EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A) Schematic of the mouseTrem2 locus and the TREM2protein. Sequence alignment of wild-type N9 (N9 wt) and TREM2 mutant N9 (N9 mu) surrounding the gRNA target site. The gRNA sequence is in cyan and protospacer‐adjacent motif (PAM) is marked with a line. The single nucleotide insertion is labeled in red. Schematic representation of wild-type TREM2 (NP_112544.1) and CRISPR/Cas9 modified TREM2 (N9 mu). TM, trans-membrane domain; SP, signal peptide. (B) Western blot analysis of lysates and media from wt and mutant N9 cells (N9 wt /mu) using antibody anti-murine TREM2 (clone 5F4), which is raised against the murine TREM2 extracellular domain. sTREM2, soluble TREM2* Indicate unspecific bands. Anti-calnexin antibody was used as loading control. (C) Phagocytosis of 1 µM HiLyte™ Fluor 488 Aβ1-42 (fAβ42) by N9 wt and N9 mu in the presence or absence of antibody 2D8 or the non-binding antibody 6687. Cytochalasin D (CytoD, 10 mM) was used as control to verify phagocytic uptake. (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.61, genotype P<0.0001, treatment P=0.0001; post hoc tests wt vs. mu for the following conditions: fAβ42 P=0.0043, fAβ42-2D8 P=0.0436). (D) Western blot of BMDM derived from wt and Trem2 knockout (ko) animals using antibody 5F4. (E) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of 2D8, or the non-binding control antibody 6687. (n = 3, +/- SEM; 2 way ANOVA, interaction P=0.0005, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42 P=0.0021, fAβ42-2D8 1 µg/ml P<0.0001, fAβ42-2D8 5 µg/ml P<0.0001, fAβ42-2D8 10 µg/ml P<0.0001, fAβ42/6687 10 µg/ml P=0.0007)(F) Quantification of relative fAβ42uptake to lowest antibody concentration used. (G) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of mAb11, or an isotype control antibody (IC). (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.0223, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42-mAb11 1 µg/ml P=0.0391, fAβ42-mAb11 5 µg/ml P=0.0069, fAβ42-mAb11 10 µg/ml P<0.0001, fAβ42-mAb11 20 µg/ml P=0.0001, fAβ42-mAb11 50 µg/ml P<0.0001).(H) Quantification of relative fAβ42 uptake to lowest antibody concentration used. (I) Recombinant mousesTREM2 does not rescue fAβ42 uptake in Trem2-deficient BMDM. Increasing amounts of sTREM2 were added to the media of wt or Trem2 ko BMDM in the presence or absence of mAb11 (10 µg/ml). (J) Western blot of primary microglia from wt or Trem2 ko animals using antibody 5F4. (K) Phagocytosis of fAβ42 by primary microglia from wt and Trem2 ko animals in the presence or absence of mAb11, or an isotype control antibody (IC). (n = 5, +/- SEM; 2 way ANOVA, interaction P=0.4797, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42-mAb11 5 µg/ml P=0.0449, fAβ42-mAb11 10 µg/ml P=0.0370, fAβ42-mAb11 20 µg/ml P=0.0299, fAβ42-mAb11 50 µg/ml P=0.0120).(L) Quantification of relative fAβ42uptake to lowest antibody concentration used.
Effect of GS inhibitor on NF54 and NF135 A) Normalized percentage +/- s.Figure 6A) Normalized percentage +/- s.d. of intracellular schizont size of NF54 (blue) and NF135 (green) after treatment of GS inhibitors at different concentrations of AIP. Median size of the schizonts were normalized to that of H2O-treated NF135 (which is set at 100%). AIP was added one day post invasion and kept on for 48 hours. Each dot represents a biological replicate where ≥100 schizonts were measured on day 5 p.i.. A two-way ANOVA was performed followed by a Tukey's multiple comparison test with the **** < 0.0001 to compare conditions within the same parasite strain. A two-way ANOVA was performed followed by a Sidak's multiple comparisons test with **** <0.0001 to compare conditions between parasite strains). B) Size difference between the GS positive (closed circle) and negative (open circle) NF135 schizonts after treatment of AIP. Each dot represents the median of a biological replicate where ≥100 schizonts were measured. Three biological replicates were performed with the median from each plotted +/- s.d.. A two-way ANOVA was performed followed by a Dunnett's multiple comparison test with *** = 0.0002 and **** = 0.0001. C) Number of schizonts per well for NF135 (green) and NF54 (blue). Three biological replicates (circle, square and triangle), each with two technical replicates were performed. D) Average +/- s.d. (dot) host cell numbers per well for three independent biological replicates under the different AIP concentration. E) The difference in size between GS positive (closed circle) and negative (open circle) NF135 schizonts (day 5 p.i.) after treatment of AIP (360 µM) on different days pre - and post-invasion. Each dot represents the median size of ≥100 schizonts +/- s.d.. The dotted blue line shows the average median schizont size of NF54 treated with H2O on day 5 post invasion of the same three biological replicates. A two-way RM ANOVA was performed followed by a Tukey's multiple comparison test: * p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001). F) Summary of observed effects of hGS on parasite development. Sporozoites can invade Z1, 2 and 3 hepatocytes but disproportionally prefer Z3 hepatocytes to make use of exclusive presence of hGS. Ability to uptake functional hGS early after invasion results in relatively larger schizonts; uptake of inhibited hGS (ihGS) reduces schizont size.
Figure 2(A) Npm1 expression levels (relative quantitation, RQ) in BM-MNCs of young (3-6 months old) and old (12-18 months old) wild type (WT) mice (n = 3 mice per group). (B) Representative flow cytometry analysis of phenotypic HSCs [c-Kit+Sca-1+Lin- (KSL) CD135-CD48-CD150+] in the bone marrow of 4-month-old Ctrl (Npm1F/FVav1Cre-), 4-months old Npm1 cKO (Npm1F/FVav1Cre+) and 2-year Old WT mice (left). Mean percentages of the indicated fraction are shown (right, n = 4-9 mice per group). (C) Representative distribution of Tubulin and CDC42 in Ctrl, 4-months old Npm1 cKO and 2-years old WT HSCs determined by IF. Scale bars, 5 μm. (D) Quantification of percentage of cytoskeletal polarized HSCs (n = 79 cells in Ctrl, n = 120 cells in cKO, n = 82 cells in Old, from 4 Ctrl and Old mice and 3 cKO mice). (E) In vitro colony replating ability of HSCs from Ctrl, 4-months old Npm1 cKO and 2-years old WT mice determined by limiting-dilution assay. Overall test for differences in stem cell frequencies between any of the groups calculated by Chisq test, * p<0.05, ***p<0.001, **** p <0.0001. The slope of the line is the log-active cell fraction. The dashed lines give the 95% confidence interval. (F ) HSCs of Ctrl, 4-months old Npm1 cKO and 2-years old WT mice were sorted and stained for TOM20. Representative images (top) and quantification of mitochondrial volume and number (bottom) in single cells (n = 58 cells in Ctrl, n = 70 cells in cKO and n = 23 cells in Old, from 3 mice per group), identified by volume rendering of TOM20. Scale bars, 2 μm. (G) Representative flow cytometry histogram (left) and quantification of TMRM median intensity (right, n = 3 mice per group) in HSCs from the indicated genotyped mice. (H) Representative flow cytometry histogram (left) and quantification of mitoSox median intensity (right, n = 4 mice per group) in HSC fraction. (I) NPM1 expression levels (relative quantitation, RQ) in MDS-L cells transfected with siNPM1 or control (siCTRL) (n = 3 mice per group). (J) Representative images (top) and quantification of mitochondrial volume and number (bottom), identified by volume rendering of TOM20, in MDS-L cells transfected with siNPM1 or control (siCTRL) (n = 58 cells per group, from 3 transfected MDS-L cultures). Scale bars, 2 μm. (K) Representative flow cytometry histogram (left) and quantification (right, n = 3 transfected MDS-L cultures) of TMRM median intensity in MDS-L cells transfected with siNPM1 or control. (L) Representative flow cytometry histogram (left) and quantification (right, n = 3 transfected MDS-L cultures) of mitoSox median intensity in MDS-L cells transfected with siNPM1 or control.
Figure 3A,B Plates with several layers that are not closely appressed. C-E Large multilayer plates having the size of human metaphase chromatids [~0.6 µm diameter ] (C); the inset schematically shows the perpendicular orientation of chromatin layers with respect to the chromatid axes proposed in the thin-plate model In other slices (D,E), the multilayer structures shown in (C) are more compact and the individual layers are not visible as separate elements. Data information: Scale bars: 50 nm.
Figure 4Three orthogonal views of gp105 dimer. The uncleaved N-terminal tag is in cyan. Residue numbers are given in strategic positions. Three orthogonal views of T4 gp8 dimer extracted from the T4 baseplate structure in the same orientations as gp105 in panels (A through C). The fragment of T4 gp7 that spans the gp8 dimer is in magenta. Superposition of T4 gp8 and gp105 dimers. The color scheme is unchanged. Gp7 (magenta) nearly perfectly matches the gp105 N-terminal tag (cyan) upon the superposition of T4 gp8 and gp105 dimers.
a,b) mRNA levels of the indicated genes involved in lipid metabolism (a) and in the autophagy and lysosome pathway (b) were quantified by quantitative rtPCR of total RNA isolated from liver samples of mice infected with the HDAd-TFEB virus.figf2(a,b) mRNA levels of the indicated genes involved in lipid metabolism (a) and in the autophagy and lysosome pathway (b) were quantified by quantitative rtPCR of total RNA isolated from liver samples of mice infected with the HDAd-TFEB virus. Gapdh was used as a control (Ctrl). Values are mean ± s.d for n = 3 mice and are expressed as fold increase compared with control mice (injected with transgeneless viral vector). *P0.05; **P0.01; ***P0.001. Control levels are indicated by dashed lines. (c) The 124 genes with a known role in the lipid metabolic process, whose expression was perturbed by TFEB overexpression, are represented as coloured circles and assigned to specific lipid breakdown (left) or lipid biosynthesis (right) sub-categories. The percentages of upregulated (red circles) and downregulated genes (green circles) are shown both for the two main groups and for each sub-category. *Note that in calculating these percentages, genes assigned to the negative regulation of lipid catabolic process and to the negative regulation of lipid biosynthetic process have been included in the lipid breakdown and in the lipid biosynthesis groups, respectively.
Figure 4(A) Binding of antibodies TB24PB037 and TB33PB123 to LAM purified from MTB as determined by Western Blot. The negative isotype control (mGO53) and a commercially available anti-LAM antibody (positive control) are shown for comparison. (B) Anti-BCG specificity of the LAM-reactive antibody TB24PB037 (black) compared to E.coli as determined by ELISA with whole bacteria (left) and flow cytometry (right). (C) Anti-LAMELISA for TB24PB037 or negative control antibody at the indicated concentrations. The mean ±SD of the absorbance was calculated. (D) Antibody TB24PB037 binds to MTB-LAM but not to LAM from M. smegmatis. ELISA performed as described in (C). (E) Representative HBHA ELISA for antibodies cloned from HBHA-reactive memory B cells of patient TB35 and HCW1. Dashed red line indicates the threshold OD405nm for positive reactivity. Green line indicates negative control antibody (mGO53; (Wardemann et al, 2003)). (F) Reactivity to HBHA was confirmed by Western Blot for a selected set of antibodies with HBHA ELISA reactivity. (G) Fluorescence microscopy shows BCG- and MTB-reactivity of representative anti-HBHA antibodies compared to a non-reactive isotype control antibody. Scale bar 10 μm (top/bottom).
Figure 4Knockdown efficiency of Eif4ebp1 and Eif4ebp2 shRNAs in 2iL (mean values ± SD). n = 3 biological replicates. Images of mESCs transduced with Eif4ebp1 and Eif4ebp2 shRNAs in 2iL, alone or in combination, in control or INK128 (200 nM) treated for three days. Scale bar, 400 µm. Relative proliferation of mESCs as in (B) (mean values ± SD, P value was calculated using two-tailed unpaired Student's t test, **P < 0.01, also hereafter in similar experiments). n = 3 biological replicates. Knockdown efficiency of Larp1 shRNAs in 2iL (mean values ± SD). n = 3 biological replicates. Images of mESCs transduced with Larp1 shRNAs in 2iL, in control or INK128 (200 nM) treated for three days. Scale bar, 400 µm.   Relative proliferation of mESCs as in (E) (mean values ± SD). n = 3 biological replicates.
Accumulation of p62 in endothelial cells lining human CCM lesions.Figure 4p62 immunohistochemistry (IHC) staining in humanbrain tissue.A, B Normal vascular endothelium of autoptic brain parenchyma samples is lacking the typical autophagic p62 granules as shown by the negative staining for p62. Scale bars: (A) 200 μm; (B) 100 μm. p62immunohistochemistry (IHC) staining in humanbrain tissue.C-F Two different representative samples of CCM lesions with a thin, single layer brain endothelium displaying either moderate (C, D) or marked (E, F) positive perinuclear "pearl necklace-like" immunostaining for p62 granules. (C, D), case n° 4 (p62++), and (E, F), case n° 8 (p62+++) are representative of CCM cases listed in Appendix Table S1. Scale bars: (C, E) 200 μm; (D, F) 100 μm. Arrows indicate endothelial p62 positive staining. p62immunohistochemistry (IHC) staining in humanbrain tissue.G-I Hematoxylin and eosin (H&amp;amp;E) staining (G) and p62immunohistochemistry analysis (H, I) of a CCM surgical sample (case n° 6 in Appendix Table S1) containing normal vessels in the peri-lesional area, which served as an internal control. Notice marked p62-positive staining in endothelial cells lining a CCM lesion (H, arrows) and p62-negative staining in endothelial cells lining a normal peri-lesional vessel (I, arrows). Scale bars: (G) 300 μm; (H, I) 100 μm. Background staining in brainparenchyma surrounding CCM lesions may be attributed to either cell debris or p62 immunoreactivity in neuronal cells and glial cells.
Aggresome-like structures are formed in SGNs and HCs from Elp3cKO animals.Figure 3(A) Proteostat staining of WT and Elp3cKO E13.5 cochlear sections reveals the presence of numerous aggresome-like structures in Elp3-deficient SGNs. Scale bar =100µm. (B) Transmission electron micrographs from E13.5 Elp3cKO SGNs confirm the presence of apoptotic neurons (marked by a red arrowhead) and a large electron dense juxtanuclear structures typical of aggresome (dashed circle). Scale bars =10µm and 2µm (last panel). (C) Chop and Chac1 transcripts are upregulated in E14 Elp3cKO compared to WT cochleae (n=8/6 for WT/KO; unpaired t-test two-tailed; p=0.0001/0.0015/0.0010; t=9.535/4.090/4.307; DF=12/12/12 for Elp3, Chop and Chac1 respectively; mean±SEM presented) indicating that the pro-apoptotic arm of the unfolded protein response (UPR) is activated in Elp3-deficient SGNs. (D-E) Proteostat staining of whole-mounted P0 organs of Corti reveal the presence of aggregates in HCs upon loss of Elp3. Orthogonal slices (Orthog) reveal aggregated proteins localize at the apical surface of Elp3cKO HCs. Scale bars =5µm. #IHC; *OHC1; **OHC2; ***OHC3. (F) Protein extracts from P1 cochleae show increased levels of polyubiquitinated conjugates upon loss of Elp3, confirming proteostasis disruption. (G) The levels of Chop and Chac1 transcripts are unaffected by Elp3 depletion in P0 organs of Corti (n=3 animals per genotype; unpaired t-test two-tailed; p=0.0003/0.4296/0.056; t=11.62/0.8778/2.662; DF=4/4/4 for Elp3, Chop and Chac1 respectively; mean±SEM presented) suggesting that proteostasis disruption in Elp3cKO HCs is not sufficient to induce UPR-mediated apoptosis.
Figure 1A, Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times. B, H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20) and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) were treated with 12 μM erastin (+) or untreated (-) for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01. C, Lipid ROS levels were assessed by flow cytometry after C11-BODIPY staining in cells treated Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01. D, H1299 cells treated were subjected to transmission electron microscopy. Representative images are shown. E, Control H1299 cells (transfected with siCont. or H2BWT) and H2Bub1-depleted H1299 cells (transfected with siRNF20 or H2BK120R) were treated with erastin either with or without a ferroptosis-specific inhibitor, ferrostatin-1 (2 μM), for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.
Global analysis of transcript profiling.Figure 2(A) Heat maps for three subsets of the pattern clustering analysis generated using the Euclidean Distance metric. Upregulated genes are in red and downregulated genes are in green. The downregulating transcriptional activity increases with time. The main branch in clustering of the different Atro mutants is along time points. See also Supplementary Figure 4. (B) Venn diagrams for the subset of genes affected at the two time points for the different Atro versions with respect to control flies. The overlap between all Atro forms and the two polyQ versions increases with time. The genes in common for all Atro versions or between the polyQ Atro mutants at both times are listed.
Figure 1(A) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in wild-type or SAS6 monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m. (B) Schematic overview of the screen design. (C) Schematic showing the procedure for a CRISPR/Cas9 positive selection screen to identify gene knockouts that increase the proliferation of cells with extra centrosomes. (D) Top hits that emerged from the screens ranked by MaGeCK FDR value. Blue hits are genes required for centriole duplication or stability. Green hits are genes predicted to be required to arrest the growth of cells with extra centrosomes. (E) Hits with known role in centriole duplication or stability. (F) Candidate hits responsible for arresting the proliferation of cells with centrosome amplification. Purple hits correspond to PIDDosome genes. Red hits correspond to downstream effectors. Green hits are novel regulators. (G) Top: Graph showing the efficiency of frameshifting INDELs measured using TIDE. N.D. = Not determined. Data shown is from n = 1 biological replicate. Bottom: Graph showing the relative growth of doxycycline treated PLK4Dox cells expressing an sgRNA targeting the indicated genes. Each dot displays measurements from a single experiment. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m. (H) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.
Influence of Spindly on corona assembly and kinetochore recruitment (A) Size-exclusion chromatography and SDS-PAGE of elution fractions of the indicated samples.Figure 7(A) Size-exclusion chromatography and SDS-PAGE of elution fractions of the indicated samples. Each Spindly construct was incubated with RZZ in absence (dotted lines) or in presence (continuous lines) of farnesyl transferase and farnesyl pyrophosphate. Elution shifts of SpindlyF and of the resulting RZZSF complexes is indicative of successful interaction. Two technical replicates were performed. (B) MPS1-induced filamentation experiments on the indicated mChRZZSF complexes (4 µM, further diluted to 0.5 µM for imaging) using a confocal spinning disk fluorescence microscope at 561 nm. Scale bar: 5 µm. Two technical replicates were performed. (C) Schematic of the cell synchronization and imaging experiment shown in D. After electroporation, cells were allowed to recover for 8 hours. Subsequently, cells were synchronized in G2 phase with 9 µM RO3306 for 15 hours and then released into mitosis by inhibitor washout. Before fixation, cells were treated with 3.3 µM nocodazole for 1 hour. (D) Representative images of fixed HeLa cells electroporated with full-length mChSpindly and mChSpindly354-605 constructs in cells depleted of endogenous Spindly by RNAi. Spindly localization was detected with an antibody against the C-terminal region of Spindly. Corona expansion or lack thereof were monitored through CENP-E. CREST serum was used to visualize kinetochores, DAPI to stain for DNA. Scale bar: 10 µm. Experiments were replicated three times. (E) Scatter dot plots representing normalized intensity ratios of the indicated Spindly constructs over CREST for individual kinetochores of cells from the experiment shown in panel D. Red lines indicate median with interquartile range. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000. (G) Kinetochore intensities were quantified like in panel E. Red lines indicate median with interquartile range. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000. (F) Cells treated like in C-D were electroporated with the indicated mChSpindly constructs. Corona expansion was evaluated through the appearance of Zwilch. Experiment replicated three times. Scale bar: 10 µm.