EMBO/sd-character-level-ner huggingface dataset:
Dendritic branching abnormalities in superficial and deep radial layers of CA1.Figure 2A: Schematic representation of radial layers in the hippocampus. B: left: Biocytin-filled pyramidal cells in the CA1 region. DAPI staining of CA1 bundle allows differentiation between deep (bottom) and superficial (top) CA1 neurons (scale bar: 100 µm). right: Schematic representation of Sholl spheres around the soma. C: Representative 3D reconstructions of CA1 pyramidal neurons from deep (left, dark blue) and superficial (right, light blue) radial layers. D: Sholl analysis reveals no genotype effect on basal dendritic segments of deep CA1 pyramidal neurons (dCA1; LM: n=21, N=7; cTKO: n=21/N=6). E: Compared to littermate controls, dCA1 neurons of NexCre cTKO animals show no significant reduction in total dendritic length in basal dendrites (unpaired Student's t-test, nsp = 0.4979). F: The number of primary basal dendrites is significantly reduced in NexCre cTKO animals compared to LM in dCA1 (Mann-Whitney test, **p = 0.0032). G: G: Sholl analysis reveals no genotype effect on apical dendritic segments of deep CA1 pyramidal neurons (dCA1; LM: n=21, N=7; cTKO: n=21/N=6). H: Compared to littermate controls, dCA1 neurons of NexCre cTKO animals show no significant reduction in total dendritic length in apical dendrites (unpaired Student's t-test, nsp = 0.1053). I: Sholl analysis reveals no genotype effect on basal dendritic segments of superficial CA1 pyramidal neurons (sCA1; LM: n=24, N=7; cTKO: n=24/N=6). J: Compared to littermate controls, sCA1 neurons of NexCre cTKO animals show no significant reduction in total dendritic length in basal dendrites (Mann-Whitney test, nsp = 0.5601). K: The number of primary basal dendrites is significantly reduced in NexCre cTKO animals compared to LM in sCA1 (Mann-Whitney test, *p = 0.0232). L: Sholl analysis reveals a significant genotype effect on apical dendritic morphology of superficial CA1 pyramidal neurons (sCA1; two-way repeated measurements ANOVA: genotype F (1, 40) = 4.643, *p = 0,0373, with post hoc Sidak's multiple comparisons test, *p < 0.05; LM: n=21, N=7; cTKO: n=21/N=6). M: Compared to LM controls, sCA1 neurons of NexCre cTKO animals show a significantly reduced total dendritic length in apical dendrites (unpaired Student's t-test, *p = 0.0358).
Transcriptional profiling of polyQ Atrophins.Figure 1(A) Illustration of the crossing and ageing scheme used to obtain total RNA extracts from fly heads for the transcriptional profiling and all successive qPCR assays. Expression of different Atrophin forms with the GMR driver was induced, owing to a temperature‐sensitive mutant Gal80 repressor. F1 flies were allowed to develop at 18°C; at this temperature the Gal80 repressor keeps transgenes silent. Newly eclosed flies (0-48 h) were collected and killed immediately (0d) or aged for 2 or 14 days at 29°C. This inactivates Gal80 and transgenes are switched on by GMR‐Gal4. This protocol allows comparing siblings that differ exclusively in their age and transgenes expression. Control flies crossed to GMR‐Gal4; UbiGal80ts in all experiments are from the w1118 stock in which all UAS transgenes have been generated. (B) Tangential eye sections of flies representative of all the different populations used in the microarray analysis at all different time points. Weak degeneration is only visible after 14 days with polyQ Atro; in particular with Atro75QN there is an initial loss of photoreceptors (PR, arrow), 30.7% of the ommatidia has lost at least 1 PR, that is only 5.1% of all neuronal PR have been lost at this stage (N=333).
Figure 3Recombination frequencies measured with the pTINV system in WT, rpb1‐1, mre11Δ and rpb1‐1 mre11Δ. Rad52 focus formation in asynchronously growing cells at 30°C. Viability after different times in 10 mM HU. The same cultures without HU were used as a control. The average values and standard deviations from three independent experiments are shown. Rad52 focus formation in asynchronously growing cells expressing normal or high levels of RNH1.
uORF regulatory activity is conserved across vertebrates.Figure 6(A) Cartoon shows strategy for investigating conservation of uORF regulatory activity The ratio of translation between the TLS and CDS is calculated and compared between 1-to-1 homologs in the same tissue type across species.uORF activity is correlated across species. Scatterplots display the translation ratio comparison between the same transcripts (TLS length > 100nt, 1-1 mouse-human homology) in fibroblasts (BJF cells in human - sample bjf2, MEF cells in mouse - sample mef2wt) (B) and brain (samples hbrainwt and brainwt) (D). The correlation between species is not due to CDS signal correlation - scatterplots (C,E) display the translation ratio comparison between homologous transcripts (TLS length > 100nt, 1-1 mouse-human homology) in fibroblasts (B) and brain (D), maintaining CDS pairings while shuffling which TLS is associated with each CDS.
Figure 7(A) Xbp1 splicing analysis (left panel) and quantification (right panel) of bone marrow-derived macrophages (BMDM) cultured in conditioned medium (CM) of Rv-treated DLD1 and SKOV3 cells or respective control medium (culture medium of cancer cells not treated with Rv), and CM of fused B16 melanoma cells or their nonfused parental cells. (B, C) mRNA expression (RT-PCR) of Il6 (B) and Arg1 (C) in BMDM cultured in the CM of Rv-treated SKOV3 cells (0.5µM for 3 days) or control cells, and CM of fused B16 cells or their nonfused parental cells. Data points refer to triplicate experiments expressed as means ± SD. (D) mRNA expression of Il6 (upper panel) and Arg1 (lower panel) in BMDM cultured in complete medium spiked with Rv (0.5 µM). Untreated BMDM were used as control. Data representative triplicate samples run in duplicate at the same time point repeated at least twice and expressed as means ± SD. (E) Flow cytometry analysis of IFNγ and Granzyme B production by human T cells activated by anti-CD3/anti-CD28 Dynabeads in the presence of CM from Rv-treated SKOV3 cells or control SKOV3 cells (cultured without Rv). Column graphs represent the percentage expression of IFNγ (upper panel) and Granzyme B (lower panel). Percentages in parenthesis refer to change from control. Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD. (F) Flow cytometry analysis of IFNγ and Granzyme B production by human T cells activated by anti-CD3/anti-CD28 Dynabeads in complete medium spiked with Rv (0.5µM). Column graphs represent the percentage expression of IFNγ (upper panel) and Granzyme B (lower panel). Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD. (G) IFNγ (left) and Granzyme B (right) production by human T cells activated by anti-CD3/anti-CD28 Dynabeads cultured in CM from fused B16 melanoma cells or their parental non-fused cells. Percentages in parenthesis refer to change from parental non-fused cells. Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.
Understanding transcriptomics changes following a TF perturbation with scRNA-Seq landscapes.Figure 7A A toy example with visual explanation of the Direction of state Transition (DoT) calculation. Three lineage-specific genes are used (full-scale DoT analysis is unbiased and uses all available genes): erythroid Klf1, neutrophilic Elane and lymphoid Dntt; their scaled expression is plotted on the reference landscape (annotation in (B, E)). Upon treatment changes in expression are observed for each gene. We calculate contributions from each gene as the product of its scaled expression and changes in expression (log2(Fold Change)). DoT score is the sum of these components, and indicates direction of cell state transition with respect to the chosen viewpoint (point of origin). B, C Annotated UMAP projections of scRNA-Seq landscapes - mouse LK + LSK populations (Dahlin et al, 2018) and Human Cell Atlas bone marrow mononuclear cells (Regev et al, 2017). Dashed lines indicate cluster positions, which mean expression values were used to designate points of origin for the DoT score analysis. D,E DoT scores calculated using genes differentially expressed after Hoxb8 or Ikzf1 loss in Hoxb8-FL cells in the context of mouse LK/LSK landscape (Dahlin et al, 2018). F, G DoT scores calculated using genes differentially expressed after Hoxb8 or Ikzf1 loss in Hoxb8-FL cells in the context of the human BMMC landscape. Arrows indicate the HSCs and megakaryocytic trajectories.
Figure 5(A) Immunoblot analysis and quantification of pTFEB S211 in HeLa cells stably expressing TFEB-GFP. Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas ). GAPDH immunoblotting was performed as a loading control. (B) Immunoblot analysis of TFEB shift in ARPE-19 CLN3 KO cells. β-actin immunoblotting were performed as a loading control. (C) Representative confocal image and quantification of TFEB localization in HeLa TFEB-GFP transfected with RagC for 48h and treated for the last 3h with DMSO, Torin1, Tamoxifen or Ospemifene. Ratios of nuclear to cytosolic TFEB localization in RagC non-expressing (RagC-) and RagC-expressing cells (RagC+) are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm. D) Representative confocal images and quantification of STX in ARPE-19 CLN3 KO cells transfected with empty vector or HA-RagC for 48h and treated for 48h with DMSO or Tamoxifen. STX average spot area presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.
a, b) Human neuroblastoma (SH-SY5Y), hepatoma (HuH-7) and breast adenocarcinoma (MCF-7) cell lines were transiently transfected with KFERQ–PS–CFP2, photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h.f6(a, b) Human neuroblastoma (SH-SY5Y), hepatoma (HuH-7) and breast adenocarcinoma (MCF-7) cell lines were transiently transfected with KFERQ-PS-CFP2, photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Images show representative cells after immunostaining for LAMP-2 (red). (b) Quantification of the number of green fluorescent puncta per cell under basal conditions (black bars) and after serum removal (gray bars). Values of NIH3T3 are included as reference. Values are mean+s.e. of three-to-five different experiments with >50 cells counted per experiment. Differences with (*) NIH3T3 or (§) with cell supplemented with serum are significant for P0.05 (range 0.002-0.03, ANOVA-Bonferroni). (c) Primary mouse skin fibroblasts were transduced with a lentiviral vector for expression of KFERQ-PS-CFP2, photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Left: representative image in the green channel. Right: Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence (black bars) or absence (gray bars) of serum. Values are mean+s.e. of three different experiments with >50 cells counted per experiment. (P=0.002, ANOVA-Bonferroni). (d) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were plated in 384-well plates, maintained in the presence (white bar) or absence (black bars) of serum and subjected to treatment with 3-methyladenine (3-MA), the proteasome inhibitor MG-132 or H2O2. In both (d, e), after fixation, images were subjected to high content image analysis and the percent of responders was calculated in >200 cells per condition. Values are mean+s.e. of results in three different experiments. *P0.001 (range 0.0002-0.0007, t-test). Scale bars, 5 μm. (e) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were plated in 384-well plates, maintained in the presence of serum supplemented media and leave untreated (white bar) or subjected to the indicated treatments (black bars). In both (d, e), after fixation, images were subjected to high content image analysis and the percent of responders was calculated in >200 cells per condition. Values are mean+s.e. of results in three different experiments. *P0.001 (range 0.0002-0.0007, t-test). Scale bars, 5 μm.
UBTD1 promotes YAP degradation by the UPS independently of the Hippo signaling pathway Immunoblot of DU145 and A549 cells at various cell densities ranging from 20% to 100% confluence showing the increased expression of UBTD1, p-YAP (ser127) and YAP levels with the cell confluency.Figure 3Immunoblot of DU145 and A549 cells at various cell densities ranging from 20% to 100% confluence showing the increased expression of UBTD1, p-YAP (ser127) and YAP levels with the cell confluency. Tubulin was used as a loading control. DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (B) Immunoblots of p-YAP (ser127), YAP and UBTD1 (left) and quantification of YAP and p-YAP/YAP levels (right) in control or UBTD1-depleted DU145 at low and high cell density. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading controls. , immunoblots for the loading control (actin) and UBTD1 knock-down efficiency (UBTD1) are duplicated because they belong to the same experiment shown in both panels. DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (C) Immunoblots of the Hippo signaling pathway using phosphorylated (p-ser909LATS and p-thr183MST1/p-thr180MST2) and total LATS, MST at low and high DU145 cells density. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin and Rho-GDI were used as loading controls. , immunoblots for the loading control (actin) and UBTD1 knock-down efficiency (UBTD1) are duplicated because they belong to the same experiment shown in both panels. DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) of YAP levels in confluent (D) DU145 UBTD1 depleted cells treated or not with cycloheximide (CHX) for 2h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control. A549 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) of YAP levels in confluent (E) A549 UBTD1 depleted cells treated or not with cycloheximide (CHX) for 2h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control. DU145 cells or A549 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) showing YAP levels in UBTD1 depleted confluent DU145 A549 cells treated or not with MG132 for 6h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.
Micropeptide encoding transcript expression is posttranscriptionally regulated by miRNAs.Figure 3(A) Distribution of the translational efficiency, in mESCs, of mRNAs (n=7156, red), cytosolic (n=341, blue) and nuclear (n=1915, grey) lncRNAs. Two-tailed Mann-Whitney U test: **-p-value<0.01 and ***-p-value<0.001. Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution. (B) Fraction of cytosolic lncRNAs with experimental evidence for ribosomal binding with (red) or without (blue) an overlapping conserved short open reading frame. (C) Distribution of the translational efficiency, in mESCs, of mRNAs (n=7156, red), micropeptide encoding transcripts (n=43, pink) and bona fide cytosolic (n=298, blue) and nuclear (n=1857, grey) lncRNAs. Two-tailed Mann-Whitney U test: *-p-value<0.05, **-p-value<0.01 and ***-p-value<0.001. Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution. (D) Distribution of the fold-change (FC) in degradation rate of mRNAs (n=13296, red), micropeptide encoding transcripts (n=43, pink), bona fide cytosolic (n=759, blue) and nuclear (n=4299, grey) lncRNAs in 4-OHT treated (KO) relative to ethanol treated (WT) cells after 8 days treatment, horizontal dashed line represents a KO/WT FC in degradation rate of 1. Statistics: NS-p>0.05, *-p-value<0.05. Two-tailed Mann-Whitney U test: p-value=0.044. Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution.
Figure 3Oxygen consumption (VO2) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons). carbon dioxide production (VCO2) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons). respiratory exchange ratio VCO2/VO2 (RER) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons). physical activity of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).
Figure 4A Transmigration of mouse neutrophils towards CXCL-1 through TNF-α-stimulated bEnd.5 cells transfected with control or SHP2-specific siRNA and pre-treated with anti-endomucin (7C7.1) or anti-PECAM-1 (Mec13.3) antibodies for 30 min at 37°C before addition of neutrophils. The transmigration rate is presented relative to that of control cells, set as 100%. B Transmigration of mouse neutrophils towards CXCL-1 through TNF-α-stimulated WT or Y731F primary mouse endothelial cells pre-treated with isotype-control or anti-PECAM-1 (2H8) antibodies for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to that of control cells, set as 100%. Extravasated leukocytes (C) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy. adherent leukocytes (D) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy. (D) and rolling flux fraction of leukocytes (E) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy.
Figure 2A. Schematic representation of the wing imaginal disc and its relationship to the adult wing. The cell fate map of the wing disc is adapted from (Bryant, 1975). B. Subset of 6,983 epithelial cells in UMAP plot of the reference single cell atlas. C. Approximate map of the epithelial clusters identified in the reference single cell atlas dataset to distinct position over the disc proper. D. Average expression level of the genes used as known markers to assign each epithelial cluster in the reference atlas dataset. ﻿Cells colored by the expression of nub, wg, zfh2, Sox15, grn, bnl, eyg, Ance, Idg4 and drm. E. Dot plot showing the expression levels of the top marker genes identified for the epithelial cells across 17 clusters of the reference cell atlas dataset. F. Confocal single plane image of third instar larval wing disc stained with anti-Nub, anti-Wg, anti-Svp, anti-Hairy (red), reporting expression of dpp-lacZ, Dad-lacZ (anti-β-gal, red) and counterstained with DAPI (cyan). Scale bars: 100 μm. Full genotypes: P{CaryP}attP2 (top and bottom panels), P{PZ}dpp[10638];+, and y- w-;+; P{lacW}Dad[j1E4].
Figure 3A Schematic representation of AAV.Syne4 and AAV.GFP constructs. B Whole mount immunofluorescence of a P9 organ of Corti of a mouse injected with AAV.GFP at P1 showing complete transduction of both inner and outer HC. Myosin VIIa was used to label the hair cells. C Examples of 8, 16, and 32 kHz regions of an organ of Corti of a mouse injected with AAV.GFP. Top shows OHC plane, bottom shows IHC plane, right shows YZ orthogonal projection. Black asterisks show bright Deiters cells. D Quantification of GFP intensity of inner and outer HC from 3 injected mice, normalized to the average intensity of HC in a control, un-injected mouse. E Transduction rates of AAV9-PHP.B at 8, 16, and 32kHz regions based on GFP fluorescence. A total of 162 IHC and 841 OHC were analyzed from 3 injected mice and 1 control littermate. F Staining for FLAG at at P14 of the organ of Corti of a mouse injected at P1 with AAV.Syne4. G Quantification of FLAG and DAPI fluorescence intensity along a line centered at the nuclear envelope. Eight OHC were measured. H 3D surface projection of two adjacent OHC from a mouse injected with AAV.Syne4. In the left cell myosin VIIa and DAPI signals were removed to only show FLAG staining.
Figure 1(A, B) Poly(I:C) treatment inhibits axonal growth at 3 DIV (A) and dendritic growth at 6 DIV (B) in WT neurons. Neurons were transfected with GFP at 1 or 4 DIV. One day later, neurons were treated with poly(I:C) for 24 h before harvest. (C) Axon and dendrite morphology of Tlr3-/- neurons after poly(I:C) treatment. Data in A,B,C were analyzed by unpaired t-test. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. ** P < 0.001, *** P < 0.0001. Scale bar, 20μm in A and B. (D, E) Neuronal morphology in saline- and poly(I:C)-treated Tlr3+/+;Thy1-Yfp (D) and Tlr3-/-;Thy1-Yfp (E) mouse brains. Total dendrite length, dendritic tip number and Sholl analysis of dendritic processes were used to examine the dendrite phenotype. Data of dendrite length and tip number were analyzed by unpaired t-test. The Sholl data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. The numbers of analyzed neurons and mice are presented in each column. Mean values SEM are shown. * P < 0.05, ** P < 0.001, *** P < 0.0001. Scale bar, 50 μm in D and E.