EMBO/sd-character-level-ner huggingface dataset:
Figure 3A) Gene expression of three biological replicates of HeLa cells transfected with Control, H2A.Z.1 or H2A.Z.2 siRNA was analysed by RNA sequencing. Venn diagram shows the number of significant (p<0.01) differentially expressed genes and the overlap between each set of genes. B) Pie charts displaying the percentages of H2A.Z peaks at or nearby transcription start sites (TSS) (dark blue), within the gene body (grey) or elsewhere in the whole genome (purple) for the differentially expressed genes following H2A.Z.1 or H2A.Z.2 depletion. A 2-sample test for equality of proportions was used for the statistical analyses. *=p<0.05; ns=not significant. C) Downregulated genes following H2A.Z.1 depletion were analysed by STRING. The image shows the cluster of genes with gene onthology (GO) terms related to cell cycle. PPI=Protein-protein interaction. D) IGV analyses of H2A.Z localisation (from ENCODE) on Aurora B (green) and c-MYC (blue) genes showing H2A.Z enrichment at the TSS and at the upstream region respectively (bottom panels). IGV analyses of ATAC seq peaks for Control si and H2A.Z.1 si for Aurora B and MYC genes. The number in red represents the log2Ratio H2AZ.1 si/Control si. E) Plot representing the average value for all the target regions around the TSS obtained by ATAC seq after Control, H2A.Z.1 or H2A.Z.2 siRNAi. F) Plot representing the number of differential chromatin accessibility regions in pairwise comparison. Green (up) represents regions with increased accessibility and red (down) with decreased accessibility. G) Distribution of the position within the nucleus of the centromere of Chr17 from the experiment in (H) calculated as the ratio d2/d1. The ratio between d2 and d1 gives the position of the centromere relative to the center of the nucleus. The graph represents the percentages of centromeres with distances following within the 5 binning categories. At least 500 nuclei were analysed per condition. Data sets were statistically analysed using Chi-square test for the distribution of signals among the 5 categories. ***=p<0.001. H) Representative image of a HeLa nucleus after FISH with Chr17 centromeric probe (green). The distance of the FISH signals from the nucleus periphery was calculated as follows: the distance from the centre of the nucleus to the periphery (d1) and the distance from the centre of the nucleus to the FISH signal (d2) were measured. (scale bar 5 μm).
Figure 1(A) Stacked column depicts proportions of cell types in the TME of 99LN-BrM analyzed by flow cytometry (n=4). (B) Stacked column depicts the relative amount of myeloid and lymphoid cells in 99LN-BrM analyzed by flow cytometry (n=5). (C) Stacked column depicts the relative amount of myeloid and lymphoid subpopulations in 99LN-BrM based on three flow cytometry panels: myeloid cells (n=5), dendritic cells (n=7), lymphoid cells (n=6). (D) Principal component analysis of tumor-associated myeloid cells vs blood monocytes and microglia from tumor-free mice. (n=3 per condition) (E) Principal component analysis of tumor-infiltrating lymphocytes (TIL) vs blood lymphocytes from tumor-free mice. (n=3 per condition) (F-I) Functional gene annotation of altered cellular pathways in (F) TAM-MG, (G) TAM-MDM, (H) TIL-CD4 and (I) TIL-CD8 compared to control cell types from tumor-free animals. Cutoffs: basemean >20 and adjusted p-value (padj) <0.05. Adjusted p-values were obtained by Wald test and corrected for multiple testing using the Benjamini and Hochberg method. All DEGs based on padj were subjected to analysis.
Human ferritin expressed in S.Figure 5(A) Δccc1/pGAL‐H+L cells were transformed with a plasmid containing a methionine regulated CCC1 (pMET3CCC1). Cells were grown in medium with galactose for 20 h, washed and incubated in galactose (•), glucose with 10 × methionine (▵), glucose (○) or glucose without methionine (▾) for 10 h. Cells were then harvested and ferritin levels determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate. The absence of methionine leads to expression of the vacuolar iron transporter Ccc1p. (B) erg6‐2 and erg6‐2 pGAL‐H+L strains were grown in medium with galactose and 250 μM FeSO4 for 24 h. Cells were then washed and incubated in galactose or glucose in the absence or presence of 50 μM MG132 for 7 h. (C) Cells were then harvested, ferritin levels determined by ELISA and immunoprecipitated using anti‐ferritin antibodies and the immunoprecipitate examined for the presence of ubiquitin by Western analysis. Cells with the erg6‐2 allele permit the entry of MG132 ( Lee and Goldberg, 1996).
Photoperiodic regulation of PIF activity.Figure 4Schematic of transcriptional and post-transcriptional regulation of PIF activity. PIF4, IAA29 and ATHB2 mRNA levels in SDs in WT (data: black lines, filled squares; simulation: black lines). As in (B-D), for LDs. Data from Nomoto et al (2012b). ATHB2 mRNA at dawn (ZT0) across a range of photoperiods. Data from Kunihiro et al (2011). Comparison of model simulations with microarray time course data from the two largest clusters of PIF-induced targets (Cluster 1: 107 genes, Cluster 2: 84 genes), in SDs (I, K) and LDs (J, L) [data from Michael et al (2008b)].
Figure 7A Dot plot presentation of the chemotaxis analyses using the Boyden chamber. CXCL12 (+) and (-) indicate the presence and absence of CXCL12 as an attractant in the bottom chambers. ENTREP-knockout enhanced the chemotaxis toward CXCL12, which was blocked by pre-treatment with AMD3100 (upper panel). The doxycyclin (DOX)-induced ENTREP expression suppressed the chemotaxis of mouse 4T1-Luc cells toward CXCL12 (lower panel). Ratio of migrated cells into the bottom chamber against the applied cells on the top chambers was normalized by the results of non-treated cells (n=7, upper panel, and n=9, lower panel, from at least two independent experiments, respectively). P-values were obtained by Student's t-tests and P < 0.005 was considered as statistically significant. Insert, the immunoblot analysis of the DOX-induced ENTREP expression in 4T1-Luc cells. NS, not significant. B Dot plot presentation of mammosphere assay (left panel) and the images of mammosphere (right panel). Each mark in dot plot presentation indicates the number of mammosphere from 3,000 cells per well in an ultra-low attachment plate from two independent experiments. To estimate the effect of CXCL12/CXCR4 pathway, 100 ng/ml of CXCL12 and 12 μM of AMD3100 were applied as indicated. For statistical analysis, variance was assessed using two-way ANOVA and significance was calculated using Tukey post hoc test correcting for multiple comparison. NS, not significant. Scale bar in the images of mammosphere 100 mm.
Loss of MpFGMYB function results in female-to-male conversion A MpFGMYB gene structure and locations of Mpfgmyb mutations.Figure 3A MpFGMYB gene structure and locations of Mpfgmyb mutations. Gray line, 5′- and 3′-flanking sequences; light purple box, UTR; dark purple box, coding region; red box, MYB domain-coding region; arrowheads, mutation positions; black arrow, transcriptional direction; dotted line, splice patterns. B Diagnosis of genetic sex using Y- and X-chromosome-linked rbm27 and rhf73 markers, respectively. Two biological replicates were analyzed for each genotype. C-L Gametangiophore morphology (C-G) and gamete development (H-L) of wild-type and mutant plants. Scale bars, 5 mm (C-G), 10 µm (H and J), 100 µm (I, K, and L).
Figure 4Membrane lipid and TAG content, and ergosterolester content (EE, inset) per OD600 unit of the yeast strains indicated after culture to mid-log phase in SD with or without 1 mM choline (C); * p < 0.05, ** p < 0.01, unpaired two-tailed t-test of the indicated bar compared to the C+ condition. Data information: All data were obtained by mass spectrometry and are presented as mean ±SD (n=3 biological replicates); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired two-tailed t-test of the indicated bar compared to the cho2opi3 parent unless indicated otherwise. Membrane lipid class composition of classes contributing at least 1% of total membrane lipids, the inset shows CDP-DAG and the separate lyso(L)-phospholipids (lyso-PL); Data information: All data were obtained by mass spectrometry and are presented as mean ±SD (n=3 biological replicates); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired two-tailed t-test of the indicated bar compared to the cho2opi3 parent unless indicated otherwise. Fatty acyl chain profiles of the total lipid, the membrane glycerolipid (ML) and the TAG fraction showing acyl chains that contribute at least 1% of total, with the C10-C14 acyl chains in the insets; Data information: All data were obtained by mass spectrometry and are presented as mean ±SD (n=3 biological replicates); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired two-tailed t-test of the indicated bar compared to the cho2opi3 parent unless indicated otherwise. PE molecular species profile (sum of carbon atoms in the acyl chains : sum of double bonds in the acyl chains) showing species that contribute at least 2% of total PE; Data information: All data were obtained by mass spectrometry and are presented as mean ±SD (n=3 biological replicates); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired two-tailed t-test of the indicated bar compared to the cho2opi3 parent unless indicated otherwise. (E-F) Percentage of molecular species containing more than 32 carbon atoms in both acyl chains (C34+C36) (E) and of saturated acyl chains (SFA) (F) in the membrane glycerolipids (ML) and the major membrane lipids; of the indicated strains cultured to mid-log phase in SD C+/-. Data information: All data were obtained by mass spectrometry and are presented as mean ±SD (n=3 biological replicates); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired two-tailed t-test of the indicated bar compared to the cho2opi3 parent unless indicated otherwise.
Figure 4A. Example traces of presynaptic Ca2+-currents recorded in patched calyx terminals as induced by a 50 ms step depolarization from −50 mV to +40 mV in 10 mV increments at a holding potential of −80 mV. B. Summary graphs of the I-V relationship for the peak Ca2+-current amplitude (left) and of the peak Ca2+-current density (right). C. Example traces (left) and summary graphs (right) of the Ca2+-current activation and deactivation kinetics. Currents were induced by a 50 ms step depolarization from -80 mV to +10 mV. D. Example traces of Ca2+-currents evoked by an action potential-equivalent depolarization (APe, from -80 mV to +17 mV for 1 ms; left), and summary graph of the APe-evoked Ca2+-current peak amplitude and charge (right). E. Example traces of presynaptic Ca2+-currents induced by a 50 ms step depolarization from −50 mV to +40 mV in 10 mV increments at a holding potential of −80 mV before and after perfusion of 200 nM agatoxin (Agtx), a selective P/Q-type Ca2+ channel blocker. F. Summary graph of the relative contribution of P/Q-type Ca2+ channels to the total presynaptic Ca2+ currents, as quantified by the relative reduction in Ca2+ currents evoked by step depolarization to +10 mV, in control and Nrxn123 TKO synapses.
Figure 7(A) Overexpression of WT DISC1, but not the DISC1 L604F mutant, in cultured neurons suppresses dendrite withdrawal induced by TLR3 activation. 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. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm. * P < 0.05, ** P < 0.001, *** P < 0.0001. (B-C) Overexpression of DISC1 in cortical neurons of mouse brain is resistant to poly(I:C)-triggered reduction of dendritic arborization. Vector control (Ctrl) (B) or Myc-DISC1 (C) was co-expressed with GFP in cortical layer 2/3 neurons in mouse brain. After poly(I:C) injection at P4 and P5, the neuronal morphology was analyzed at P7 by tracing the GFP signals. The 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 collected from 3-4 mice of each group are indicated. Mean values SEM are shown. Scale bar, 30 μm. * P < 0.05, *** P < 0.0001.
a) Immunoblot for the indicated Cx of homogenates (Hom) and autophagic vacuoles (AV) isolated from fed or 6 h starved (Stv) mice.figf1(a) Immunoblot for the indicated Cx of homogenates (Hom) and autophagic vacuoles (AV) isolated from fed or 6 h starved (Stv) mice. Quantification of enrichment is shown in Supplementary Fig. 1a. (b) Immunofluorescence for LC3 and Atg16 in normal rat kidney (NRK) cells expressing GFP-Cx43 maintained in the presence or absence of serum for 4 h. Single channels are shown in reverse black and white, and magnification of single and merged images are shown in colour. Arrows: co-localization of Atg16/Cx43 (yellow) or Atg16/Cx43/LC3 (white). Quantification is shown in Supplementary Fig. 1c. (c) Immunofluorescence for the indicated endogenous proteins in NRK cells maintained in serum-supplemented media without additions (None) or treated with chloroquine (CQ) for 4 h. Full fields are shown in Supplementary Fig. 1d. (d) Maximum projection confocal microscopy 3D reconstruction (left) followed by a single-plane time-lapse acquisition of the region outlined by the white square in HeLa cells expressing EBFP2-Cx43 (pseudocoloured in red) and sfGFP-ATG16. (e) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP-Cx43 but knocked down for Eps15. Single channels are shown in reverse black and white, and merged images in colour. Inset shows higher magnification. Bottom: Quantification of co-localization between Atg16 and Cx43 (n = 3 wells, 4 independent experiments, >20 cells per experiment). Values are mean + s.e.m. * P 0.05. (f,g) Immunoblot for the indicated proteins of immunoprecipitates (IP) of endogenous Cx43 and Atg16 from WT MEFs maintained in the presence (f,g) or absence of serum (4 h; g). I: input; FT: flow through. (h) Immunoblot for the indicated proteins of immunoprecipitates (IP) of endogenous Cx43 from untreated WT MEFs, on incubation with 3-methyladenine (3MA) or in MEFs from Atg5-null mice (Atg5−/−). (i) Immunoblot for Flag and Cx43 of immunoprecipitates (IP) of endogenous Cx43 in WT MEFs transfected with the indicated Flag-tagged constructs of truncated Atg16.
Enhanced sensitivity of atg5, atg7 and nbr1 mutants to heat and oxidative stresses.Figure 5(A) Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed in 22°C and 45°C growth for 10 hours and then moved to room temperature for 3-day recovery or (B) sprayed with 20 µM methyl viologen (MV) and kept under light for two days before the picture of representative plants was taken. The experiments were repeated three times with similar results.
Figure 4(A) Metabolomic analysis of CRISPR-generated cystinotic cells (CTNS-/-) treated with cysteamine (100 µM), bicalutamide (35 µM), and a combination of cysteamine and bicalutamide (100 µM and 35 µM, respectively) (n (B) Heatmap analysis of the measured metabolites in CTNS-/- cells upon treatment with cysteamine or cysteamine-bicalutamide combination treatment (n = 3). Metabolites significantly decreased were displayed in green, while metabolites significantly increased were displayed in red. Heatmap and REACTOME analysis of the altered proteins in CTNS-/- cells upon treatment with cysteamine, bicalutamide, and cysteamine-bicalutamide combination treatment (n = 3). Proteins significantly decreased were displayed in green, while metabolites significantly increased were displayed in red. Heatmap and REACTOME analysis of the altered proteins in CTNS-/- cells upon treatment with cysteamine, bicalutamide, and cysteamine-bicalutamide combination treatment (n = 3). Proteins significantly decreased were displayed in green, while metabolites significantly increased were displayed in red. (E), Proteomic analysis of CTNS-/- cells treated with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide, respectively (n = 3). AKGDH; Alpha-ketoglutarate dehydrogenase, GLUD1; GLUD2; Mitochondrial glutamate dehydrogenase 1/2, IGF2R; Cation-independent mannose-6-phosphate receptor, GSTK1; Glutathione S-transferase kappa 1, COX6B1; Cytochrome c oxidase subunit 6B1, ACACA; Acetyl-CoA carboxylase 1-Biotin carboxylase, GLS; Glutaminase kidney isoform, mitochondrial, CASP3; Caspase-3, NPC1; Niemann-Pick C1 protein.
Figure 8(A) Retinal sections from mice injected with AAV2 viruses as indicated, immunolabeled for phospho-S6. Blue colour is DAPI to indicate nuclei. Inset images show individual colours at the same scale as the full image. (B) Percentage of phospho S6 positive cells in the retinal ganglion layer of mice injected with AAV2.shScramble.GFP or AAV2.shPTEN.GFP. (C) Number of pS6-postive RGCs of mice injected with AAV2.GFP or AAV2.p110δ. (D) Representative images of retinal whole mounts from AAV-injected mice stained for the RGC marker Brn3A to indicate RGC survival. (E) RGC survival 28 days after optic nerve crush. (F) CTB-labelled RGC axons 4 weeks after optic nerve crush. Boxes i. to iv. show regenerating axons. Dashed line indicates crush site. White arrows mark the end of axons. Graph shows quantification of regenerating axons at different distances distal to the lesion sites.
Changes in the expression of 20S proteasome catalytic subunits modulate 20S-associated regulators.Figure 5The two HEK EBNA cell lines express only standard proteasome or immunoproteasome subunits. Western blots against the immuno- (β1i, β2i, β5i) and standard (β1, β2, β5) catalytic subunits of the 20S proteasome. Calnexin is used as a loading control. Black lines delineate the boundary between vertically sliced images that juxtapose lanes that were non-adjacent in the gel. Importantly, the bands were assembled from the same blot. Relative normalized abundance indexes of proteasome regulators in HEK EBNA cells containing only immunoproteasome compared to HEK EBNA cells containing only standard proteasome. The normalized abundance indexes for each regulator were set to 1 for standard proteasome conditions (n = 4). Kinetics of IFN-γ treatment on HeLa cells. HeLa cells were stimulated for 0, 24, 48, or 72 h with IFN-γ. Western blots were performed on total cell lysates with antibodies against the β2i, α2, and α5 subunits. IRF-1 was used to control IFN-γ treatment efficiency, and GAPDH was used as a loading control. For each time point of the IFN-γ treatment, proteasome complexes were purified and analyzed by LC-MS/MS. Proteasome complexes dynamics was measured by label-free quantitative proteomics. The normalized abundance index of each protein or protein complex obtained at each time point was compared to the one obtained at the 0 h time point to obtain a regulator relative normalized PAI (n = 3).
Impairment of mitochondria and mitophagy in cells harboring α-synuclein inclusions.Figure 6(A) α-Synuclein fibrils were introduced into HEK293 cells stably expressing both DsRed-LC3 and EGFP-parkin. After 24 h, the cells were immunostained with anti-phosphorylated α-synuclein (P-αSyn). There was no alteration in the localization of EGFP-parkin. The arrows indicate α-synuclein inclusion. Scale bar, 10 µm. (B) Mitochondrial clearance was confirmed in mock- (upper panels) or α-synuclein fibrils-introduced cells (lower panels). After introduction, EGFP-parkin cells were treated with DMSO (left) or 10 µM CCCP for 4 h (middle) or 16 h (right). Cells were examined immunocytochemically with anti-Tom20 (red) and anti-phosphorylated α-synuclein antibody (P-αSyn; aqua). Figures were presented as merged images. Individual images were provided in supplemental figure (Fig. S8). Scale bar, 10 µm.
Figure 1A Schematic setup of the screen for PD-L1 regulators in the presence and absence of IFN-γ. RKO reporter cells expressing PD-L1-eGFP fusion protein (blue) were mutagenized with an sgRNA/Cas9 library. PD-L1low cells were enriched by FACS and sgRNA target genes were identified by deep sequencing. An enrichment analysis determined frequencies of different sgRNAs and their target genes. B sgRNA frequencies (log2 of fold change) in PD-L1low cells unstimulated (left) or IFN-γ stimulated (right). Enriched sgRNAs (dashed line) are highlighted; hit genes (black, red) and controls (green, yellow or purple) are shown and labeled with target gene names. C sgRNA frequencies (log2 of fold change) in viability screen. sgRNAs targeting controls are highlighted; essential (orange), non-essential (blue). Genes attributed to PD-L1 modulation are shown in different colors (red, yellow and purple).
Figure 5A. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. The phosphorylation status of GSK3β (Ser9), ERK1/2 and EGFR (Tyr1173) was assessed by western blotting and normalized according to the total protein levels. RHOB overexpression was also monitored by western blotting. B. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 72 h with erlotinib alone (black and red curves) or in combination with the AKT inhibitor G594 at 100 nM (green and blue curves). The surviving cell fraction was determined by an MTS assay. C. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) and treated for 72 h with increasing concentrations of erlotinib in the absence or presence of increasing doses of G594. The surviving cell fraction was determined by an MTS assay, and erlotinib IC50 values were determined for each condition. (**: p<0.001 vs. AdCont cells; ***: p<0.0001 vs. AdCont cells). HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. Apoptosis was then determined by quantification of the subG1 cell population (D). HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. Apoptosis was then determined by detection of cleaved PARP and caspase-3 (E).
The regulatory connections of the osmotic stress-responsive GRN.Figure 3A The significant regulatory interactions identified by nCounter Nanostring at 1 h, 2 h, 4 h, 8 h and 24 h after induction of overexpression of a TF. B The confirmed regulatory interactions between the 20 TFs part of the GRN, according to transient expression assays (n = 4 biological repeats). Green arrows represent activation and red arrows repression.  C Heatmap of the significant regulations upon induction of overexpression of the five members of the core network, the activators (green) ERF6, ERF59, ERF98 and the repressors (red) ERF8 and ERF9. Color code represents FDR-corrected p-values with thresholds at FDR=0.01, 0.05 and 0.1. Data information: In A, data are extrapolated from estimated averages, n = 3 independent experiments, FDR-corrected p-values < 0.1 (mixed model analysis, user-defined Wald tests). The thickness of the arrows represents the FDR value. In B, data are presented as averages, n = 3 independent experiments. The intensity of the color of the arrows represents the strength of the regulation according to the TEA values and the thickness the FDR value of the nCounter Nanostring experiment. In C, data are represented as FDR-corrected p-values, n = 3 independent experiments.