A) As a protein folding sensor, we constructed a tripartite fusion protein in which the test protein (shown in blue) is inserted between the N-terminal portion of the selectable marker, β-lactamase (shown in pink), and the C-terminal portion of β-lactamase (shown in peach). Instability of the test protein leads to either degradation or aggregation, resulting in lower ampicillin resistance (AmpS) of strains containing this biosensor compared to those in which the test protein is stable. If a host factor or chaperone such as Spy stabilizes the test protein, higher levels of ampicillin resistance (AmpR) will be exhibited. First, we generated a transposon (Tn) library in strains containing either a protein folding sensor or a β-lactamase glycine-serine linker construct (see Methods for details). Secondly, ampicillin resistance selections were performed, and colonies resistant to various levels of ampicillin were pooled and subjected to deep sequencing. This enabled the genomic mapping of the position and frequency of transposon insertion sites. Genes showing an altered transposon insertion frequency in the presence of a protein folding sensor were selected as candidates for host factors that affect the stability of that protein folding sensor. (B) When transposon insertion libraries subjected to ampicillin resistance selection, a substantial increase in transposon insertion frequency in the pstSCA genes was observed in strains containing the protein folding sensor (pBR322 bla::Im7 L53A I54A) relative to a strain containing just pBR322. Transposon insertion frequency increased 21-, 24-, and 8-fold in the pstS, pstA, and pstC genes, respectively, compared to the insertion frequency found with no ampicillin selection.
Characterization of SFT2D2 Function in Endosome-to-Golgi Retrieval.Figure 3(A) Primary screen anti-CD8 antibody-uptake images for control cells (top) and SFT2D2 KD cells (bottom). (B) Cells stably expressing SFT2D2-Myc show localization of SFT2D2 to perinuclear membranes and endosomes, including VPS35-positive endosomes (arrowheads in inset). Treatment with nocodazole disperses the endosomes and even more clearly shows colocalization of SFT2D2-Myc and VPS35 (arrowheads in inset). (C) Cells stably expressing SFT2D2-Myc were costained with antibodies against various post-Golgi SNARE proteins. Colocalization was quantified by Pearson's correlation coefficient (right-hand graph) and indicates very extensive colocalization with STX6, STX7, and VAMP8. The image panels illustrate colocalization of SFT2D2-Myc and STX5 at the Golgi (top, arrowhead) but much more extensive colocalization of SFT2D2-Myc and STX6 (bottom, arrowheads). In (B) and (C), the white dashed box delineates the area magnified in the insets. (D) SNARE protein staining was compared for control and SFT2D2 KD HeLa cells and quantified. The graph shows the change in cellular intensity measured for each post-Golgi SNARE investigated. Images illustrate the increased cellular intensity of VAMP3 in SFT2D2 KD cells. (E) Control and SFT2D2 KD HeLa cell lysates were separated by LDS-PAGE and blotted for the indicated SNARE proteins or actin.
Figure 1A. In absence of netrin-1, DCC induces apoptosis, unless mutated on its D1290 caspase cleavage site. B. DCC and netrin-1 expression (FACS analysis) in B-cells lymphocytes isolated from DCC+/+ and DCCmut/mut mice spleen and lymph nodes. Results are presented as expression ratios between means expression levels in mutant animals (n=3) and control ones (n=3). No significant difference was observed between wild-type and mutant animals. C. DCCmut/mut mouse with splenomegaly and systemic lymphadenopathy. Left panel: black and white arrows indicate respectively the inguinal and mesenteric lymph nodes. Right panel: enlarged spleen in a lymphoma-bearing mutant mouse (left) as compared to control organ (right). D. Incidence of lymphoid proliferations and lymphoma in DCC+/+ (n=19) and DCCmut/mut (n=29) mice. Tumors classification was performed blinded to genotype according to anatomopathologists consensus (29). *: p=0.036; one-tailed Fisher's exact test. E. Histology analysis and immunophenotyping of lymphoid proliferations in DCC control and mutant mice. Sections from control spleen, low-grade FL and high-grade DLBCL were stained with hematoxylin-eosin-safran or with antibodies to B220 or Ki67. w: white pulp, r: red pulp, cr: compressed red pulp.
Figure 1A. Overview images of hematopoietic sites in an E 9.0 embryo depicting the yolk sac (YS) and para-aortic splanchnopleural region (P-Sp) with phase contrast (upper image) and direct fluorescence microscopy (lower image). Stars indicate CX3CR1+ cells (green) and arrow head points to c-kit+/CD31+ EMPs (blue/white) in the YS. Scale bar represents 100 µm. One representative picture out of five independent experiments is displayed. B. Higher magnifications of the YS and P-Sp. CX3CR1-GFP expression (green) are found in the YS but not in the P-Sp, where only background staining is present. F4/80 (red), CD31 (blue), c-kit (white). Scale bars represent 50 µm. Representative pictures out of five independent experiments are shown. C. Flow cytometry analysis of MΦ precursors in the YS of E 9.0 Cx3cr1GFP/WT animals (A1: CD45+c-kitloCX3CR1loF4/80lo, A2: CD45+c-kit-CX3CR1+F4/80+). Mean fluorescent intensity (MFI) is presented. Four mice were investigated showing similar results. D. Fate-mapping strategy to target the A2 progenitor population preferentially in the YS. Male Cx3cr1CreER:R26-yfpmice were bred to wild-type animals and plug positive mice were injected at E 9.0 with tamoxifen (TAM). Offspring was analyzed at E 16.0, P0 or P42. E. Flow cytometry of F4/80hiCD11blo and F4/80loCD11bhi tissue MΦ in adult (P42) Cx3cr1CreER:R26-yfpmice. Cre negative mice served as a control. Representative pictures from three independent experiments are displayed.F. Quantification of YFP labeling in tissue MΦ at E16.0, P0 and P42 after TAM application at E 9.0 in plug positive Cx3cr1CreER:R26-yfpmice. Data are normalized to YFP+ microglia and presented at mean ± s.e.m. At least one experiment out of two is shown and each symbol presents one mouse. G. Hierarchical clustering of the 1.000 most variable probes within the dataset of embryonic development of yolk sac-derived tissue MΦ (EMP: erythromyeloid precursor, A1/A2: yolk sacMΦ progenitors, embryonic microglia (Emb F4/80 MG), embryonic Kupffer cells (Emb F4/80liver), embryonic (Emb) F4/80kidney cells. Heat map displays z-transformed log2-expression values from red to blue via white. H. Above: Sample-wise co-regulation network of embryonic MΦ development. Pearson correlation threshold of 0.92 was used to generate the network. Below: scheme thereof.
Figure 4A KEGG pathway enrichment analysis of the virus-host PPI network was conducted, and RNA transport related proteins were highly enriched. The color of a node corresponds to the frequency of occurrence of the prey that has been detected in the virus-host interactome. B In total, 693 proteins were connected by both viral baits and host receptor baits in the combined interactome. KEGG pathway enrichment analysis of these 693 proteins was conducted, and three pathways, namely, RNA transportation, endocytosis, and protein processing in the ER, were highlighted. C,D Enriched proteins were illustrated by the schematic diagram relevant to Endocytosis (P-value: 5.84×10-15) (C) and Protein processing in ER (P-value: 1.80×10-22) (D). The color of each slice in a node corresponds to the frequency of occurrence of the prey that was detected in virus-host interactome (left half slice) and host-receptor interactome (right half slice).
Fig.Figure 3A-D. CMTs visualized with MAP4-GFP in root epidermal cells of the cre1-12 mutant at the transition (TZ) and the elongation (EZ) zones at time point 0 and 60 min after mock (DMSO) (A), cytokinin (CK) (B), auxin (C) and CK plus auxin (D) treatments. As CK and auxin sources 10 µM BAP and 0.1 µM NAA were used, respectively. For the double CK plus auxin treatment roots were pretreated for 60 min with CK, whereafter they were transferred to medium supplemented with both compounds. Scale bar 10 µm. E. Histograms of CMT orientation distributions at time points 0 and 60 min in epidermal cells of cre1-12 at the TZ and the EZ treated as for (A-D). n = 14-29 cells per root growth zone in 4-8 roots per condition in 3 independent replicates. F, G. Analysis of the CMT plus-end growth with the EB1b-GFP reporter in cre1-12. F. Z-stack maximum image projection of EB1b-GFP tracked for 30 seconds (on the left) and single trajectories of EB1b-GFP signal tracked over 60 sec (on the right) in root epidermal cells of the EZ in the wild type and cre1-12 treated with CK for 60 min. Scale bars 10 µm and 0.5 µm, respectively. G. CMT plus-end growth rates (µm/min) quantified from the EB1b-GFP trajectories tracked during 5 min in epidermal cells of the EZ treated with mock (DMSO) or CK. In the boxplots, the center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software; whiskers span minimum to maximum values, individual data points are represented by dots. (ns = non-significant, *** P < 0.001 by Student's t-test, n= 7-9 cells with 7 roots per condition in 3 independent experiments). Concentrations and treatment conditions (F, G) were as for (A, B). H, I. CMTs visualized with MAP4-GFP in root epidermal cells of the cre1-12 mutant. Cells at the TZ, the EZ and the DZ were monitored at time point 0 and 60 min after treatment with oryzalin (1 µM) (H), and CK plus oryzalin (I). As CK source 10 µM BAP was used and 1 µM oryzalin. For double (CK and oryzalin) treatment roots were pretreated for 60 min with CK and then transferred to medium supplemented with both compounds. Scale bar 10 µm.
Figure 3A, Scatter plot of RNA and protein expression. B, Venn diagram showing the overlap of rhythmic RNA and proteins. C, Cumulative distributions of Pearson correlation coefficients for the indicated groups of RNA-protein pairs. RNA-Prot-, neither circadian; RNA+Prot+, both circadian; RNA-Prot+, RNA not circadian and protein circadian; RNA+Prot-, RNA circadian and protein not circadian (Wilcoxon sum rank test). Heatmap representations of transcripts and protein accumulations for the RNA-protein pairs with rhythmic transcripts Distribution of phase difference between RNA-protein pairs for those with rhythmic transcripts Heatmap representations of transcripts and protein accumulations for the RNA-protein pairs with rhythmic proteins Distribution of phase difference between RNA-protein pairs for those with rhythmic proteins
Figure 1(A) Quantitative MS-based phospho-proteomic experiments used in this study. Experiments performed in this study compared the phosphoproteome of a wild type and a CDC55 deletion (n = 8) or depletion strain (n = 4), and the effect of hyperosmotic stress (0.5 M NaCl, ten minutes) on global phosphorylation patterns between a wild type strain and a CDC55 (n = 2) or an IGO1/IGO1 deletion (n = 3) strain. Published MS datasets that were integrated (indicated in the upper right) capture the early response to hyperosmotic stress (0.5 M NaCl, five minutes) and the effect of Hog1 inhibition on the early response to hyperosmotic stress (0.5 M NaCl, five minutes) (Romanov et al., 2017, Data ref: Romanov et al., 2017), the effect of a deletion of RTS1 (Hollenstein et al., 2020) and the effect of Cdc28 inhibition (Kanshin et al., 2017) on the global phosphoproteome. All SILAC ratios represent knockout versus wild type, stressed versus unstressed, or inhibited versus not inhibited. After integration of the datasets further analyses were performed using motif & GO enrichment, as well as in vivo biochemical validation.
Figure 3(A) Imaging of RAD21KD-MCF7 and -SNU16 cells to assess cell morphology. Depletion of RAD21 caused a conversion from an epithelial-like to fibroblast-like morphology. Immunofluorescence staining for (B) VIM and (C) β-catenin in RAD21KD-SNU16 cells. The indicated cells were immunostained with antibody specific for VIM or β-catenin, and DAPI. Each image represents three individual experiments. (D) Western blots for EMT-markers TGFBR1, TGFBR2, TGFB1, ITGA5, and β-catenin in RAD21KD-MCF7 and -SNU16 cells. (E) Stable transfection of RAD21 shRNA in MCF7 cancer cells increased migration capabilities revealed by a wound-healing assay. Wound size was measured at 0, 24, and 48 h to calculate open wound rate (%). Data are presented as the means ± S.D. for three independent experiments. *P < 0.01 and; **P < 0.05. Statistical significance was validated by Student's t-tests. qPCR was performed to measure the expression of EMT markers VIM, ZEB2, CDH2, ZEB1, and FN1 along with the epithelial marker CDH1 in (F, G) shR#1_MCF7 or (H, J) shR#1_SNU16 cells after transfection with 20nM negative control, TGFB1 (siTGFB1#3 and siTGFB1#4) or ITGA5 (siITGA5#1 and siITGA5#2) siRNA for 48 h. Values were normalized relative to 18S transcription. Gene expression values in the samples were divided by those in the controls (i.e. each gene in the control is '1'). Data are presented as the means ± S.D. for three independent experiments *P < 0.01. Statistical significance was validated by Student's t-tests.
Figure 3(A-B). Oxygen consumption in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group). (C). Body temperature of male mice after 11 weeks of AKG supplementation (n = 9 per group). (D-E). Representative images (D) and quantification (E) of BAT thermogenesis induced by 6 hr cold exposure at 4℃ in male mice supplemented with AKG for 11 weeks (n = 9 per group). The mRNA expression of thermogenic genes (F) in BAT of male mice after 11 weeks of AKG supplementation (n = 3-6 per group). Immunoblots and quantification (G) of UCP1 protein in BAT of male mice after 11 weeks of AKG supplementation (n = 3-6 per group). (H-I). DAB staining (H) and quantification (I) of UCP1 in BAT of male mice supplemented with AKG for 11 weeks (n = 9 per group). (J). Serum levels of NEFA in male mice supplemented with AKG for 11 weeks (n = 9 per group). (K). The mRNA expression of CD137, CD40, TBX1, TMEM26, CITED1 and slc27a1 in iWAT of male mice supplemented with AKG for 11 weeks (n = 8 per group). (L-M). Respiratory exchange ratio (RER) in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group). (N-O). Immunoblots (N) and quantification (O) of p-HSL and ATGL protein in gWAT of male mice after 11 weeks of AKG supplementation (n = 3 per group). (P-Q). Representative images (P) and quantification (Q) of p-HSL DAB staining in gWAT and iWAT of male mice after 11 weeks of AKG supplementation (n = 9 per group). (R). The mRNA expression of PPARγ, FASN and ACC in the gWAT and iWAT from male mice supplemented with AKG for 11 weeks (n = 6 per group). (S-V). Serum levels of E (S), NE (T), T3 (U), and T4 (V) in male mice supplemented with AKG for 11 weeks (n = 8-9 per group).
SCOC is required for autophagy.Figure 4(A) SCOC is required for autophagy in HEK293 cells. Anti‐ULK1, ‐Actin and ‐LC3 blot after siRNA treatment in HEK293 cells in FM, ES or EL. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RF EL versus siSCOC‐03 EL, *P=0.0419. (B) SCOC is required for p62 degradation. p62 levels were determined by western blot (Supplementary Figure S5) after incubation in FM, ES for 2 or 4 h, or ES with BafilomycinA1 (ES+BAF) for 2 and 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); RISCfree FM versus RISCfree ES 2 and 4 h, ***P>0.0001 and **0.0014, respectively. (C) SCOC-GFP and SCOC-myc levels are reduced by SCOC siRNA duplex‐03. Anti‐GFP, ‐Myc and ‐Actin blots of HEK293 cells after treatment with ULK1, RISCfree or SCOC siRNA and overexpression of GFP, SCOC-myc or SCOC-GFP. (D) Overexpression of SCOC-myc increases autophagy. Anti‐Actin, ‐LC3 and ‐Myc blots of HEK293 cells after transfection of pcDNA or SCOC-myc. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=5). SCOC-myc EL versus pcDNA EL, *P=0.0215. (E) Endogenous SCOC colocalises with endogenous FEZ1 in HEK293 cells. Arrows show partial colocalisation. Boxed region is at higher magnification in merge panel on bottom right. Scale bars=20 μm.
Figure 2(A) UMAP plot of cells integrated from datasets of GW7, GW8, GW10, and GW11 samples (GW7S-NC, GW8S-02NC, GW10C-01NC, GW10T-01NC, GW10L-01NC, GW10C-02NC, GW10T-02NC, GW10L-02NC, GW11C-NC, GW11T-NC, GW11L-NC) showing neuronal differentiation tendency. Cell types are labelled (left). Heatmap showing differentially expressed genes (DEGs) in neuronal restricted progenitors (NRP) and immature dorsal neurons (IDN) (right). (B) Visualization of selected top gene ontology (GO) enrichment terms for NRP and IDN clusters based on DEG analysis. (C) Immunofluorescence staining of the spinal cord from GW7, GW8, and GW10 for a dorsal marker (PAX7, green), neuronal precursor marker (ASCL1, red), and nuclei marker (DAPI, blue). Images show the neuronal differentiation process on the dorsal side of the spinal cord. Scale bar: 100 μm. (D) Stacked proportional changes of neural progenitor cells (NPC) and neuronal cell classes from GW7-GW11. Cell types were identified as: proliferative neural progenitors (Pro-NPC), glial restricted progenitors (GRP), IDN, DI4 interneurons (DI4), DI5 interneurons (DI5), motor neuron (MN), DI1 and DI2 interneurons (DI1/2), DI3 interneurons (DI3), DI6 interneurons (DI6), and V0 and V1 interneurons (V0/1). (E) Three-dimensional developmental trajectories for snRNA-seq samples from the GW10 cervical sample (top view in the upper right corner). Cells are classified by differentiation stage according to expression of specific markers (neuronal progenitor: PAX3 and PAX7; DI5 interneuron: POU4F1 and TLX3; V3 interneuron: SIM1; and motor neuron: MNX1). Cells in the neuronal development trajectory are coloured by gene expression. Distances from progenitor cells to differentiated neurons represents the differing maturation status between dorsal neurons (DI4, DI5) and ventral neurons (MN, V3). Cells with no detectable expression for a given gene were omitted from the plot.
Figure 7A. Kinase assays against Histone H1. Top and middle panels indicate shorter and longer exposures respectively of the same kinase assays. Bottom panel is a CBB staining of Histone H1 showing equal loading of the protein. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. B. Kinase assays against GIP1. Top and and middle panels indicate shorter and longer exposures respectively of the same kinase assays. Bottom panel is a CBB staining of GIP1 showing equal loading of the protein. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. C. Western blot against StrepIII-tagged proteins to show loaded amounts of the CDKs. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. Double CDKB2;2 bands are likely due to a truncation of the expressed protein. D. Time-lapse of confocal microscope pictures of root meristematic cells tagged with GFP-GIP1 in Col-0 (top panel) and cycb1;1 cycb1;2 (bottom panel). GIP1 localizes at the nuclear polar caps, followed by co-localization with microtubules at the spindle and phragmoplast arrays. In cycb1;1 cycb1;2 double mutants, GIP1 exhibited an abnormal localization, being found at the spindle (magenta arrowheads) and phragmoplast (white arrowheads) midzones, which are normally devoid of the protein. Scale bars: 5 μm.
Figure 3A. PHFs GOLPH3 OE (left), or KD (right), were fixed and processed for IF: Endogenous LCS (green), GOLPH3 (red), TGN46 or DAPI (blue). Dashed lines indicate cells overexpressing or interfered for GOLPH3. Scale bar, 20 µm. Quantification of the amount of endogenous LCS (right graph) at the Golgi; data are means ± SEM derived from two biological replicates, n=42 in CTRL, n=49 in GOLPH3 KD and n=27 in GOLPH3 OE; ***p <0.001 [Student's t-test]). B. GOLPH3-OE HeLa cells were fixed and processed for IF: Endogenous LCS (green), GOLPH3 (red), and TGN46 (blue). Dashed lines indicate cells overexpressing GOLPH3. Scale bar, 20 µm. Quantification of the amount of endogenous LCS (right graph) at the Golgi (data are means ± SEM derived from two biological replicates, n=21 in CTRL, n=11 in GOLPH3 OE; ***p <0.001 [Student's t-test]). C. GOLPH3 OE or KD HeLa cells expressing LCS-HA were fixed and processed for IF: HA (green). Asterisks indicate GOLPH3 OE cells. Scale bar, 20 µm. D. Assessment of LCS-GR synchronized transport in GOLPH3 OE or KD HeLa cells. Micrographs show cells at the indicated trafficking time points. Scale bar, 20 µm. E. Representative WB of cells treated as in (D) (n = 3). F. Plots of the densitometric quantification of the amount of LCS-GR (Golgi mature band) over time, starting from 2 hr chase (data are means of at least 3 experiments ± SD). G. GOLPH3 OE or KD HeLa cells expressing LCS-HA were treated with bafilomycin A1 (BafA1, 10 nM) for 16 hr, fixed, and processed for IF labelling: LCS (green), LAMP1 (red), and DAPI (blue). Arrowheads indicate LCS and LAMP1 co-localisation. Scale bar, 20 µm. H. WB of cells treated as in (G). Densitometric quantification of the blot is plotted in the top panel (data are means of at least 3 experiments ± SD). I. GOLPH3 OE or KD HeLa cells expressing the indicated HA-tagged glycoenzymes were fixed and processed for IF. Asterisks indicate GOLPH3 OE cells. Scale bar, 20 µm. J. Number of glycoenzymes-HA positive spots per cell in GOLPH3-KD cells (left panel) and percentage of cells with glycoenzymes-HA in the ER in GOLPH3-OE cells (right panel) are plotted (n is indicated in the graph; data are means ± SEM; ***p <0.001 [Student's t-test]).