EMBO/sd-character-level-ner huggingface dataset:
Acute Ece2 KD in the developing mouse cortex causes ectopic rosettes and nodules.Figure 2Quantification of distribution of GFP+ transfected cells 1dpe upon CTRL and Ece2 KD by IUE (n=3 CTRL and 4 Ece2 KD brains; *P=0.044 in One-way ANOVA and Tukey's Multiple Pairwise Comparison; data shown as mean±SEM). IHC staining of GFP (transfected cells) and Pax6 1 dpe showing ectopic Pax6+ progenitors (red circle) and a reduction in their apical localisation (yellow circle). High magnification images of GFP and Pax6 showing that ectopic progenitors are mostly not transfected. D-J, E13-16 Ece2 KD. GFP-Pax6+ progenitors delaminate upon Ece2 KD (red arrows), leaving behind a region free of Pax6+ cells in the VZ (yellow circle) and forming ectopic rosettes (yellow box). Reduction in apical Arl13b in the electroporation site (white arrowheads) indicates loss of apical belt integrity and disruption of apico-basal polarity upon Ece2 KD. Inside the nodule, which is surrounded by thickened GFP+ processes (pink arrows), Arl13b staining shows the formation of an ectopic apical surface Reduction in β-catenin in the electroporation site (white arrowheads) indicates loss of apical belt integrity and disruption of apico-basal polarity upon Ece2 KD. , Deep layer and I, upper layer neurons localise next to ectopic rosettes, forming nodules of ectopic neurons (blue arrowheads for single ectopic neurons and blue circle for large nodule). Quantification at 3 dpe, counting the proportion of brains with reduced density of Pax6 in the VZ, ectopic progenitors or neurons, and changes in apico-basal polarity (including ectopic apical surface formation and reduction in apically localised apical markers; n=6 brains per condition; Exact binomial test: ****P < 0.0001). Scalebars in A, B, D, E, H, I=100 µm; in C, F, G Scalebar = 50 µm. V marks the position of the ventricle.
Figure 6Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 (A) GFP+ Ghost X4R5 cells obtained after treatment with supernatant from infected SERINC3 KD cells. Results are expressed as a ratio to the number of GFP+ cells after treatment with supernatant from infected control cells. . Mean ± SD of three independent experiments is shown. Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (B) Number of particles secreted were quantified by NTA. Results are expressed as a ratio to the number of particles secreted by uninfected luciferase shRNA cells. Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (C) 100K pellets from infected cells were subjected to iodixanol velocity gradient separation. Six fractions were recovered and analyzed by Western blot for the presence of SPN, SERINC3 and p24. (T = top; B = bottom). (D) Quantification of SPN and p24 signals in the different fractions of two independent iodixanol fractionations (exp 1: open symbols, exp 2: closed symbols). AU = band intensity in a given pellet / sum (band intensity in all 12 pellets). Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). 2K, 10K, 100K obtained from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4-3 EGFP-Nef+ were analyzed by Western blot with antibodies against SPN, and p24. The 2K, 10K, and 100K pellets obtained from 20 x 106 cells were loaded on the gel. Representative images (E) Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). 2K, 10K, 100K from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4-3 EGFP-Nef+ were analyzed and quantification of SPN and p24 signals (F) are shown. For each pellet, arbitrary units (AU) represent the ratio of signal intensity in the given pellet to the total amount of protein in the same given pellet and relative to the total amount in 2K+10K+100K from control cells. Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (G) Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with EV samples. Captured EVs were detected with a mixture of APC-labeled anti-CD9, anti-CD63, and anti-CD81. Quantification of the median APC fluorescence values for all bead populations after background and isotype antibody correction. Results were relativized to the values obtained with control sample for each experiment. Dotted line represents values obtained for EVs isolated from control cell CM.
a) Mitochondrial respiratory chain complexes activity values normalized to citrate synthase (CS), and (b) mitochondrial inner membrane potential (Δψm) in WT and Pink1 KO mice MEF.f4(a) Mitochondrial respiratory chain complexes activity values normalized to citrate synthase (CS), and (b) mitochondrial inner membrane potential (Δψm) in WT and Pink1 KO mice MEF. (c) Western blot against PDK1 and HIF1α in WT and Pink1 KO MEF transfected with siHif1α (or its siControl); ß-actin was used as loading control. d) PDH complex activity in WT and Pink1 KO MEF transfected with siHif1α or siControl. Rate of whole-cell H2O2 (e) and mitochondrial O2·− (f) detection in WT and Pink1 KO MEF (g) Detection of mitochondrial O2·− using the mitochondrial-specific probe MitoSox in MEF incubated in the absence (−) or in the presence (+) of the plasma membrane permeable GSH-EE, or by expressing mitochondrial-tagged forms of glutamate-cysteine ligase, (+mitoGCL; empty vector, pIRES2-EGFP, denoted as −) or catalase (+ mitoCAT; empty vector denoted as −), shows effective mitochondrial ROS removal. (h) Western blot against HIF1α in Pink1 KO MEF incubated with (or without) GSH-EE, or transfected with empty vector, mitoGCL or mitoCAT, showing reduction in HIF1α protein abundance by the treatments that remove mitochondrial ROS; α-TUBULIN was used as loading control. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).
| The iron chelator deferoxamine (DFOA) promotes amoxicillin (AMX) tolerance in vivo.Figure 7(A) Schema of experimental design. Briefly, mice were pretreated with streptomycin (5 mg/mL) for 2 days and then intragastrically inoculated with V. cholerae (~1.97x1010 cells in 200 µl PBS). After 24 h, mice in each group received water (H2O), amoxicillin (50 mg/kg), deferoxamine (200 µl of a 100 µM solution), or both amoxicillin and deferoxamine. (B) Excretion of V. cholerae. Fecal samples were collected every two hours and assessed for their viable cell content (Colony Forming Units, CFU) to monitor the level of V. cholerae excretion. Each symbol represents fecal samples from an individual mouse (n=4). The number of bacterial CFU was normalized per milligram of feces (CFU/mg). Error bars represent standard deviation. ∗∗ P<0.005; ∗∗∗ P<0.001 (two-way ANOVA).
Figure 2Kinase activation enrichment analysis phosphorylation status of specific phosphosites associated with known or predicted upstream kinases biological pathway enrichment analysis phosphorylation status of proteins involved in Ca2+ signaling in human, rat and mouse models of exercise.
Transcriptome analysis of in vitro activated B cells shows accelerated plasma-cell differentiation and compromised activation of MYC-target genes in the absence of DOT1L.Figure 6A. MA-Plot of normalized RNA-Seq data generated from three independent biological replicates for each genotype showing differential (false discovery rate (FDR) < 0.05) expression of genes between Dot1L-KO and WT B cells after two days of in-vitro activation with LPS+IL-4. B. Differential (FDR < 0.05) expression of Bach2, Prdm1 (encoding BLIMP-1), Cd138 (a plasma-cell marker) and Myc transcripts as indicated by counts per million after TMM normalization from WT and KO. Data was generated from three independent biological replicates for each genotype. Bars and error bars indicate mean ± SD. Statistical significance is indicated by FDR after the Benjamini-Hochberg multiple testing correction performed by edgeR package using R language. C-D. Enrichment of plasma-cell signature genes in KO as compared to WT activated B cells (C) and Enrichment of MYC-target genes in WT as compared to KO activated B cells (D). Enrichment of gene sets is depicted by a BARCODE plot; P-value calculated via FRY test show the statistical significance of enrichment of each gene set. A p-value of less than 0.05 was considered as significant.
Figure 3A. Host C57Bl/6 mice were injected with CD45.1+ splenocytes, isolated from SWHEL x Cyp1a1Cre R26R eYFP mice together with SRBC-HEL or SBRC-mock. The transferred mice were treated with 3-MC or vehicle. Readout at d7 post-challenge was eYFP expression in transferred CD45.1+ cells. B. Representative flow cytometry analysis of Cyp1a1 expression, reported by eYFP, in CD45.1+ cells harvested from mice challenged as in A. Gating is indicated above the plots. Representative data of n = 3 independent experiments. GC cells: CD95+ GL-7+; non-GC cells: CD95- GL-7-.C, D. Summary of percentage of GC (C) and non-GC eYFP+ (D) cells from flow cytometry analysis. n = 3 independent experiments; lines indicate mean; one-way ANOVA, Tukey's multiple comparison test.
The IFT22A86R mutant displays a severe retrograde IFT phenotype in vivo A.Figure 6A. Western blot analysis of the IFT22RNAi+GFP::IFT22A86R cell line probed with the anti-IFT22 antibody (bottom) and with an anti-BiP as loading control (top). B. IFA with the indicated cell lines using the mAb25 (marker for the axoneme, central panels) and an anti-IFT172 antibody (marker for IFT, bottom panels). The top panels show the phase contrast images merged with DAPI (cyan) that stains nuclear and mitochondrial DNA. The arrowheads indicate the presence of short flagella stained with the Mab25 antibody in the IFT22RNAi cells. Scale bars correspond to 5 µm. C. Sections of IFT22RNAi+GFP::IFT22rescue (top panels) or IFT22RNAi+GFP::IFT22A86R cells (bottom panels) were analysed by transmission electron microscopy. Sections through the flagellar pocket, the transition zone and the flagellum are shown. Scale bars are 500 nm (flagellar pocket sections) or 200 nm (transition zone and flagellum sections). The white arrow indicates an endocytic vesicle budding off the flagellar pocket whereas the black one points at an IFT train. (Axo = axoneme, TZ = transition zone, BB = basal body)
Effect of the β7 mutant E239K on KIF1A motility and kinetics in vitro.Figure 4(A) Representative kymographs of tetramethylrhodamine-labelled microtubule movement driven by the WT and the E239K mutant. The arrowheads represent the end positions of the WT and E239K. (B) Movement of microtubules on glass driven by the WT and E239K. ****, p < 0.0001, chi-square test. (C) Microtubule gliding velocity of the WT (green) and E239K mutant (magenta) determined by kymograph slope. Data and error bars in the inserted graph are presented as the mean ± SD. n = 269 microtubules for WT. n = 263 microtubules for E239K. ****, p < 0.0001, unpaired t test­. (D) ATPase activity of WT (green) and E239K mutant (magenta) activated by microtubules. Data are represented as the mean ± SD. The data from at least three independent experiments were analysed. ****, p < 0.0001, two-way ANOVA. (E) Measurements of the association and dissociation of mant-ATP. Nucleotide-free KIF1A(WT) and KIF1A(E239K) (final concentrations 2 µM) were rapidly mixed with varying concentrations of mant-ATP (2.5-25 µM) in a stopped-flow fluorometer. Representative transients at 25 µM, 12.5 µM and 2.5µM mant-ATP and fitted single-exponential function curves are shown. Data are represented as the mean ± SD. The data from three independent measurements were analysed. (F) Rate constants (kobs) determined for the reaction of mant-ATP with nucleotide-free KIF1A(WT) and KIF1A(E239K) were plotted against concentration of mant-ATP. Data are represented as the mean ± SD. The data from three independent measurements were analysed. (G) Measurements of the dissociation of mant-ADP for 80 s (left panel) and the enlarged view for the first 8 s (right panel). KIF1A(WT) and KIF1A(E239K) (final concentrations 50 µM) preloaded with mant-ADP were mixed into an excess of unlabeled ATP (5 mM) with or without taxol-microtubule. Fluorescence decreases were observed upon the dissociation of mant-ADP. (H) Summary of the experimentally determined kinetics parameters of KIF1A(WT) and KIF1A(E239K). Data are represented as the mean value ±SD for mant-ATP measurements.
Hh autonomously regulates cortex gliogenesis and non-autonomously regulates NB proliferationA-D) Representative images showing that upon knockdown of Hh in cortex glial cells (NP2222-GAL4>mGFP with UAS-dcr2), cortex glial membrane and overall Repo+ glial cell number are significantly reduced, quantified in (C) (n=7, 8 brain lobes) and (D) (n=7, 8 brain lobes), respectively.Figure 2A-D) Representative images showing that upon knockdown of Hh in cortex glial cells (NP2222-GAL4>mGFP with UAS-dcr2), cortex glial membrane and overall Repo+ glial cell number are significantly reduced, quantified in (C) (n=7, 8 brain lobes) and (D) (n=7, 8 brain lobes), respectively. E-G) Hh knockdown in glia (repo-GAL4>GFP) results in niche disruption and clustering of NBs (circled with yellow dashed line), as well as an increase in the percentage of NBs in M phase (pH3+), quantified in (E) (n=12, 10 brain lobes). H-K) Representative images showing that Hh overexpression using pan-glial (repo-GAL4) and cortex-glial (NP2222-GAL4) drivers both result in a decrease in NB EdU index, quantified in (J) (n=16, 20 brain lobes) and (K) (n=11, 14 brain lobes), respectively. L-M'') Representative images showing ci-lacZ is expressed in NBs (yellow arrows). (M-M'') are zoomed in images of (L). N-P) Overexpression of ciACT in NBs (dnab-GAL4) reduces EdU index, quantified in (P) (n=12, 10 brain lobes).
Figure 4A. DRG neurons expressing Myc-Syt11 were immunostained for Myc-Syt11, dynamin (Dyn), AP-2, clathrin (CL), TGN46, synaptobrevin 2 (Syb2), and LAMP1. For dynamin, the box along the plasma membrane was straightened, enlarged, and is shown in the upper right panels with arrows indicating co-localized puncta. An enlarged inset of the inside of the cell (box) is shown in the lower panel. For localization of the transferrin receptor, EGFP-transferrin receptor (TfR) was expressed in DRG neurons. Scale bars, 10 μm. B. Analysis of co-localization of Myc-Syt11 with dynamin, AP-2, clathrin, TGN46, synaptobrevin 2, transferrin receptor, and LAMP1. N = 3 independent experiments.
a) Immunoblot for the indicated proteins in control MEFs or those knocked down (KD) for the three different Cx.figf8(a) Immunoblot for the indicated proteins in control MEFs or those knocked down (KD) for the three different Cx. (b) Immunofluorescence of LC3 in control MEFs (Ctr) or those knocked down (KD) for three different Cx maintained in the presence or absence of serum. Left: representative fields and higher magnification insets. Nuclei are highlighted with DAPI. Right: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment). (c) Immunoblot for LC3 in the same cells maintained in the presence of serum untreated (−) or treated with inhibitors of lysosomal proteases (PI) for the indicated times. GAPDH is shown as the loading control. (d) Representative images of control MEFs or those knocked down (KD) for the indicated proteins transfected with the mCherry-GFP-LC3 tandem reporter. Right: quantification of the average number per cell of puncta positive for GFP and mCherry (autophagosomes; APG) and those positive only for mCherry (autophagolysosomes; APL; n = 3 wells, 3 independent experiments, >50 cells per experiment). (e) Electron micrographs of control MEFs or those knocked down for the indicated Cx. Insets show autophagic vesicles. Bottom: Quantification of the average number of AV (left) and percentage of cytosolic area (right) occupied by AVs (n = 4 wells, 4 independent experiments, >10 micrographs per well). (f) Immunoblot for indicated proteins in immunoprecipitates (IP) for Cx26 or Cx32 (left) or Atg16 (right) from liver homogenates. Inp: input, FT: flow through. BIP is shown as the negative control for immunoprecipitation. All values are mean + s.e.m and significant for *P 0.01 (analysis of variance + Bonferroni test was used in b). Scale bars: 5 μm in fluorescence images; 1 μm and 0.1 μm in top panels and insets of e. Uncropped images of blots are shown in Supplementary Fig. 9.
Promoter archetypes change upon modulation of p53 dynamics through small molecule inhibitors.Figure 4Chk2 inhibition with the small molecule BML-277 induces transient p53 dynamics with only one pulse after 10 Gy IR . A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and addition of 10 µM BML-277 by immunofluorescence staining (see Methods section for details) are shown as box plots (see Data Visualization section). We quantified promoter activity of BAX (B, sustained archetype) and PPM1D (C, pulsatile archetype) before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after irradiation with 10 Gy IR and inhibition of the second p53 pulse by Chk2 inhibition. Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The fraction of active promoters was reduced at 6 h and 9 h after irradiation; the transcription rate was not notably affected. Sequential treatment with Nutlin-3 converts pulsatile p53 dynamics into sustained nuclear levels. A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and sequential treatment with 0.75 µM Nutlin-3 at 2.5 h, with 2.25 µM at 3.5 h and 4 µM at 5.5 h post IR based on immunofluorescence staining (see Methods section for details) are shown as box plots (see Data Visualization section). We quantified promoter activity of MDM2 (E, transient archetype) and CDKN1A (F, transient archetype) before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after irradiation with 10 Gy IR and sequential Nutlin-3 treatment. Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The relative fraction of active promoters strongly increased, changing transient to sustained archetypes. The transcription rate increased as well both compared to basal levels and to previous experiments with pulsatile p53 dynamics (inset, fold change relative to IR alone for each time point).
Figure 11(A) Growth curves of (i) BW25113(ycbB, relA') and its ΔsltY derivative; and (ii) strain M1 and its derivatives (M1.1 to 1.4) harboring mutations in sltY. The growth medium contained (i) 40 µM IPTG, 1% L-arabinose, and 8 µg/ml ceftriaxone for BW25113(ycbB, relA') and its ΔsltY derivative or (ii) 50 µM IPTG and 16 µg/ml ampicillin for M1 and its derivatives. (B) Ceftriaxone-resistance depends upon ycbB and relA' expression. The disk diffusion assay was performed with BW25113 ΔrelA ΔsltY harboring pKT2(ycbB) and pKT8(relA') or pKT8(relA') only. Induction was performed with 40 µM IPTG and 1% L-arabinose for ycbB and relA', respectively. Disks were loaded with 30 µg of ceftriaxone.
Figure 1(A-B) Live third instar discs incubated in DiBAC. DiBAC fluorescence is observed in the wing imaginal disc in both apical (A-A'), and basal (B-B') optical sections. Increased fluorescence is observed in the center of the pouch (white arrow, B'), and at the dorsoventral (D-V) compartment boundary in the anterior compartment (yellow arrow, B', and inset, D). (C-E) Comparison of DiBAC with the voltage-insensitive dye FM4-64. Incubation of live discs in FM4-64 (E) shows more uniform fluorescence when compared to DiBAC (D). Quantitative comparison of fluorescence in the two white boxes in each panel is shown in (C). N=7 discs for each treatment, data compared using an unpaired t test, ** indicates p<0.001, error bars are standard deviations. The region boxed in yellow in (D) is shown at higher magnification to show DiBAC fluorescence at the D-V compartment boundary. (F, G) Incubation in ouabain results in brighter and more uniform DiBAC fluorescence. (H-H'') Live wing discs expressing UAS-RFP anterior to the compartment boundary, under the control of ptc-Gal4, were incubated in DiBAC, showing that the stripe of increased DiBAC fluorescence coincides with the posterior edge of ptc expression. White arrowhead in (H) indicates the stripe in the wing pouch; yellow arrowheads indicate the stripe in the dorsal and ventral hinge. (I, I') Early L3 discs incubated in DiBAC. Patterned depolarization is evident throughout the third larval instar, with the stripe of increased fluorescence becoming narrower in more mature discs with developmental time (Compare I with A, bracket in I indicates width of increased DiBAC fluorescence).
Figure 4(A, B) Representative (A) photographs of fly eyes and (B) quantitation with siRNA mediated knockdown of SRPK1 (dSRPK1); n=30-34/genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (C) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC) 28-EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion. (D) Quantitation of rough eye phenotypes. t-test with Welch corrections for comparisons with the control; n= 31-34 flies/ genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (E) Representative external eye imaging for detection of GFP aggregates caused by (CGG) 90-EGFP transgene expression (top). Converted images used to quantify total intensity of GFP puncta (bottom). (F) Depletion of SRSF1 or SRPK1 by RNAi results in reduced (CGG) 90-EGFP puncta compared to control siRNA as quantified by total intensity (a.u. = arbitrary unit). n=13-15 flies/genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.
Figure 5Construction of hybrid systems through cell lysate mixing. Principal components analysis of transcriptional profile similarity for individual and hybrid lysates with different mixing ratios for (b) E. coli + B. subtilis Principal components analysis of transcriptional profile similarity for individual and hybrid lysates with different mixing ratios for (c) E. coli + C. glutamicum hybrids. Pairwise correlation of RS7003 transcriptional profiles between hybrid lysate and single constituent species lysates for (d) E. coli + B. subtilis Pairwise correlation of RS7003 transcriptional profiles between hybrid lysate and single constituent species lysates for (e) E. coli + C. glutamicum hybrids with different mixing ratios.