EMBO/sd-character-level-ner huggingface dataset:
EHD1 binds PS in vitro A His‐tagged EHD1 was mixed with PC‐based liposomes containing the indicated % (mol/mol) of PS or PE.Figure 5A His‐tagged EHD1 was mixed with PC‐based liposomes containing the indicated % (mol/mol) of PS or PE. After 15 min, the mixture was spun at 100,000 g for 30 min, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE. The proteins and lipids were then stained with Coomassie blue.B, C The intensities of individual bands in (A) were quantified with ImageJ, and the percentage of bound proteins was calculated. In (B), the data represent the mean ± SD of n = 3 independent experiments and were analyzed with one‐way analysis of variance (ANOVA) followed by Tukey-Kramer post hoc test. *P 0.01; NS, not significant. In (C), each point represents the mean of two independent experiments with an error bar showing the range of values obtained. The sigmoidal fitting was applied. D, E His‐tagged EHD1 was mixed with PC‐based liposomes (20, 40, or 60% PS) of decreasing size produced by extrusion through filter pores of the size indicated. After 15 min, the mixture was spun at 100,000 g for 30 min, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE. The proteins and lipids were then stained with Coomassie blue. In (E), the intensities of individual bands were quantified (mean ± SEM of three independent experiments). See Supplementary Fig S8 for the size distributions of the prepared liposomes.
Kinetic characterization of the rhodopsin dark state.Figure 2Kinetic characterization of the rhodopsin dark state. Thermal decay of dark state rhodopsin: A) WT, B) T94I and C) G90D rhodopsin. The spectra are normalized to the OD280 and OD500/OD480 at t=0. D) Absorption maximum plotted as a function of time and fitted to a single exponential decay. Retinal isomerization in the rhodopsin dark state: E) WT, F) T94I and G) G90D rhodopsin, H) The fraction of 11-cis retinal is plotted as a function of time and fitted to a single exponential decay. SB stability in dark state rhodopsin: I) WT, J) T94I and K) G90D rhodopsin. L) OD440 is plotted as a function of time and fitted to a single exponential decay. All decay rates were measured at 55 C.
Figure 5A, B UMAP plots of Dataset 3 which contains five WT (DN3a, DN3b γδ+, DN4 γδ+, DN3b γδ- and DN4 γδ-) and two Lat-/- (DN3 γδ- and DN3 γδint) scRNAseq datasets. The UMAP plot is colored according to sorted samples (A), and to expression levels of the specified genes (B). C Heatmap showing scaled expression of top 25 DEG characterizing each of the seven sorted samples of Dataset 3. The names of the sorted samples are specified at the top of the heatmap, and 3,000 cells were randomly selected for each sample. Each column represents the expression profile of a single cell. The sample from which a DEG is derived is indicated by colored bars on the left. Bar superpositions specify DEG that are found in more than one sample. The genes displayed in panel B are highlighted with a star. Gene expression is color-coded with a scale based on z‐score distribution, from −2 (purple) to 2 (yellow).
dEsyt localizes to MCS and determines RDGB localization.Figure 4(A) Confocal images showing the localization of endogenous dEsyt protein in photoreceptors of one day old dark reared flies probed with antibody against dEsyt. Scale bar: 5µm. dEsytKO shows no staining when probed with dEsyt antibody. (B) Confocal images showing the localization of exogenously expressed dEsyt::mCherry protein expressed using the eye specific Rh1-Gal4 in one day old dark reared flies. Rh1-Gal4 is shown as a control. A single ommatidium is shown. Scale bar: 5 µm. Phalloidin marks F-actin staining and highlights rhabdomeres R1-R7. (C) Western blot from the head extracts of wild type and dEsytKO probed with the antibody against RDGB. Rearing conditions, age of the flies and genotype is indicated on top of the blot. Tubulin was used as the loading control. (D, E) Confocal images showing the localization of RDGB in wild type and dEsytKO photoreceptors of flies which are (D) 1 day old-dark reared and (E) 6 days old- exposed to constant illumination. For (D) and (E) RDGB visualized using an antibody against the endogenous protein. Rhabdomeres are outlined using phalloidin which marks F-actin. Scale bar: 5 µm.
FARP2 activation and Myosin-II inhibition cooperate to induce collective invasion downstream of ROCK2 inhibition.Figure 8A) Caco-2 cysts stably expressing GFP (treated or not with Y27632), or Cherry-FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH 730/733 (FARP2-del/Mut) were allowed to invade for 2 days, fixed and stained for F-actin. Boxed regions i, ii, iii, iv and v are shown at high magnification. Arrowheads point to non-protruding cells, arrow points to protruding cells, white star shows nucleus that engages in protrusion. Scale bars: 20μm. B) Bargraph representing the percentage of protrusive cysts expressing GFP (treated or not with Y27632) or the mentioned GFP-FARP2 constructs from 3 independent experiments (Means ± SEM, paired t-test, **p<0,01, n.s: non significant). C) Representative images of Caco-2 cysts treated with Y27632 or Blebbistatin or transfected with siRNA targeting Myosin-IIA and allowed to invade for 2 days. After fixation, the cysts were stained for F-actin (Phalloidin) and nuclei (DAPI). Representative Y27632-induced « invasive » phenotype and Blebbistatin-induced « dendritic » phenotype are displayed. Arrows point to protruding cells, white star shows nucleus that engages in protrusion and arrowheads point to "dendritic" protrusions. D) Bar graph representing the percentage of cysts with the "invasive" or "dendritic" phenotypes after treatment with Y27632, Blebbistatin or siMyosin-IIA or siMyosin-IIB from at least 3 independent experiments (Means ± SEM, unpaired t-test,*p<0,05, n.s: non significant). E) Mature Caco-2 cysts were transduced with GFP or Cherry tagged FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH/730/733 (FARP2-del/Mut), and treated (or not, NT) with Blebbistatin. 2 days after invasion,the cysts were fixed and stained for F-actin. Boxed regions i, ii, iii and iv are shown at high magnification. Arrowheads point to non-protruding cells; arrow points to protruding cells and white stars show nucleus that engage in protrusions. Scale bars: 20 μm. F) Bargraph representing the percentage of protrusive Caco2 cysts expressing the mentioned constructs , treated or not with blebbistatin, from at least 3 independent experiments (Means ± SEM , t-test,**p<0,01, n.s: non significant).
Inhibition of autophagy restores A2 pathogenicity.Figure 5(A,B) Agar plugs containing actively growing cultures of the OA deficient A2 mutant were inoculated onto leaves of Arabidopsis Col-0 and select Arabidopsis autophagy mutant plants. These mutants showed enhanced susceptibility to the normally non-pathogenic A2 strain. Lesion diameter was monitored over time and all images were recorded 48 hours post inoculation. (C) Tomato leaves were either pre-infiltrated with water (control) or autophagy inhibitors Wortmannin, LY294002, Chloroquine (CQ), and 3-methyladenine (3-MA). Agar plugs containing actively growing A2 were placed on the infiltrated leaves to initiate infection. (B) 48 hours post inoculation; Trypan blue was used to determine the extent of fungal colonization and cleared with acetic acid and ethanol (1: 3, v/v). Images were taken 48 hours post inoculation.
Figure 2(A) Illustration of the expected distribution of data points combined from independent genetic reporter screens. Each data point represents the directionality score of a deletion mutant. Mutants altering directionality positively by increasing divergent YFP levels are expected to be found in quadrant I. Mutants decreasing YFP with negative directionality scores are found in quadrant III. Data points closer to the diagonal (grey region) have high reproducibility between the screens. (B) Distribution of mutants by directionality scores from the GCG1pr and ORC2pr genetic screens. Quadrants I and III reveal the non-essential mutant subunits of several protein complexes (highlighted in colors) and mutants involved in CAF-I pathway (square data points) altering the directionality. The top candidate repressors and activators of DNC are labeled. (C) Flow cytometry quantification of mCherry/YFP fluorescence in the mutants with GCG1pr FPR background. The data are normalized to the signal values of the wild type (FPR) strain. (D) Endogenous transcript analysis by RT-qPCR in the mutants. The bars represent fold gene expression ratio of coding and divergent transcript normalized to the expression of the reference gene, UBC6.
Blue-native gel electrophoresis (BNGE) mass spectrometry and immunodetection analysis of cIII2-related proteins.Figure 3(A) Complexome profiles of cIII2 structural subunits generated by analyzing the peptide content in each of the 64 slices in which the gel lanes were excised The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1.0, vs. the molecular mass calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines was determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and ∆4-CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments. (B) Second-dimension BNGE of digitonin-solubilized samples from WT and ∆4-CYB cells, western blot and immunodetection of the indicated cIII2 structural subunits with specific antibodies. The immunodetection patterns were equivalent to the complexome profiles. (C) Quantification of the total peak area under the curves (AUC) defined by the peptide intensity peaks for the indicated cIII2 subunits. The x-axis values were the slice number (1-64) and the y-axis values were the relative peptide intensity. The graph shows the mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0083 (UQCRC2); **p=0.0033 (UQCRFS1); *p=0.0224; n.s.=non-significant. (D) Complexome profiles of two cIII2 assembly factors (BCS1L and LYRM7 or MZM1L) generated The represented values are the mean ± SEM of the two reciprocal labeling experiments.
Figure 2A. Absolute copy number of protein in kidney tissue and human embryonal kidney cells (HEK293), liver tissue and liver cancer cell line (HepG2), lung tissue and lung cancer cell line (A549), and breast tissue and breast cancer cell line (MCF7). The order of proteins are the same in the tissue and corresponding cell line and the proteins have been order according to the abundance in the respective tissue. B. The direct correlation between RNA (TPM) and protein abundances (copy number) for all quantified genes in the same tissues and cell lines. Spearman (ρ) and Pearson (r) correlation between the two values across the quantified genes are shown. The other seven tissues and five cell lines are shown in Figure EV4.
Figure 3Enriched pathways comparing BALB/c thymic NKT1 and NKT2 transcriptomes; ATP content from sorted thymic T cell populations; oxygen consumption rate (OCR) from sorted thymic T cell populations measured in a Seahorse Mitostress test; basal OCR were calculated based on data from (c). spare respiratory capacity (SRC) were calculated based on data from (c). (f) maximal glycolytic activity and (g) spare glycolytic capacity were calculated as in Appendix. Fig. S6.
Figure 8(A) Am80 selectively activates RARα and RARβ to alter expression of RA-target genes, leading to modest myeloid expansion and effective neutrophil differentiation. Up arrow, increased expression of cell cycle inhibitor p21Cip/Kip; down arrow, decreased expression of p21Cip/Kip. (B) GCSF has limited effect on mediating transcription of RA-target genes that promote granulocytic differentiation. Dot line, non-direct effect on gene transcription; white arrow, limited effect on gene transcription. (C) Am80-modulated selective activation of RARα and RARβ synergizes with GCSF induction of C/EBPα-dependent and -independent transcriptional regulation, resulting in effective differentiation of large amounts of granulocytic precursors into functional neutrophils during myeloid expansion.
Rheotaxis of human sperm.Figure 4Trajectories of human sperm in the absence of a fluid flow. Sperm were tracked for 1.86 s. The starting point of each trajectory was centered to the origin of a coordinate system, represented by the intersection of the dotted lines in the center of the circle. Spider-web plot of the mean (± SD) angular frequencies (binning: 315°-45°, 45°-135°, 135°-225°, and 225°-315°; 1,301 sperm; 7 experiments) in the absence of a fluid flow. Representative trajectories of human sperm in the presence of a fluid flow. Spider-web plot of the mean (± SD) angular frequencies (1,083 sperm; 7 experiments) in the presence of a fluid flow. The red arrow indicates the flow direction. Representative trajectories of CATSPER2-/- sperm in the absence of a fluid flow. Spider-web plot of mean (± SD) angular frequencies of CATSPER2-/- sperm in the absence of a fluid flow (1,068 sperm; 6 experiments). Trajectories of CATSPER2-/- sperm in the presence of a fluid flow. Spider-web plot of the mean (± SD) angular frequencies of CATSPER2-/- sperm in the presence of a fluid flow (1,068 sperm; 7 experiments). The red arrow indicates the fluid flow direction. Representative trajectories of CATSPER2-/- sperm in Ca2+-free buffer in the absence of a fluid flow. Spider-web plot of the mean (± SD) angular frequencies (442 sperm; 4 experiments) of CATSPER2-/- sperm in Ca2+-free buffer in the absence of a fluid flow. Representative trajectories of CATSPER2-/- sperm in Ca2+-free buffer in the presence of a fluid flow. Spider-web plot of the mean (± SD) angular frequencies (620 sperm; 4 experiments) of CATSPER2-/- sperm in Ca2+-free buffer in the presence of a fluid flow. The red arrow indicates the flow direction.
Figure 7(A) Representative micrograph of an eye from day 7 after glutamate intoxication, from a mouse that was i.v.-injected with monocytes, showing monocyte localization to the SRS after this mode of application, and indicating a potential homing route for the cells (IL-10, red; CX3CR1-GFP, green). (B-E) Mice were injected i.v.t. with either monocytes or PBS 1 day after GT intoxication, and ocular sections from 7 days after the injury were analyzed. (B, C) Representative micrographs of noninjured and injured retinas stained for the RGC marker, Brn3a (red; scale bar, 100 µm), and quantification of RGC survival. Arrows indicate Brn3a+ cells. Bar graph shows mean ± SE of each group. *, P < 0.05. (B-E) Mice were injected i.v.t. with either monocytes or PBS 1 day after GT intoxication, and ocular sections from 7 days after the injury were analyzed. (D, E) Representative micrographs of GT-intoxicated eyes that were i.v.t.-injected with monocytes, showing the expression of an array of cytokines and neurotrophic factors (red) by the injected monocytes (CX3CR1-GFP, green). The injected cells were found in the vitreous and in proximity to injured RGCs (left panels), as well as in the SRS, adjacent to the RPE (right panels). Insets show higher magnification of representative cells (scale bar, 100 µm; inset scale, 20 µm). n = 4-5 per group. GCL - ganglion cell layer; GT - glutamate; mon - monocytes; RGC - retinal ganglion cell; RPE - retinal pigment epithelium; SRS - subretinal space; vit - vitreous.
Human NDP52 binds nsP2, localizes near CHIKV RCs, and restricts cell shutoff promoting cell survival in HeLa cells.Figure 4Human NDP52 binds nsP2, localizes near CHIKV RCs, and restricts cell shutoff promoting cell survival in HeLa cells. Yeast cells expressing Gal4 DNA BD fused alone (empty vector) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to NDP52 or the indicated NDP52 deletion mutants (top). Yeast cells expressing BD fused to nsP2 or the indicated nsP2 deletion mutants were cotransformed with a plasmid encoding the Gal4 AD fused alone (empty vector) or to NDP52 (bottom) (A). Cells were infected for 15 h, then labeled using antibodies to NDP52 and nsP2 (B), or to TGN46, nsP2 and nsP3 (C). Cells were treated with control (CTRL) (left) or NDP52 (right) siRNA, infected for 15 h, then labeled using antibodies to NDP52, nsP2 and puromycin. Quantitative analysis was performed by counting the percentage of infected cells with TGN‐associated RCs (n=30 cells per experiment) (D). Cells were transfected with empty plasmid (CTRL) or nsP2wt‐3XFLAG or nsP2R606A‐3XFLAG. Cell mortality (that is, % of dead cells) was measured 24 h post transfection (E). Cells were treated with control (CTRL) or NDP52 siRNA, then mock infected (M) or infected (C) for 24 h. Cytoplasmic and nuclear fractions were isolated and immunoblotted for nsP2 and NDP52. The amounts of the cytoplasmic and nuclear fractions of nsP2 were quantified by densitometry and expressed as the ratio of nuclear to cytoplasmic fraction of nsP2 (F). Cells were treated with control (CTRL) or NDP52 siRNA, infected for 15 and 24 h and treated with puromycin. Whole‐cell lysates of siRNA‐treated cells were immunoblotted for NDP52, capsid or actin. Puromycin incorporation into newly synthesized protein was revealed using antibodies to puromycin (G). Cells were treated with CTRL or NDP52 siRNA, then transfected with NT or nsP2‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 48 h post transfection (H).
Deletion of concavin leads to rounding of sporozoites A.Figure 1A. Blood-stage growth rate of concavin(-) parasites in comparison to wild type. Data points represent parasites growing in individual mice, n indicates total number. P-value is calculated using the Mann Whitney test. Shown is the mean ± SEM. B. Gametocyte to ookinete conversion of 3 biological replicates. P-value calculated using the Mann Whitney test. Shown is the mean ± SEM. C. Average speed of moving ookinetes. Data points represent individual ookinetes, n indicates total number of cells from 3 biological replicates. P-values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison. D. Ookinete images of wild type and concavin(-) parasites revealing similar cell shapes. Scale bar 5 µm. E. Oocyst development of concavin(-) parasites compared to wild type. Data points represent individual midguts observed between d12-17 post infection from 3 independent cage feeds. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison. F. Mosquito infections resulted in deformed sporozoites in the salivary gland. Shown are example images of different sporozoites arranged to illustrate their rounding up over time. G. Quantification of sporozoite rounding up over time in the midgut (MG) the haemolymph (HL) and salivary gland (SG) at the indicated days post infection from three biological replicates. Sporozoites were classified as either normal, deformed or round as illustrated in F. H. Average speed of salivary gland sporozoites. Data points represent individual sporozoites, n indicates total number from 3 biological replicates. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test. I. Selected trajectories of manually tracked sporozoites.
ZEB1-dependent epigenetic modifications.Figure 2A Schemes for the genomic loci of the miRNA and E-cadherin genes, showing regions of the CpG islands (yellow), of the qRT-PCR amplicon for chromatin immunoprecipitation (ChIP) analysis (blue) and of the bisulfite sequencing (red). B, C Histone marks were analyzed using ChIP coupled to qRT-PCR for Panc1 control versus shZEB, MDA-MB-231 control versus shZEB (B), and BxPC3 control versus gemcitabine resistant (gr) (C). In MDA-MB-231 and Panc1, the active histone marks H3K4me3, H3ac, H4ac, and H3K9ac were enriched. Vice versa, in the drug-resistant clones of BxPC3, the active marks were reduced in the CpG islands. The repressive histone mark H3K27me3 was not detectable in the miR-200 loci, but in the loci of miR-203 and E-cadherin in Panc1 and MDA-MB-231. DNA methylation status was determined by bisulfite sequencing. Depletion of ZEB1 in MDA-MB-231 resulted in almost complete demethylation, whereas the selection of drug-resistant, ZEB1-expressing clones in BxPC3 induced complete methylation. n = 2 (Panc1) or 3 (MDA-MB-231 and BxPC3), mean ± SEM; unpaired Student's t-test.
mTORC1 regulates TFEB.Figure 2(A) Lysosomal stress inhibits mTOR signalling. Immunoblotting of protein extracts isolated from HeLa cells treated overnight as indicated. Membranes were probed with antibodies against p‐T202/Y204‐ERK1/2, ERK1/2, p‐T389‐S6K, and S6K to measure ERK and mTORC1 activities. (B) Torin 1 induces TFEB dephosphorylation and nuclear translocation. FLAG immunoblotting of cytosolic and nuclear fractions isolated from TFEB-3 × FLAG HeLa cells starved in amino acid‐free media and subsequently stimulated as indicated for at least 3 h. Correct subcellular fractionation was verified with H3 and tubulin antibodies. (C) Effects of ERK and mTOR inhibitors on TFEB nuclear translocation. TFEB-GFP HeLa cells were seeded in 96‐well plates, cultured for 12 h, and then treated with the indicated concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2. After 3 h at 37°C, cells were processed and images were acquired using the OPERA automated confocal microscope (Perkin‐Elmer). Scale bars represent 30 μm. (D) Dose-response curves of the effects of ERK and mTOR inhibitors on TFEBnuclear translocation. TFEB-GFPHeLa cells were seeded in 384‐well plates, cultured for 12 h, and treated with 10 different concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2 ranging from 2.54 nM to 50 μM. The graph shows the percentage of nuclear translocation at the different concentrations of each compound (in log of the concentration). The EC50 for each compound was calculated using Prism software (see Materials and methods for details). (E) Immunofluorescence of HEK‐293T cells treated with DMSO or Torin 1 and stained with antibodies against endogenous TFEB and the lysosomal protein RagC (green and red, respectively, in the merge). DAPI is included in the merge. Scale bars represent 10 μM. (F) Rag GTPase knockdown induces TFEB nuclear translocation. HeLa cells stably expressing TFEB-3 × FLAG were infected with lentiviruses encoding Short hairpin (Sh‐) RNAs targeting luciferase (control) or RagC and RagD mRNAs. In all samples, 96 h post infection, cells were left untreated (N=normal media), starved (S=starved media) or treated with Torin 1 (T=Torin 1) for 4 h and then subjected to nuclear/cytosolic fractionation. TFEB localization was detected with a FLAG antibody, whereas tubulin and H3 were used as controls for the cytosolic and nuclear fraction, respectively; levels of S6K phosphorylation were used to test RagC and RagD knockdown efficiency. (G) Loss of mTORC2 does not affect TFEB phosphorylation. Mouse embryonic fibroblasts (MEFs) isolated from Sin1−/− or control embryos (E14.5) were infected with a retrovirus encoding TFEB-3 × FLAG; 48 h post infection, cells were treated with Torin 1 (T) for 4 h where indicated, subjected to nuclear/cytosolic fractionation and immunoblotted for FLAG, tubulin, and H3. (H) Binding of TFEB to mTORC1. HEK‐293T cells that express TFEB-3 × FLAG were lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR, the mTORC1 subunit raptor and the mTORC2 components rictor and Sin1. FLAG-Rap2A served as negative control.
Figure 8When cancer cells enter mitosis, BCL9 will bind to the β-catenin/Axin1 complex via its N-terminal, which includes HD2. As CDK14 and CDK1 kinases are activated by Cyclin Y/Y-L and Cyclin B1, LRP6 is phosphorylated by CDK14, and BCL9 is phosphorylated by CDK1 on its N-terminal. These changes will stabilise BCL9 and LRP6 signalosome components and prevent clathrin from binding and subsequent clathrin-mediated-endocytosis with mechanisms not fully explored, which further maintains mitotic Wnt/STOP signalling, modulates efficient cell division and promotes cancer cell growth.