EMBO/sd-character-level-ner huggingface dataset:
Layout and analysis of the RNAi screen used to identify immune-modulatory tumor genes.Figure 2Layout and analysis of the RNAi screen used to identify immune-modulatory tumor genesA Workflow and experimental layout of the RNAi screen which was performed thrice, each time in duplicates, along with an additional CTG-based viability screen to filter out lethal genes from the final hit list. Hits were analyzed after data normalization using the cellHTS2 package. B Correlation between replicates for screen 2 for both viability and toxicity sets, as determined by the Pearson rank correlation test. C-E Graphical summary of gene function related to modification of T cell-mediated tumor lysis and cellviability for screens 1, 2, and 3, respectively. Positive score = reduced cancer cellviability; negative score = increased viability. X-axis: influence on cellviability without addition of T cells. Y-axis: influence on cellviability with addition of T cells. Appropriate immune-suppressive (PD-L1, CEACAM-6, GAL-3) and lethality (UBC, PLK-1) controls, along with few positive (GRM4, GRK5) and negative (CCR9, IL8) candidate immune-modulatory hits are highlighted herein. C-E Graphical summary of gene function related to modification of T cell-mediated tumor lysis and cellviability for screens 1, 2, and 3, respectively. Positive score = reduced cancer cellviability; negative score = increased viability. X-axis: influence on cellviability without addition of T cells. Y-axis: influence on cellviability with addition of T cells. Appropriate immune-suppressive (PD-L1, CEACAM-6, GAL-3) and lethality (UBC, PLK-1) controls, along with few positive (GRM4, GRK5) and negative (CCR9, IL8) candidate immune-modulatory hits are highlighted herein.
Figure 2A, Sample collection procedure and labelling scheme using tandem mass tag (TMT) proteomics B, Heatmap representation of 342 rhythmic proteins (JTK-Cycle, p < 0.05). C, Phase distribution of the circadian proteins shown in B D, Validation of TMT quantitation using an alternative method to label samples E, Distribution of amplitudes of the 342 circadian proteins. Amplitudes were calculated by taking the ratio between the maximum and the minimum of each protein profile. F, Cumulative density of protein abundances of circadian proteins vs. all proteins (n = 4759 proteins quantified; Wilcoxon sum rank test, p < 10-4) G, Scatterplot representation of the GO analysis of the rhythmic proteins
null embryos and exogenous ER Stress induction in wildtype embryos upregulates Chac1 and Trib3 (A, B) Western blot of individual E9.Figure 6Western blot of individual E9.5 wildtype (n=4) and Slc7a5-null (n=4) embryo lysates immunoblotted using an antibody against phospho-GCN2 (Thr899) and total GCN2 (loading control), ***p = 0.0001 ; band intensities measured with FIJI and analysed with unpaired t-test Data information: All error bars indicate SEM. Western blot of individual E9.5 wildtype (n=4) and Slc7a5-null (n=4) embryo lysates immunoblotted using an antibody against phospho-eIF2α (Ser51) and total eIFα (loading control), *p = 0.0482, band intensities measured with FIJI and analysed with unpaired t-test Data information: All error bars indicate SEM. (C-F) phosphorylated eIFα was assessed by immunofluorescence in E9.5 embryos in the forebrain, wildtype (C,c1) (n=2 embryos, 0/14 sections) and Slc7a5-null (D, d1) (n=2 embryos 16/16 sections), and in hindbrain/anterior spinal cord, wildtype (E-e2) (n= 4 embryos, 0/62 sections) and Slc7a5-null (F-f2) (n= 4 embryos, 54/54 sections) including in the otic vesicle (compare e2, f2). Scale bars in (C-e1, D-f1) 100 µm and in (e2 and f2) 25µm. Arrowheads indicate ventral midline. (G) An increase in Trib3 protein was detected by Western blot in Slc7a5-null embryos, each lane represents an E9.5 embryo lysate (wildtype n=3, Slc7a5-null, n=4) with α-Tubulin loading control, unpaired t-test *p = 0.0153 Data information: All error bars indicate SEM. (H-L) TUNEL assay to detect apoptotic cells in wildtype (n=3 embryos, 32 sections) or Slc7a5-null (n=5 embryos, 47 sections); (H, I) transverse sections through the head at level of the forebrain and (J, K) the spinal cord, (H-K) scale bars 50 µm, arrowheads indicate ventral midline. (L) Quantification of TUNEL positive cells within spinal cord sections was performed Each dot represents the average apoptosis cell count for a single embryo, Welch's correction unpaired t-test, p = 0.250, F-test to compare variances, p = 0.0003 Data information: All error bars indicate SEM.
Figure 1(A) Top hit selected candidates are listed. The plot shows relative S9.6 nuclear intensity values for cells transformed with each of the indicated pool of siRNAs. (B) Representative images of HeLa cells immunostained with S9.6 and nucleolin antibodies after transfection with at least a pool of two siRNAs of each original pool. (C) Relative S9.6 signal intensity per nucleus after nucleolus signal removal in HeLa cells transfected with the indicated siRNAs. More than 250 total cells from three independent experiments were considered. The median of each population is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. ***p<0.001 (Mann-Whitney U test).
Figure 2A. Serum, plasma or urine levels of vitamin D, vitamin A, folate, zinc, iron and iodine status biomarkers in diet groups. Solid lines indicate cut-offs for deficiency, and dashed lines indicate cut-offs for insufficiency. The RBP limits for vitamin A insufficiency and deficiency are validated for the method at 1.17 µmol/l and 0.83 µmol/l, respectively. Vitamin A status was calculated based on RBP, transthyretin, and CRP.(Talsma et al, 2015). B. Cholesterol metabolism biomarkers in the diet groups. The subfigure summarizes cholesterol metabolism in humans and highlights cholesterol-related metabolites measured in this study with black font. (Nelson & Cox, 2012; Risley, 2002).
Figure 7Schematic representation of the UBQLN4 deletion mutant proteins used in this study. (B, C and D) The wild-type (WT) form of Flag-tagged UBQLN4 and its truncated derivatives were expressed in HeLa cells with T7-tagged IL-2RαΔSS (B and C). At 4 h after the addition of 10 μM MG-132, UBQLN4 was immunoprecipitated with an anti-Flag antibody and the precipitates were blotted with an anti-T7 antibody. (B, C and D) The wild-type (WT) form of Flag-tagged UBQLN4 and its truncated derivatives were expressed in HeLa cells with T7-tagged IL-2Rα ΔSS (B and C) and T7-tagged ubiquitin (D). At 4 h after the addition of 10 μM MG-132, UBQLN4 was immunoprecipitated with an anti-Flag antibody and the precipitates were blotted with an anti-T7 antibody. (E) GST-tagged and purified UBQLN4 and its truncated derivatives (ΔUBA and ΔSTI-II) were incubated with K48-linked polyubiquitin chains. After in vitro GST pull-down, the precipitates were probed with an anti-polyubiquitin (FK2) antibody.
Figure 7A) HEK293A cells treated with transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 and RAB11A and B (knockdown confirmed by immunoblot analysis) were fed with Alexa-647 Transferrin for 15 minutes in full medium, and the fluorescent transferrin chased out for the indicated time periods. At the end of the time course, cells were trypsinised, fixed and the Alexa-647 fluorescence analysed by flow cytometry. Results are plotted as percentage of transferrin fluorescence at t=0 for the RISC free control cells, expressed as the mean of three independent experiments ± s.e.m. B) Cells transiently transfected with RISC Free control siRNA, or siRNA duplexes directed against TRAPPC8 or RAB1A and B were stained for the cis-Golgi markers GM130 or RAB1B and analysed by confocal microscopy. Scale bars = 20 μm in main panels, 10 μm in inset panels. Bar graph: > 100 cells per siRNA were scored for fragmented or normal juxtanuclear Golgi stacks from three independent experiments. Error bars ± s.e.m., * p<0.05, ** p<0.01, one way ANOVA with Sidak's multiple comparison test. C) HeLa C1 cells transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 or RAB1A and B (knockdown confirmed by immunoblot analysis), were treated with D/D solubiliser for the indicated time periods, trypsinised, fixed and their GFP content analysed by flow cytometry. The line graph shows the GFP fluorescence as a percentage of t=0, ± s.e.m. from three independent experiments.
Schematic Representation of the Wild Type (WT) and Mutant vBcl-2 Constructs Interacting with Beclin1 in the Yeast-two-hybrid System.Figure 1The 171-amino acid sequence of vBcl-2 is indicated at the top with the α-helical structures numbered according to previous publication [25]. Colored boxes denote the BH 1-4 domains in vBcl-2. "+" : positive interaction, "−" : no interaction. L: linker region; TM: transmembrane domain; Triple arrows denote the alanine substitutions at Ser85-Gly86-Arg87 within the BH1 domain of vBcl-2.
Figure 2The number of TFs shared between metabolic pathways is greater than expected by chance for all combination of pairs of metabolic pathways (Dataset EV4). The size of the nodes corresponds to the number of TFs identified for each pathway. The width of the edge linking two metabolic pathway indicates the number of TFs shared between the two pathways.
Figure 3A. DRIP-qPCR analysis reveals accumulation of R-loops across the BU-1 locus in primpol cells. The DRIP signal was calculated as enrichment over RNase H-treated samples and was normalised to -0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired t-test was used to compare differences between matched amplicons in primpol BU-1A(GAA) 10 and the other cell lines indicated. **** p ≤ 0.0001, ns = not significant B. DNA:RNA hybrids in primpol BU-1A(GAA) 10:GgRNase H and primpol BU-1A(GAA) 10:hPrimPol. An unpaired t-test on three biological replicates was used to compare differences to primpol BU-1A(GAA) 10 for each matched amplicon. The bar represent the mean and whiskers the SD. *** p ≤ 0.001, ** p ≤ 0.01, ns = not significant C. Overexpression of chicken RNase H1 prevents (GAA) 10 induced BU-1A epigenetic instability in primpol cells. Fluctuation analysis was performed on three primpol BU-1A (GAA) 10 clones. One-way ANOVA was used to calculate the significance of differences in BU-1 instability between primpol BU-1A ΔG4 and other cell lines. **** p ≤ 0.0001, ns = not significant D. Diagram of the RNase H1 hybrid binding domain (HBD) - mCherry fusion and flow cytometry expression profiles of the construct in four clones E. R-loop stabilisation induces epigenetic instability of BU-1. Bu-1a fluctuation analysis of wild type cells expressing HBD-mCherry. The scatter plots pool results from at least two different clones with matched HBD-expression. Mean ± SD reported. **** p ≤ 0.0001, *** p ≤ 0.001, ns = not significant; one-way ANOVA.
Fig.Figure 3A: Cal27 or JHU029 cells were transfected with control or ELDR siRNA, and EGFR mRNA expression was examined by qRT-PCR. 18S gene was used as internal control. n = 3 biological replicates and 3 technical repeates. B: Cell lysates from control and ELDR depleted Cal27 or JHU029 cells using two different siRNAs to ELDR were subjected to Western blot analysis for the EGFR using specific antibodies. The membrane was reprobed with an antibody against Actin as an internal control. The same actin blot was used in Fig. 2D (for PCNA). The bottom panel shows a quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD, C: Cal27 and JHU029 cells were transfected with siELDR, ELDR plasmid or both siELDR and ELDR plasmid. Cell lysates were analyzed by Western blot analysis for EGFR expression using specific antibody. The membrane was reprobed with an antibody against Actin as internal control. The same actin blot was used in Fig. 2E (for PCNA). n = 2 biological replicates. D: Cell lysates from control and ELDR overexpressed NOK were subjected to Western blot analysis for the EGFR using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. The right panel shows a quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD, E: Cell lysates from control and ELDR-depleted Cal27 and JHU029 cells were subjected to Western blot analysis for phsospho-STAT3 (pSTAT3 Y-705) and total STAT3 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Right panel shows a quantitative representation of Western blot band intensities. n = 2 biological replicates. Data are represented as the mean ± SD,
Recycling endosomes provide membranes for SNX18-mediated GFP-LC3 membrane trafficking and remodeling.Figure 9.Recycling endosomes provide membranes for SNX18-mediated GFP-LC3 membrane trafficking and remodeling. (A) HeLa cells were starved or not starved for 2 h before fixation and immunostaining against endogenous SNX18, Rab11, and TfR. (B) Cells were transfected to express flag-Atg16L1, starved, and immunostained against Rab11 or TfR and flag. (C) Cells were starved and immunostained against TfR, Atg16L1, and SNX18. (D) Cells were transfected with control or SNX18 siRNA, starved, and immunostained against SNX18 and Rab11. Inset panels show enlarged views of the boxed regions. (E) Cells were transfected with control or Rab11 a+b siRNA, starved, and immunostained against SNX18 and Atg16L1. Cells were imaged by confocal microscopy. Bars, 5 µm.
a) Oil red O staining of liver sections isolated from mice with the indicated genotype fed ad libitum and 24 h fasted.figf4(a) Oil red O staining of liver sections isolated from mice with the indicated genotype fed ad libitum and 24 h fasted. Original magnification ×40. (b) Toluidin blue staining of liver sections isolated from fed and 24-h-fasted Tcfeb-LiKO and control mice (Tcfeb flox/flox mice). Arrows indicate lipid droplets. Original magnification ×100. (c) Bar graphs show the quantification of the number of lipid droplets per hepatocyte from electron microscopy analysis. A representative image is shown on the right. Values are mean ± s.d. of at least 10 cells per mouse (n = 3 mice per group). *P≤0.05. (d) The oxygen consumption rate (OCR) in primary hepatocytes isolated from control and Tcfeb-LiKO mice was measured with an XF24 analyser (Seahorse) before and after the addition of palmitic acid (0.2 mM) conjugated with BSA. The vertical red line indicates the time at which palmitate was added to cells. Values are mean ± s.d. for 3 independent experiments *P≤0.05. (e,f) Total FFA (e) and glycerol (f) in the serum isolated from 6-h-fasted Tcfeb-LiKO and control mice. Values are mean ± s.d. (n = 5 mice per group) *P≤0.05 compared with controls. (g) Total serum ketones in fed and fasted Tcfeb-LiKO and control mice. Bars are mean ± s.d. for n = 10 mice per group. **P≤0.01 compared with fed control mice. (h) EchoMRI measurement of fat and lean mass in fed and in 24- and 48-h-fasted mice expressed as relative percentage to fed (100% in the graph). Indicated values are mean ± s.d. for n = 5 mice per group. *P≤0.05;**P≤0.01 compared with fed control mice. (i) Visceral fat pad mass isolated from 2-month-old-mice with indicated genotypes. Indicated values are mean ± s.d. for n = 5 mice per group. *P≤0.05; compared with fed control mice.
Molecular census of myeloid cells in different compartments of the healthy mouse eye.Figure 1Scheme of the murine eye. Schematic diagram depicting the workflow for the isolation of single CD45+CD3-CD19-Ly6G- cells from different eye compartments (retina, ciliary body, cornea) under healthy conditions for unbiased single-cell RNA-seq (scRNA-seq). Morphological heterogeneity of sorted myeloid cells revealed by May-Grünwald-Giemsa stained cytospins. Scale bars represents 10 µm. t-SNE representation of individual hematopoietic cells from all eye compartments measured by scRNA-seq. Each dot represents an individual cell. Color code indicates the respective cell types. Unbiased cluster analysis of subpopulations of cells found in the steady-state adult retina (cluster 13, 14, 15) , ciliary body (cluster 1, 5, 10, 11, 12), cornea (cluster 2, 3, 6, 9, 16) and of mixed composition (4, 7, 8, 17) could be identified as microglia (cluster 13, 14, 15), macrophages (cluster 1, 2, 3, 6, 7, 8, 9, 10, 16) or peripheral blood leukocytes (cluster 4, 5, 11, 12, 17).   t-SNE representation of single cells based on the tissue of origin.   Stacked bar plot (Marimekko chart) representation of macrophage subsets to a given cluster demonstrating pure microglia populations in clusters 13, 14 and 15. Hypergeometric testing revealed significantly enriched macrophage clusters in the cornea (2, 3, 6, 9, 16) and the ciliary body (1, 5, 10, 11, 12) as indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Heat map of the most regulated genes per cluster (adjusted p-value < 0.05 based on the negative binomial distribution). Clusters are arranged from the left to the right and represent microglia (light green), macrophages (purple) and monocytes /granulocytes/lymphocytes (grey). The scale bar represents color-coded z-scores. Genes that were subsequently confirmed on protein level in h) and i) are highlighted by asterisks. Above: t-SNE representation of P2ry12 and Tmem119 expression. Below: representative immunofluorescence images for P2ry12 (red) and Tmem119 (red) on CX3CR1+ retinal microglia (outer plexiform layer) but not on ciliary body and CX3CR1+ cells in the peripheral stroma and epithelium of the cornea (green). Representative images out of three animals are shown. Scale bars represent 50 µm. Above: t-SNE plots for Cd74 and H2-Aa expression. Below: Typical immunofluorescence pictures for CD74 (red) and H2-Aa (red) in CX3CR1+ cells in the ciliary body and the peripheral stroma and epithelium of the cornea (green). Two animals were examined. Scale bars represent 50 µm.
Figure 3Schematic of TAM treatment and behavioral tests. Open field (OF) test. (B) Color-coded distribution of the exploratory path in the open field test. X-Y axes of squares correspond to the OF arena. Old WT mice as well as Lmnb1-cKO mice spend more time in the periphery of the OF arena. Open field (OF) test. (C) Total distance traveled during the OF test. No significant difference among groups. F(2, 25) = 2.23, P = 0.13, ANOVA. (D) Velocity of travel during the OF test. F(2, 25) = 1.60, P = 0.22, ANOVA. N.S. (not significant). (E) Fraction of time spent in the periphery. F(2, 25) = 10.71, P = 0.00044, ANOVA followed by Tukey Kramer, ** P < 0.01. (F) Fraction of traveled distance in the periphery, F(2, 25) = 3.84, P = 0.035, ANOVA followed by Tukey Kramer, * P < 0.05 (n: WT = 10, WT-old =7, Lmnb1-cKO = 11 for the OF test). G. Principal components analysis of behavior from the OF test (n: WT = 10, WT-old =7, Lmnb1-cKO = 11 for the OF test). Circles indicate 66% confidence levels for each group. H-J. Novelty suppressed feeding. (H) Total feeding time during the NSF test, P = 0.0068, Kruskal-Wallis test followed by Dunn's test. ** P < 0.01. (n: WT = 6, WT-old =7, Lmnb1-cKO = 6 for novelty suppressed feeding). (I) Food consumption during the NSF test, P = 0.014, Kruskal-Wallis test followed by Dunn's test. * P < 0.05. (J) Total number of feeding instances during NSF test. N.S. (not significant).
Figure 4A. Miro1 K572R mutant interacts with HA-Parkin similarly to Miro1 WT. HEK cells overexpressing myc-Miro1 WT or K572R mutant were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. Myc-Miro1 was immunoprecipitated and probed for myc-Miro1 and HA-Parkin. Note the diminished degradation of Miro1 K572R in input and the additional band above Miro1 WT in the IP, which is missing in the case of the K572R mutant B. Miro1 K572R ubiquitination by Parkin is reduced. HEK cells overexpressing HA-ubiquitin, myc-Miro1 or myc-Miro1 K572R and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. HA-ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin-HA and myc-Miro1 C. Representative images of PC6 cells overexpressing Miro1 K572R or Miro1 WT together with EYFP-Parkin. K572R mutant induces Parkin translocation similar to WT in basal conditions but not after treatment with A&O (both 7.5 µM) for 3 hours. Note the spatial heterogeneity values shown in the images D. Quantification of EYFP-Parkin signal heterogeneity in PC6 cells demonstrates that WT Miro1 overexpression amplifies Parkin translocation in response A&O (both 7.5 µM) treatment for 3 hours, but K572R mutation of Miro1 abolishes this effect. ***P < 0.001 and ****P < 0.0001, n = 152-172 cells from 3 dishes per group, Kruskal-Wallis test. Data are presented as means ± SEM.
Figure 7A Mass spectrometry identification of the Atp1 family of proteins captured by biotinylated OC43p peptide in rat dorsal spinal cords from female rats (n=2/groups). Negative log10P - confidence score and peptide sequence coverage (%) were calculated in PEAKS Studio™ 8.5 (BSI). B Relationship of ATPase subunits identified in string-db.org. Lines indicate evidence-based interactions. C ATPase gene family expression in sciatic nerves, DRG and spinal cords of naïve rats. TPM (Transcripts Per Kilobase Million) measures the transcription frequency of a specific gene by RNA-seq (n=3/group). Blue to red color intensity corresponds to Log2(TPM). D, Affinity capture of proteins by OC43p and OC43-SCR bound magnetic beads. Specific reactivity to Atp1a1 and Atp1a2 was detected by protein immunoblotting using specific primary antibodies. OC43p-SCR-coated and uncoated beads (Beads) were used to control non-specific binding. Spinal cords were isolated from male (M) and female (F) rats (n=2/group). Input protein lysates (right side, in duplicates) served as loading controls. Approximate molecular weights (in kDa) are indicated by arrows.