EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A) Genome-wide screen and internalization assay workflow. Library-transduced Ramos Cas9 cells were incubated with fluorescent, biotinylated surrogate antigen, anti-IgM F(ab')2 (total antigen) and subsequently stained with fluorescent streptavidin (surface antigen). Flow cytometry plots show a control sample incubated on ice preventing anti-IgM F(ab')2 endocytosis and an experimental sample from which cells displaying high and low anti-IgM F(ab')2 internalization were flow sorted and analyzed for gRNA abundance. (B) GeCKO library results showing mean internalization score per gene versus statistical significance. Whole-genome data come from two independent repeats. Data information: genes with annotation related to endocytosis or intracellular trafficking are highlighted. Red denotes examples of positive regulators; green denotes examples of negative regulators of internalization. (C) Brunello library results. Whole-genome data come from two independent repeats. Data information: genes with annotation related to endocytosis or intracellular trafficking are highlighted. Red denotes examples of positive regulators; green denotes examples of negative regulators of internalization. (D) Minilibrary validation results from four independent experiments. Validated candidates with internalization score <-0.4 or >0.35 and q<0.05. Data information: genes with annotation related to endocytosis or intracellular trafficking are highlighted. Red denotes examples of positive regulators; green denotes examples of negative regulators of internalization. (E) Selected validated candidates grouped by functional annotation.
Figure 2A-C. Representative photomicrograph of young (6-8 weeks) and aged (>12 months) livers 40 h post PH stained with Ki67 and visualized by IHC and H E for histology. Sections were scanned using Histech Pannoramic MIDI Scanner and Ki67 positive cells were quantitated with Quant Center 2.0 software. A proliferation index (p.i.) was calculated based on the number of Ki67 positive hepatocytes to total hepatocytes and livers were classified as (+) regenerating, p.i.10% or (-) non-regenerating, p.i.10%.D. Number of young (6-8 weeks) and aged (12 months) mice with (-) non-regenerating and (+) regenerating or livers based on p.i., 40 h post PH. Animals deemed non-surviving for ethical reasons were euthanized 24 hours post PH. Representative results of a single experiment with n=9 aged and n=6 young animals per group.
Depletion of Rab7 GAP TBC1D15 disrupts the bilateral architecture, morphology and dynamics of late compartments.Figure 6Identification of a Rab7 GAP that promotes endolysosomal system's bilateral architecture along the PN/PP axis. A. Graphical summary depicting the functions of 3 known TBC domain-containing GAPs for Rab7. B. Representative confocal images of fixed HeLa cells depleted of TBC1D2, 1D5, or 1D15 using siRNA oligo pools and immunolabeled against CD63 (white), scale bar: 10µm. C. Plot of CD63 pixel distribution as a function of TBC1D2/1D5/1D15 depletion expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n≥8 cells per condition analysed from 2 independent experiments. D/E. Rescue of TBC1D15 depletion phenotype. D. Representative confocal images of fixed HeLa cells transfected with either control siRNA (siC) or oligo #3 targeting TBC1D15 (siTBC1D15) and ectopically expressing either GFP-EV, GFP-TBC1D5 or siRNA-resistant GFP-TBC1D15res (green), immunolabeled for CD63 (magenta), scale bar: 10µm. E. Quantification of rescue expressed as % cells (average) in the population exhibiting one of 3 phenotypes: clustered, dispersed or intermediate; total numbers of cells analysed per condition appear above each bar, n=3 independent experiments. Effects of TBC1D15 depletion on the morphology and dynamics of late organelles. F. Representative electron micrographs of fixed HeLa cells transfected with either control siRNA (siC) or a pool of oligos targeting TBC1D15 (siTBC1D15) are shown, scale bars as indicated. G. Analysis of late compartment dynamics as a function of TBC1D15 depletion. Tracks followed by SiR-lysosome-positive vesicles during a time-lapse lasting 255 sec (5 s per frame), with highest displacement rates for each track depicted on a rainbow colour scale (blue: immobile; red: maximum mobility per time interval). Zoom insets (3x) highlight select peripheral (PP) and perinuclear (PN) regions, scale bar: 10µm EV10). H. Quantification of high motility fraction (% tracks with displacement rates above 0.9µm/sec), n siC=27, n si1D15=37 images (2≥ cells per image) analysed from 2 independent experiments. Effectiveness of TBC1D15 (si1D15) depletion is confirmed by immunoblot against endogenous TBC1D15 (e1D15) following its immunoprecipitation (IP), TL: total lysate. I. FRAP of GFP-Rab7 in live HeLa cells transfected with either control siRNA (siC, black line) or a pool of oligos targeting TBC1D15 (si1D15, red line). Plotted is average GFP-Rab7 signal recovery during 200 s following bleaching, expressed as % of pre-bleach signal, n=3 bleach regions per sample.
Figure 7A Preys identified showing significant enrichment by N proteins specifically from one or more human coronaviruses. PSM, peptide-spectrum match. B Pulldown and Western blot validation of the interaction between N protein and G3BP1/G3BP2. Cells transfected with vector or construct encoding GFP or N protein from different human coronaviruses were compared in these experiments. C,D Effect of SARS-CoV-2 N protein on stress granule formation. A549 (C) and MCF10A (D) cells were transfected with pinducer20-N protein of SARS-CoV-2. All the cells are treated with sodium arsenite but with or without Dox to induce N protein expression. Cells were fixed and stained with the indicated antibodies. The green signal is G3BP1, the red signal is HA (for N protein), and the blue signal indicates DAPI/nuclei. Scale bar: 10 µm. E Quantification of the stress granule formation in A549 and MCF10A cells. Total of 30 cells were counted and a student's t test was used for the statistical analysis (***, p<0.001). Box limits represent 25th percentile and 75th percentile; horizontal line represents median. Whiskers display min. to max. values.
Figure 6Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids. Twenty-four hours after transfection, cells were treated with cycloheximide (CHX, 100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (upper panel). The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 3 for both groups). Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids or cotransfected with SOCS1-MYC + OTUB1-GFP plasmids. Twenty-four hours after transfection, cells were treated with CHX (100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (upper panel). The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 3 for both groups) (mean + SEM). Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were cotransfected with SOCS1-MYC and OTUB1-GFP/OTUB1-∆N-GFP/OTUB1-C91S-GFP plasmids. Twenty-four hours after transfection, cells were treated with CHX (100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (left panel). The right panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 4 for both groups). Data represent mean + SEM. Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were stimulated with IFN-γ (10 ng/ml) for four hours. Thereafter, cells were treated with CHX (100 μg/ml) for indicated times. Whole cell lysates were analyzed by WB with indicated antibodies. Primary astrocytes from OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids. Five hours later, astrocytes were subsequently transfected with different siRNAs or left untreated. Twenty-four hours after siRNA transfection, protein was isolated and analyzed by WB for SOCS1 expression. Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were transfected with nonsense siRNA and SOCS1 siRNA-2. Twenty-four hours after transfection, cells were stimulated with IFN-γ (10 μg/ml) for 16 hours before mRNA isolation. Quantitative real-time PCR was performed to detect mRNA levels of CXCL10, CCL2, and NOS2 (n = 4 for all groups). Data show mean + SEM.
Alteration of TMED2 expression and relationships with eAGR2-mediated monocytes attraction.Figure 6A) Representative histological H&E staining of sections of the proximal colon and ileum of WT and TMED2 hypomorph mice. B) Immunohistological analysis of AGR2 and MUC2 expression in the proximal colon of WT and TMED2 hypomorph mice. C) Semi-quantitative analysis of AGR2 and MUC2 expression in the proximal colon of WT (blank bars) and TMED2 hypomorph mice (black bars) (n=6). D) Freshly isolated PBMCs were placed in Boyden chambers towards media conditioned by cells overexpressing AGR2 WT, E60A, ∆45 or AGR2 AA mutants and incubated for 24 h. Migrating cells were then characterized and quantified by flow cytometry using FCS/SSC parameters or CD14 monocyte marker. (**): p=0.0016. E and F) Freshly isolated PBMC were placed in Boyden chambers towards media conditioned by cells overexpressing TMED2 and either AGR2 WT (E) or AGR2 AA mutant (F) and incubated for 24 h. Migrating cells were then characterized and quantified For E (*): p=0.0461 (AGR2WT/TMED2) and 0.018 (AGR2WT/TMED2 + blocking Ab) respectively; (**): p=0.0053 and (***): p=0.0048; for F (***): p=0.0007. G) Freshly isolated PBMC were placed in Boyden chambers towards media conditioned by cells silenced for TMED2 and incubated for 24 h. Migrating cells were then characterized and quantified (*): p=0.0149. H) Freshly isolated PBMC were placed in Boyden chambers towards decreased doses of recombinant AGR2 and incubated for 24 h. CCL2 cytokine was used as positive control for monocyte migration. ns: non-statistically significant, (*): p=0.0171, (**): p=0.0084, (***): p=0.0003. I) Impact of AGR2 blocking antibodies on AGR2-mediated monocytes migration was tested using Boyden chambers as described above. The concentrations of recombinant human AGR2 was of 200 ng/ml and decreasing amounts of antibodies were used from 20 µg to 1 µg. The non-relevant antibody (Isotype) was used at the maximal dose of 20 µg. Data are representative of three independent experiments. ns: non-statistically significant, (*): p=0.0165 (Ab=20μg/ml), 0.0223 (Ab=10μg/ml) and 0.0493 (Ab=5μg/ml), respectively.
Figure 3(A-C) HeLa cells were transfected with the indicated siRNAs. After 48 h, the cells were transfected with TFEB-EGFP (green) for 24 h. The cells were stained with anti-mTOR (red) antibody and DAPI (blue), and then visualized using confocal microscopy. Regions within the dotted boxes are magnified in the insets. Scale bar, 5 µm. The quantification data of TFEB puncta and nuclear localization are shown in Figure 3B and Figure 3C respectively. Data from three independent experiments represented as means ± S.E.M., **, p<0.01, one-way ANOVA. (D) HEK 293 cells were transfected with the indicated siRNAs. After 48 h, the cells were re-transfected with LAMP1-RFP (red) and constitutively active HA-GST tagged Rag GTPase mutants (RagA Q66L + RagC S75L = RagAGTP + RagCGDP) for 24 h. The cells were stained with anti-HA (green) antibody and DAPI (blue). Cells were visualized using microscope IX71. Regions within the dotted boxes are magnified in the insets. Scale bar, 5 µm. (E) HEK 293 cells were transfected with siRNA targeting TDP-43. After 48 h, the cells were re-transfected with EGFP tagged TFEB-WT, TFEB-S211A, TFEB-Q10A/L11A or TFEB-Δ30, along with LAMP1-RFP for 24 h. Cells were visualized using microscope IX71. Regions within the dotted boxes are magnified in the insets. Scale bar, 5 µm. (F) HEK 293 cells were transfected with the indicated siRNAs. After 48 h, the cells were re-transfected with EGFP tagged TFEB-WT or TFEB-R245-247A, along with LAMP1- RFP for 24 h. Then the cells were incubated with Torin-1 (250 nM) for 1 h, Earle's Balanced Salt Solution (starvation) for 2 h or incubated with both. Cells were fixed and visualized using confocal microscopy. Regions within the dotted boxes are magnified in the insets. Scale bar, 5 μm.
Effect of rpb1‐1 and rpb1‐S751F on replication velocity Analysis of replication by BrdU incorporation.Figure 5Analysis of replication by BrdU incorporation. Cells were synchronized in G1 with α‐factor and released in the presence of BrdU. Samples were taken at the indicated times and processed to measure BrdU incorporation by ChIP analysis. Replication was analyzed in two DNA regions, one covering the early origin ARS508 and the SPF1 gene and the other the late origin ARS603 and the YFL034W gene. DNA combing analysis of replication. Graphs indicate the distribution of BrdU track length (kb) at 60 and 120 min after α‐factor release in the presence of 40 mM HU, the replication speed (kb/min) determined with the two time points analyzed and the inter‐origin distance (kb) at 60 min after release. The median test was applied.
Figure 3(A) The miR-195 binding site as predicted by TargetScan. (B) MICU1 3'UTR in LightSwitch™ 3′UTR Reporter Vector (wild type or miR-195 binding site deleted) was co-transfected with either non- target miR control (miR-CTL) or different concentrations of miR-195. Post 24h transfection luciferase (Renilla) activity was measured. Relative light units (RLU) compared to miR-CTL were plotted. Data are mean ± S.D. n=3 (Biological repeats). *P<0.05, Student's t-test. CP20 cells were transfected as indicated, 48 h post transfection 4x106 cells were permeabilized, and extra-mitochondrial calcium ([Ca2+]out) clearance was measured, representative traces of [Ca2+]out‑ clearance in miR-CTL and miR-195 transfected cells. [Ca2+]out pulses and FCCP were delivered as indicated. CP20 cells were transfected as indicated, 48 h post transfection 4x106 cells were permeabilized, and extra-mitochondrial calcium ([Ca2+]out) clearance was measured, representative traces of [Ca2+]out‑ clearance in control siRNA (siCTL) and siMICU1 transfected cells. [Ca2+]out pulses and FCCP were delivered as indicated. (E) Bar graph illustrating the number of Ca2+ pulses handled by control miR, miR-195, siCTL and siMICU1 transfected cells. Mean ±SEM; n=3-4 (Biological repeats). *P<0.05, Student's t-test. (F) Representative [Ca2+](m) traces after addition of FCCP (10 µM) in permeabilized CP20 cells transfected with non-target miR-CTL or miR-195. (G) Quantification of resting matrix [Ca2+](m) after addition of FCCP. n=5 (Biological repeats) *P<0.05, Student's t-test, (H) Representative traces of mitochondrial membrane potential (∆Ψμ) in CP20 cells transfected with miR-CTL or miR-195. (I) Quantification of relative ∆Ψμ. (n=5, Biological repeats). (J) Intracellular lactate was measured in miR-CTL, miR-195, siCTL, and siMICU1 expressing OVCAR4, CP20, and OVSAHO cells. n=3, biological repeats *P<0.05, Student's t-test. (K&L) miR-CTL, miR-195, or miR-195 + pLYS1-MICU1-Flag transfected cells were stained with MitoSOX Red, and mitochondrial ROS levels were determined by flow cytometry. The histogram shows representative staining and bar graph (right) shows results of three independent experiments. Mean ± SEM; . n=3, biological repeats. *P<0.05 when comparing with miR-CTL, # P<0.05 when comparing with miR-195 by Student's t-test.
Figure 1C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. (A) Chemical structures of heme (HO-1 substrate) and CoPP (HO-1 in vivo inducer). C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Heme degradation reaction catalyzed by HO-1. C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Heme oxygenase activity is increased by CoPP and decreased by SnPP in the liver as measured by gas chromatography. C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Total leukocyte and red blood cell count in PB. CoPP increases the number of all WBC populations. Granulocyte and monocyte percentage are increasing, whereas lymphocyte percentage is decreasing among PB leukocytes after CoPP. C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Heatplot of the cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases the concentrations of a group of cytokines (red box). C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Selected cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases concentrations of G-CSF, MCP-1, IP-10, IL-6 and IL-5. Results are shown as mean + SEM, one-way Anova with Bonferroni post-test, n = 5 mice per group.
Model for DNA end resection by the Sgs1-RPA-Dna2 complex A Sgs1-Dna2 complex binds first to a 5' DNA overhang assisted by RPA.Figure 6A Sgs1-Dna2 complex binds first to a 5' DNA overhang assisted by RPA. At the DNA junction both helicases start to move unidirectionally on either of the strands. Due to the faster movement of Sgs1, this helicase powers the unwinding of the DNA duplex and causes the formation of a ssDNA loop in front of Dna2. Upon an occasional strand switch of Sgs1, the protein rewinds the DNA and moves towards Dna2. This leads to shortening of the loop. Upon encounter of Dna2, Sgs1 switches back to the original strand to power further unwinding.
Figure 2(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons transfected with GFP-RFP-LC3B and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Right panel, quantification of LC3B puncta. (B) Upper panel, immunoblot analysis of endogenous LC3B (Map1lc3b), C9orf72 and control Actin of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Lower panel, real time RT-qPCR quantification of endogenous C9orf72 mRNA expression relative to Rplp0 mRNA. (C) Left panel, representative images of immunofluorescence labeling of endogenous P62 (Sqstm1) on organotypic cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of P62 aggregates. (D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting C9orf72 and plasmids expressing control GFP, HA tagged wild type or mutant RAB39b (CA, Q68L or CN, S22N), RAB8a or RAB7. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.
Figure 5A. 20µg of the indicated bsAbs were injected i.v. into 57BL/6 mice and serum concentrations were measured at the indicated time points after injection using the Promega assay described in the methods section. Mean values and standard deviations obtained from groups of four animals per time point are indicated. B. 64Cu labeled bsAbs were injected into SCID mice carrying established DU145 (PSMA-negative, marked by "1") and 22Rv1high (PSMA-positive, marked by "2") tumors on opposite flanks. MRT and PET scans were obtained after 48 hours. C-F. 64Cu labeled bsAbs were injected into NSG mice carrying 22Rv1high tumors (5 animals per group), and uptake at the indicated locations was determined by PET quantification over time (C, D) and at termination of the experiment after 48 hours (E, F) as described in the methods section. Error bars represent standard deviation of the mean value.
Q-dependent phenotypes in cultured cell lines.Figure 4A The volcano plot shows differentially translated transcripts against adjusted p-values for SF compared to SF+q. Red dots indicate padj< 0.1 in multiple tests. Data information: S (standard medium), SF (serum free medium), q (queuine) B Comparative analysis of differentially translated mRNAs of both experimental groups (SF compared to SF+q and SF compared to SF+qR) using Ingenuity pathway analysis identified EIF2 signaling as the most significantly affected canonical pathway. Data information: S (standard medium), SF (serum free medium), q (queuine) C Representative polysome profiles of HeLa cells grown in S and SF medium in the absence or presence of q. As a measurement for the global translation rate the fraction of polysomal ribosomes was quantified; *p <0.05 (t-test); n=5. Data information: S (standard medium), SF (serum free medium), q (queuine), RO (ribosome occupancy). D Proliferation analysis of HeLa cells grown under the indicated culture conditions. Population doubling levels of three biological replicates were calculated for each time point. Error bars: ±SD; n = 3. Data information: S (standard medium), SF (serum free medium), q (queuine)
miR-155 promotes autophagosome formation.Figure 5(A and B) RAW264.7 cells stably expressing GFP-LC3 were transiently transfected with control or miR-155 mimic for 24 h, and GFP-LC3 puncta (>1 µm) were detected by confocal microscopy (A) and quantified (B). RAW264.7 cells were transiently transfected with control or miR-155 mimic for 24 h. Endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (Green), and LC3 puncta (>1 µm) were detected by confocal microscopy (C) and quantified (D). (E and F) RAW264.7 cells were transiently transfected with control or miR-155 mimic for 24 h, and then incubated with a specific fluorescent dye MDC (50 µM). The MDC-labeled autophagic vacuoles were detected by confocal microscopy (E) and cells with MDC-positive autophagic vacuoles were quantified (F). Cells treated with rapamycin were used as a positive control. Arrows indicate the GFP-LC3 puncta or autophagic vacuoles labeled with MDC; scale bar = 5 µm. Data are shown as the mean ± SEM of three independent experiments (n = 300 cells). **, p<0.01; ***, p<0.001.
Identification of spatial differential gene expression (spDEGs).Figure 6a. Normalized expression of Tox in CA1sp1, Leng8 in CA3sp3, Hecw1 in DG3, and Thra in Astro1 show variable expression throughout the hippocampus. Scale bar is 500 microns. spDEGs were computed by comparing the true variance in gene expression between cell subtype neighborhoods to that of randomly permuted cell (sub) type neighborhoods. b. Histogram of the number of statistically significant spDEGs (Benjamini-Hochberg corrected FDR < 0.05) in each subtype. c. Histogram of the number of subtypes that have an spDEG for each gene.
Figure 9Leukocytes destabilize endothelial junctions by stimulating PECAM-1. Leukocyte-induced stimulation of PECAM-1 triggers the release of SHP2 that directly interacts with and dephosphorylates VE-cadherin-Y731, which is required for the internalization of VE-cadherin. In addition to mobilizing SHP2, leukocytes stimulate Ca2+-signals in endothelial cells, and activate actomyosin-based tension across the VE-cadherin-catenin complex which is needed for Y731 dephosphorylation to occur. Since β-catenin/plakoglobin mask the accessibility of Y731 for SHP2, we propose that leukocytes destabilize junctions by PECAM-1-SHP2-triggered dephosphorylation of VE-cadherin-Y731 which becomes accessible by actomyosin mediated mechanical force exerted on the VE-cadherin-catenin complex.