EMBO/sd-character-level-ner huggingface dataset:
PNRC1 interferes with nucleolar RNA metabolism A.Figure 2A. Click-iT RNA imaging assay performed on HeLa cells expressing RFP-PNRC1 or RFP control. Confocal images of transfected cells pulsed with EU for 16 hours were collected at the indicated time points after EU removal (green: Alexa 488-EU, red: RFP, blue: DAPI, scale bar: 5µm). RFP-PNRC1-transfected cells are indicated with arrowheads. Confocal microscopy images of HeLa cells expressing GFP-PNRC1 and stained with antibodies against NPM1 (B) nucleolar proteins (nuclei are stained in blue with DAPI, scale bar: 5µm). Confocal microscopy images of HeLa cells expressing GFP-PNRC1 and stained with antibodies against UBF1 (C) nucleolar proteins (nuclei are stained in blue with DAPI, scale bar: 5µm). A magnification of the merged channel images is provided for UBF1 staining. D. Agarose/formaldehyde gel separation of neotranscribed rRNAs collected from LacZ and PNRC1-expressing cells pulsed for 15 minutes with 3H-uridine and chased for the reported time points (minutes). Samples were loaded according to the amount of incorporated 3H, quantified by liquid scintillation. FU was included as a control of a blocked rRNA processing. E. Ratios between the intensity of 47S pre-rRNA band and bands belonging to 32S or 28S rRNA species relative to panel (D) and measured at 60 minutes after the chase for both LacZ or PNRC1-expressing cells. F. Real Time PCR quantification of 5'-ETS steady-state levels in HeLa cells expressing PNRC1 or GFP control. The average ± SD of 3 biological replicates is shown.
Figure 6A-C Immunoblot analysis stability of ARR1/10/12 and ARR1T553A/10S383A/12S323A proteins under salt stress. Seven-day-old seedlings of 35S:ARR1:MYC, 35S:ARR10:YFP, 35S:ARR12:YFP, 35S:ARR1T553A:MYC, 35S:ARR10S383A:YFP, and 35S:ARR12S323A:YFP were treated with or without 200 mM NaCl for indicated time points. Actin was used as the internal reference. The relative intensity of band detected by anti-MYC or anti-GFP antibody to that by anti-Actin antibody without treatment was set to 1.0. D-I Quantification analysis of stability of ARR1/10/12 and ARR1T553A/10S383A/12S323A proteins under salt stress in (A-C).
Figure 6The top 10 Reactome terms enriched from differential splicing events regulated by PLB. To define the differential splicing events, genes with FPKM > 1 in at least one differentiation stage were selected. Among them, the differential splicing events were identified by the double cutoffs of delta PSI > 0.2 and FDR < 0.05. GSEA analysis of NOTCH signaling in isoform level between DMSO-control and 2.5 nM of PLB treated cells. The bar graph representing the percentage of CD31+CD34+ cells at day 5 of differentiation after treatment with DMSO and various concentrations of DAPT starting from day 2.5. The heatmap plotting the expression of NUMB_S and the key components in NOTCH signaling in day 2-APLNR+ cells, day 5-DMSO treated and day 5-PLB treated cells (2.5 nM), respectively. The NOTCH components were divided into groups of upregulated, downregulated, and unchanged by comparison between DMSO and PLB treated cells. The heatmap was scaled with Z-Score using the log2(FPKM) expression of indicated genes. n=2 technical replicates RT-qPCR assay showing the expression of HES1 mRNA in the GFP+ cells on day 5 of differentiation with 1.25 nM PLB supplementation alone or in combination with NUMB_S ectopic expression from day 2.5. RT-qPCR analysis confirmed the overexpression of HES1 mRNA after DOX induction. The ACTB gene was used as a control. The data was normalized to the mRNA level in empty vector control cells. Western blotting showing the level of HES1 overexpression after DOX induction. The GAPDH gene was used as a loading control. The representative FACS plots indicating the generation of CD31+CD34+ EPCs on day 5 of differentiation with or without HES1 overexpression. DOX was treated from day 2.5 to 5 to induce HES1 overexpression. Statistical analysis of the percentage of CD31+CD34+ EPCs of (H). A hypothetical model. During hematopoietic differentiation deriving from hESCs, a splicing factor switch occurs during the transition from mesoderm APLNR+ cells to EPCs. Concomitant with the switch of components in the splicing machinery, the splicing regulator SRSF2 is downregulated, leading to the enrichment of an EPC-induced NUMB_S isoform. Alternative splicing of NUMB can precisely control NOTCH activity, probably by suppressing HES1 expression and, ultimately, regulate EPC specification. The blue and red colors represent the two switching clusters within splicing factors during hematopoietic differentiation.
Figure 8(A) Scheme of the protocol to determine differential gene expression after treatment of HUVECs 24h with COCO. (B) Differential gene expression heatmap generated by DESeq2 of top-altered genes of HUVECs treated with COCO compared to PBS-treated control cells (n = 4 independent samples/group). (C) GSEA pathway analysis of control vs COCO-treated HUVECs for TGFβ, BMP or ROS pathways.
a) Induction of autophagy in response to 5-FU in the GFP::LGG-1 reporter strain fed on RNAi targeting the indicated DNA repair genes (scale bars, 10 μm).f4a) Induction of autophagy in response to 5-FU in the GFP::LGG-1 reporter strain fed on RNAi targeting the indicated DNA repair genes (scale bars, 10 μm). (b) The fraction (as % of untreated control) of hermaphrodites having embryos with excessive GFP expression following 5-FU treatment. Autophagy induction in independent experiments (circles) and the mean (line) are presented. (c) F1 survival after control (black diamonds) or bec-1 RNAi (red squares) in N2 and msh-6(pk2504) mutants. The survival curve shows the mean±s.d. for each data point from three independent experiments.
Fig.Figure 5A,B) Mean baseline-detrended bioluminescence profiles from plate wells containing either PER2::LUC U2OS cells with (WT, left panel) or without REV-ERBα (KO, right panel) recorded at 37°C before and after transfer to 18°C for 24h (A) or 5h (B) (grey region) from biological repeat 1. 6 differently colored profiles represent cells synchronized at 6 distinct phases of the circadian period prior to the start of 18°C exposure (time zero). C) Schematic showing the measurement of each profile from time zero to the peak marked "x" (this is the second peak after 18°C exposure or the corresponding peak in cells kept at 37°C Plotting these times shows the effect of 18°C exposure on the spread of these profiles. D) Boxplots and individual points for times for each plate well profile for both WT and KO cells kept at 37°C or transferred to 18°C for 24h (top panel) or 5h (bottom panel) from biological repeats 1 and 2. Points are colored according to their distinct phase prior to 18°C exposure. Colored lines (bottom panel) show the change in the mean for the points from each phase. Boxplots display median (central line), interquartile range (IQR, box edges) and range (whiskers). Number of wells: top panel WT cells: 37°C (120), 18°C 24h (119); top panel KO cells: 37°C (120), 18°C 24h (120); bottom panel WT cells: 37°C (58), 18°C 5h (120); top panel KO cells: 37°C (60), 18°C 5h (120). E) Bar graphs showing the mean amplitude change (left axis) following 18°C exposure for 5h for each group of plate well profiles (grouped according to their distinct phase (a-f) prior to 18°C exposure, 10 wells per group) for both WT and KO cells from biological repeats 1 and 2. Mean changes are plotted to approximately align with the position of the control profile at the point of rewarming within the circadian period that is represented by the PER2:LUC (dashed line) and predicted REV-ERBα expression level (right axis) (grey shaded region (light grey indicates REV-ERBα deletion)).
Figure 3Aliquots from agonist-treated samples shown in Figure 2 were deglycosylated with PNGaseF, run on SDS-PAGE and immunoblotted with antibodies detecting arrestin (upper panels, α-HA) and the GPCRs (as indicated, CRF1R is equipped with a N-terminal FLAG, V2R carries a C-terminal 1D4 epitope).
Figure 6A, B Representative kymographs of WT (A) and Arghap45−/− (B) naive CD4+ T cells on 2D surface coated with ICAM-1 and CCL21. C, D Top, representative sequences of cell contours during migration for 466 s of WT (C) and Arghap45−/− (D) naive T cells on 2D surface coated with ICAM-1 and CCL21. The color scale highlights each time-resolved shape. Rectangle length, 112,6 μm (WT) and 51,6 μm (Arghap45−/−). Bar, 10 µm. Botttom, three representative time-resolved shapes and their corresponding calculated contours are shown. Bar, 10 µm. E, F Histograms of instant eccentricities (left) and 2D plots of adherent surface versus speed (right) of WT naive T cells on 2D surface coated with ICAM-1 and CCL21. In the 2D plot, the 3 populations discussed in the results have been highlighted: a, hardly motile (instant speed <5 µm/min) cell population with low (projected adherent area < 20%,) adhesion, b, hardly motile (instant speed <5 µm/min) cell population with high (projected adherent area > 40 %) adhesion, and c, highly adherent (projected adherent area >20 %) cell population with elevated speed (ranging between 15 and 30 µm/min). Data information: In (E) and (F), histograms of instant eccentricities correspond to > 60 cells and > 2000 contours, and fitted by a double Gaussian for WT naive T cells (means = 0.54, 0.8 and SD = 0.18, 0.06) and a single Gaussian for Arghap45−/− naïve T cells (Mean=0.58, SD =0.18). 2D plots correspond to > 60 cells and > 2000 events. In (F) events which combined null motility and adherence over the recorded time have been excluded. G, H Histograms of instant eccentricities (left) and 2D plots of adherent surface versus speed (right) of Arghap45−/− naive T cells on 2D surface coated with ICAM-1 and CCL21. In the 2D plot, the single adherent (projected adherent area > 25 %) and slow (instant speed < 15 µm/min) cell population is highlighted. Data information: In (H), events which combined null motility and adherence over the recorded time have been excluded.
Figure 2A. Distribution of Oct4, Nanog, Sox2, Suz12 and Pol II ChIP-seq reads around the transcription start sites (TSS) of the mESC-specific SATS (left) and common (right) isoforms in mESCs. B. Occupancy frequency of key regulatory factors on the promoter regions of the mESC-specific SATS and common isoforms. Numbers in boxes represent the number of isoforms bound by each factor. Three stars, double stars and one star represent p-values less than 1e-5, 1e-2 and 0.05, respectively (Fisher's exact test). C, D. Venn diagrams representing the presence of Oct4, Sox2 or Nanog binding sites on the promoter regions of the mESC-specific SATS (C) and common (D) isoforms. E, F, G. Expression changes of the mESC-specific SATS isoforms and their corresponding common isoforms in Oct4 (E) knockdown mESCs detected by qRT-PCR. Variation bars represent the standard error of the mean relative expression levels (SEM) of three independent biological replicates. The double stars and one star represent p-values less than 0.01 and 0.05, respectively (Two-tailed Student's t test). E, F, G. Expression changes of the mESC-specific SATS isoforms and their corresponding common isoforms in Sox2 (F) knockdown mESCs detected by qRT-PCR. Variation bars represent the standard error of the mean relative expression levels (SEM) of three independent biological replicates. The double stars and one star represent p-values less than 0.01 and 0.05, respectively (Two-tailed Student's t test). E, F, G. Expression changes of the mESC-specific SATS isoforms and their corresponding common isoforms in Nanog (G) knockdown mESCs detected by qRT-PCR. Variation bars represent the standard error of the mean relative expression levels (SEM) of three independent biological replicates. The double stars and one star represent p-values less than 0.01 and 0.05, respectively (Two-tailed Student's t test).
Figure 2A. Tau immunoreactivity (magenta) specifies the cortical plate (CP) surrounding the luman (L) (i). Selected optic sections at high magnification (ii and iii) and highresolution imaging (iv) show Tau localization only in axons of cortical neurons. Note the somas of neurons are free from Tau protein. At least eight organoids from four different batches (n=4) are tested. Figures display scale bars. B. Tau localization in SARS-CoV-2-positive neurons (AB4, green) in selected optic sections (i). Note, in contrast, to control groups, SARS-CoV-2-exposed organoids display mislocalized Tau (magenta) majorly into the somas of neurons (arrowheads). Selected confocal slices are shown to distinguish Tau mislocalization into neuronal soma (arrowheads). At high magnification, neuronal soma is further specified by the perinuclear localization of SARS-CoV-2 (green) (ii and iii). Bar diagrams at right quantifies the percentage of neurons (Mock and SARS-CoV-2 exposed) exhibiting mislocalized Tau (iv) and the fraction of SARS-CoV-2-positive neurons exhibiting Tau-positive axons spanning different cortical areas (v). For statistics, at least 300 cells from six organoids from four different batches (n=4) were tested. Figures display scale bars. Unpaired t test with Welch's correction, ***P<0.001. Data presented as mean + SD. C. Schematic cartoon of differential Tau distribution in mock compared to SARS-CoV-2-positive neurons. In mock, Tau is sorted mainly to axons. In SARS-CoV-2 positive neurons, Tau is missorted to the soma (determined by Pan-Tau antibody). Furthermore, phosphorylated Tau (at T231) majorly localizes in the soma (bottom panel, determined by Tau AT-180 antibody, see below). D. In contrast to controls (i), Tau AT180 antibody (magenta) that specifically recognizes the phosphorylated Threonine 231 of Tau protein distinctly localizes at the somas of SARS-CoV-2-positive neurons (AB4, green) (ii). At least four organoids from two different batches (n=2) are tested. Figures display scale bars. E. Co-localization of SARS-CoV-2 (AB4, green) and phosphorylated Tau protein (magenta) at somas of cortical neurons revealed by high-resolution imaging and de-convolution. Representative images from at least 300 cells examined. Figures display scale bars. F. The bar diagram quantifies the fraction of Tau AT180-positive neurons that co-localize with SARS-CoV-2-positive neurons. For statistics, at least 250 cells from four organoids and two independent batches (n=2) were examined. Unpaired t test with Welch's correction. Data presented as mean + SEM.
Figure 5(A) LN229 cells stably transduced with Flag-tagged wild-type Merlin or the S518D mutant were treated with thapsigargin and subjected to immunoprecipitation with a Flag antibody and followed by SDS-PAGE. Outlined gel pieces were analyzed by mass spectrometry. (B) Numbers of unique peptides of Merlin or Ubiquitin from each outlined gel pieces in (A) are shown. (C) The percentage of ubiquitinated forms for each peptide from each immunoprecipitated product are shown. (D) and (E) Mass spectrometry spectrums of the indicated peptides from each immunoprecipitated product are shown. (F) Merlin-depleted LN229 cells expressing His-Ubiquitin were stably transduced with the indicated HA-tagged Merlin forms or an empty vector. These cells treated with DMSO or thapsigargin were subjected to nickel affinity purification in a denaturing step followed by western blotting for HA. Arrows indicate mono-ubiquitinated Merlin. The red asterisk indicates native Merlin and black asterisks indicate non-specific signals. The ratio of mono-ubiquitinated to native Merlin in each lane of the input blot was quantified by Image J and is shown under the blot. (G) Merlin-depleted LN229 cells were stably transduced with an empty vector, Flag-tagged wild-type (WT) Merlin or its K396R mutant. These cells were detached (S, suspension) by trypsinization using the procedure described in the Methods section and reseeded (A, attached) for two hours. Cell lysates were subjected to immunoprecipitation with a Flag antibody followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot. (H) Merlin-depleted LN229 cells stably transduced with wild-type (WT) Merlin or its K396R mutant were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. (I) Merlin-depleted LN229 cells stably transduced with HA-tagged wild-type (WT) Merlin or its K396R mutant were treated as described in (G) and subjected to western blotting. The ratio of mono-ubiquitinated to native null in each lane was quantified by Image J and is shown under the blot.
Figure 2A. Cumulative growth curves of trisomic and wild-type ES cells. The number of ES cells was counted at the indicated days in culture. The cell lines were tested in triplicates. Error bars, ±S.D. * P < 0.05, ** P < 0.01. B. Cell cycle distribution of trisomic and wild-type ES cell lines. The percentage of cells in G1, S, and G2/M phases was determined via flow cytometry. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01. C. The percentage of Annexin V+ apoptotic cells was quantified. Error bars, ±S.D. n=3. D. Trisomic and wild-type ES cells were treated with Cycloheximide or MG132 for 48 hours at the indicated doses. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.
Figure 5(A) Scheme of PhotoClick cholesterol methodology to detect C99 interaction with cholesterol. (B) Representative immunoblot to reveal C99 levels in total homogenate (TH) and MAM fractions of APP-DKO cells expressing C99WT or C99MUT before (input) and after cholesterol pull-down. Acsl4 was used as a MAM marker. Streptavidin-HRP was used to detect total biotinylation (biotin conjugated to PhotoClick cholesterol). (C) Quantification of pulled-down C99 levels versus input from the experiment in B. Two-way repeated measures ANOVA (Fraction, mutation) (n=5; * p< 0.05, ** p< 0.01). (D) Immunoblot showing the levels of pulled-down PhotoClick Cholesterol (streptavidin-HRP) in ER and MAM fractions. Note how the levels of pulled-down C99 in the ER are negligible when compared to those from MAM. Isolated MAM and ER fractions were assessed by western blot (shown in AppendixFig S1E). (E) Quantification of free cholesterol levels (FC) analyzed by lipidomics after subcellular fractionation to obtain MAM from the indicated cells. Lipid units are represented as molar mass over total moles of lipids analyzed (mol%). Graphs represent percentage over controls. Dashed line represents control levels. One-way ANOVA. (n=4; * p< 0.05) (F-G) Quantification of cholesterol uptake and esterification in the indicated cells was measured by 4h incubation with 3H-cholesterol and subsequent analysis of radiolabel incorporation. The dashed line indicates control levels. Graphs represent fold change over controls. Treatment with Sandoz 58-035, a specific ACAT1 inhibitor, caused a ~95% reduction in cholesterol esterification. EV, empty vector. One-sample t-test. (n=3-4; * p< 0.05). (H) Ratio of cholesteryl esters:free cholesterol (CE:FC) in the indicated cells.
Hormesis of low-dose 5-FU in pancreatic cancer cells and interaction with OMP.Figure 2  The cell lines ASPC-1, MiaPaCa-2 , Colo357, Panc-1, Panc89 and PancTu1 were untreated or treated with 5-FU alone or in combination with the indicated concentrations of OMP for 4 days. At lower doses of 5-FU alone (black lines, 0 µg/ml OMP) a growth-stimulatory effect (hormesis) was observed in the cell lines ASPC-1, Panc-1 and PancTu-1. The data points show the means of 8 measurements. The curves are fitted in these cell lines using the Brain-Cousens model (see sub-section 4.11). In MiaPaCa-2 and Panc-89 cells no hormesis occured, these curves were fitted using a three parameter logistic model. In Colo357 cells the curves could not be fitted by any pharmacodynamic model due to large standard errors at these lower concentrations (data points shown). The red, green and blue lines indicate the dose-effect curves of 5-FU when various concentrations of OMP were added (10, 20 and 40 µg/ml, respectively). In ASPC-1 and Panc-1 cells the hormesis of 5-FU was reversed and in PancTu-1 cell it was mitigated by OMP depending on the concentration of 5-FU. In MiaPaCa-2 cells we found an additive interaction of 5-FU with OMP. In Panc-89 cells the interaction was antagonistic.
Model of Wnt's mechanism of action on TNT formation.Figure 9In the absence of Wnt (A), dephosphorylated βCaMKII is attached to actin (G- and F-actin), helping to stabilize the actin cytoskeleton by impairing the access of actin polymerizing factors. In the presence of Wnt (B), βCaMKII gets rapidly phosphorylated and detaches from actin. Actin-polymerizing factors can now promote the remodeling of the actin cytoskeleton, which could generate new TNT structures. After dephosphorylation of βCaMKII, several hours after Wnt stimulation (C), βCaMKII reattaches to actin and it accumulates at the bases of TNTs, thus contributing to the stabilization of this protrusions. The increase in TNT number and duration, could impact the intercellular transfer of cargoes occurring through them.
Figure 1A. Confocal images of mitochondrial morphology in wild-type (WT) and Drp1-/- 293T cells transfected with empty vector (left panel) and Myc-hFis1 (right panel), stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green). B. Quantitative analyses of fragmented mitochondria size (mean area (μm2) per mitochondrion) after Myc-hFis1 overexpression in WT and Drp1-/- 293T cells using Image J software (Particle analysis) in three independent experiments. In each cell, only dispersed individual mitochondria were analyzed. The total number of mitochondria (mito) analyzed is indicated for each condition. C. Quantitative analysis of mean mitochondria number per cell in WT and Drp1-/- 293T cells overexpressing Myc-hFis1 using Image J software (Particle analysis) in three independent experiments. D. Schematic representation of hFis1 mutants used in this experiment. E. Percentages of cells with indicated mitochondrial morphologies in WT and Drp1-/- 293T cells transfected with empty vector (control), Myc-hFis1 and either Myc- or GFP-tagged mutants as indicated in three independent experiments.
Figure 4Migration assays (24 h) of MM-231-shLSD1-WT/R838A/R838K/R838F cells (A) with or without MS049 treatment. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars indicate the mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test). invasion assays (48 h) of MM-231-shLSD1-WT/R838A/R838K/R838F cells (B) with or without MS049 treatment. Representative micrographs of invaded cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars represent mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test). Migration assays (24 h) of MCF7-LSD1-WT/R838A/R838K/R838F cells (C) with or without MS049 treatment. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars indicate the mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test). D invasion assays (48 h) of MCF7-LSD1-WT/R838A/R838K/R838F cells (D) with or without MS049 treatment. Representative micrographs of invaded cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars represent mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test). E MM-231-shLSD1-WT/R838A/R838K/R838F cells (1×106) were injected into the tail veins of 5-week-old female nude mice, and 7 weeks later, mice were sacrificed with euthanasia and lungs were fixed in paraformaldehyde solutions. The number of visible surface metastatic lesions in lungs was counted (mean of 7 mice) (Data are mean ± SD, n = 7 mice for each group, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test). F Lung metastatic nodules confirmed by haematoxylin and eosin staining. Scale bars, 100 μm.