EMBO/sd-character-level-ner huggingface dataset:
Figure 2HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR) and grown in fed or starved conditions for 1h. Cells were fixed and co-labelled with ULK1 (red) antibody and A) anti-CALNEXIN (CANX) (green) antibody to highlight ER structures; (n=3 independent experiments, n≥16 fields analysed) B) GOLGIN97 (green) antibody to label GOLGI complex; (n=3 independent experiments, n=12 fields analysed) C) SEC31A (green) to point out COPII vesicles. (n=3 independent experiments, n≥16 fields analysed) D) ERGIC53 (green) to mark ERGIC compartment. (n=3 independent experiments, n≥14 fields analysed). Analysis of ULK1 localization with each compartment is reported in each graph. Co-localization analyses were performed by ImageJ plugin Jacop. Values of Mander's coefficient for ULK1 are expressed in percentage as mean ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ***, P<0,001; ****, P<0,0001; ns, not significant. Scale bars 5 μm. White arrowheads point at ULK1 puncta associated to the analysed markers; N, nucleus. E) HeLa cells were transfected as in A), labelled with the indicated antibodies and imaged by SIM microscope. Scale bar in large panels, 5 μm; in small panels, 1 μm. F) HeLa cells were co-transfected with control siRNA and an empty vector (EV), or 3'UTR TFG siRNA together with an EV or HA-TFG plasmids. Cells grown for 1h in starvation medium then fixed, permeabilized, and labelled with anti-ULK1 (green) and anti-ERGIC53 (red) antibodies respectively. Hoechst was used to stain nuclei. Co-localization analysis were performed as in D). Values of Mander's coefficient for ULK1, expressed as percentage, are mean ± SEM. Statistical analyses were performed by one-way ANOVA followed by Dunnetts's multiple comparison test. *, P<0,05. (n=3) (n=3 independent experiments, n≥9 fields analysed). Scale bar 5 μm.
Figure 6Schematic diagram of the experiment in which PS exposure was measured in the brains of 1-month- and 3-month-old wild-type mice by injecting AAV9-GFAP-secA5-mCherry in the neonatal stage (P0). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue), and secA5 (red) in the HP (hippocampus) of 1-month- and 3-month-old wild-type mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the expression of secA5 (B) and IBA1 (C) in inhibitory post-synapses (Gephyrin-positive, green) and pre-synapses (VGAT-positive, blue) in the HP of 1-month- and 3-month-old wild-type mice (n = 4 for each group, ***P = 0.0009, *P = 0.0248, n.s., not significant). Schematic diagram of the experiment in which Annexin V was injected intravenously (every two days) to block PS in the brains of 1-month- and 3-month-old wild-type mice. Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the HP of 1-month-old wild-type mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of 1-month-old mice after PBS or Annexin V injection (n = 4 for each group, ***P = 0.0013, **P = 0.0090). Schematic diagram of the experiment in which microglial Mertk KO (CX3CR1-CreERT2;Mertkfl/fl) and control (Mertkfl/fl) mice were generated. Both 1-month-old microglial Mertk KO mice and 1-month-old control mice were injected with 4-OHT for 5 consecutive days. Representative confocal z-stack images of Gephyrin (green)- and VGAT (magenta)-positive inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, **P = 0.0027, *P = 0.0062). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue) and secA5 (red) in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the relative expression of secA5 in inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, *P = 0.0015).
Figure 1Immunoblot (IB) analyses of the levels of β-catenin and RAS in DLD1, WT-HCT116, MT-HCT116, HepG2, and SW48 cells transfected with Myc-axin for 36 hr Effects of β-catenin knock-down on polyubiquitination-dependent degradation of RAS. IB analyses of MT-HCT116 cells transfected with control or two independent β-catenin siRNAs for 48 hr Polyubiquitination assays of MT-HCT116 cells cotransfected with two independent β-catenin siRNAs and HA-ubiquitin (Ub) for 72 hr in the presence of the proteasomal inhibitor MG132, and WCLs were immunoprecipitated with an anti-pan-RAS antibody IB analyses of HEK293 cells transfected with WT- or S33Y-Flag-β-catenin with or without HA-GSK3β for 36 hr IB analyses of HEK293 cells transfected with β-catenin siRNA or control siRNA for 48 h and then treated with L-cell-conditioned medium (L-CM) or Wnt3a-CM for 8 hr Effects of KYA1797K or XAV939 on degradation of β-catenin and RAS in isogenic HCT116 cells harboring WT- or MT-CTNNB1. IB analyses of β-catenin and RAS levels in WT- or MT-HCT116 cells treated with 5 or 20 µM of KYA1797K or XAV939 for 24 hr ICC analyses of WT- or MT-HCT116 cells treated with 20 µM of KYA1797K for 24 hr. Scale bars, 10 µm For polyubiquitination assays, WT- or MT-HCT116 cells were transfected with FLAG-Ub for 24 hr and then treated with 20 µM of KYA1797K for 24 hr in the presence of MG132. WCLs were immunoprecipitated with β-catenin or pan-RAS antibody and subjected to IB analyses to detect each protein The 3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazolium bromide (MTT) assays were performed for measurements of growth of WT- or MT-HCT116 cells treated with the indicated concentrations of KYA1797K. Data are presented as the mean ± SD (n = 3)
Figure 3, overview of the MetaHybridLouvain procedure. B, viSNE layout of CyTOF single-cell events colored according to the 26 canonical leukocyte phenotypes detected by MetaHybridLouvain. Unique sub-communities of each canonical phenotype are differentiated by the numerals used as point marks. The categorizations from NodLabel are provided as text labels. Note that over-plotting has been disallowed, so this is a sampling of the single-cell events. C, number of sub-communities detected by MetaHybridLouvain for each of the canonical leukocyte phenotypes. D, viSNE layout of CyTOF single-cell events with points colored according to the CHIKV channel. By qualitative comparison monocytes, myeloid dendritic cells (mDCs), and B cells (labeled) have the highest CHIKV envelope protein (E2) expression on the cell surface. E, differences in CHIKV E2 protein expression between acute-phase and convalescent-phase samples per MetaHybridLouvain sub-community. Sub-communities are ordered by largest to smallest difference and filtered to sub-communities where the median of the channel means per sample was higher in the acute-phase samples at a significance threshold of FDR < 0.05. Sub-communities are named by their parent canonical community name plus an arbitrary number F, heatmap of log10 scaled PBMC sub-community frequencies for all samples (n = 42 patients × 2 timepoints = 84 samples). Clinical variables are depicted for all samples across the top of the heatmap; convalescent post-symptom onset anti-CHIKV antibody titer and viral titer (which was measured during the acute phase) are both in units of log10 dilutions. Hierarchical clustering (using complete linkage) was applied to both samples (X axis) and sub-communities (Y axis). Four major clusters of sub-communities and two major clusters of samples (largely separating acute and convalescent samples) are highlighted. , fold changes in log10 frequencies for PBMC sub-communities contrasted between acute and convalescent phase samples, filtered to a significance threshold of FDR < 0.05 (moderated paired t-test, mixed effects model). Error bars represent the 95% confidence interval. The acute phase frequency for each sub-community is depicted with the purple heatmap. *, FDR-adjusted P (FDR P) < 0.05; **, FDR P < 0.01, ***, FDR P < 0.001.
Figure 1A) Schematic representation of the wet-spinning set-up that was used for the biofabrication of myo-substitutes. B) 3D printed co-axial nozzle. C) Detail of the bioink extrusion at the tip of the co-axial needle. D) Hydrogel fiber bundle after removal from the drum E) bright field high magnification image showing densely packed fibers. F) Effect of motor speed on fiber diameter: interestingly, the experimental values are lower than the theoretical ones due to the shrinking of the alginate-based bioink upon exposure to divalent calcium ions.
a) The number of CLSs was counted in WAT of each type of mouse as described in the Methods.f5(a) The number of CLSs was counted in WAT of each type of mouse as described in the Methods. *P0.05; one-way ANOVA, n=3. (b) Total mRNA was extracted from each tissue, and RT-PCR was done using primers specific for Tnfa, Il6, F4/80 or pro-Il1b. The expression of pro-Il1b was determined using SVF fraction of WAT tissue. (c,d) Lysate of SVF was subjected to immunoblot analysis using anti-IL-1β (c) or anti-caspase-1 antibody (d). (e) Primary peritoneal macrophages were isolated, and treated with PA of the indicated concentrations in the presence or absence of LPS. ELISA was conducted to determine IL-1β concentrations in the culture supernatant. (BSA, bovine serum albumin) ***P0.001; two-way ANOVA, n=4. (f) After incubation of primary peritoneal macrophages with PA of the indicated concentrations with or without LPS for 16 h, NAD+/NADH ratio was determined using a commercial kit. *P0.05, **P0.01, ***P0.001; two-way ANOVA, n=5. (g) After the same treatment of peritoneal macrophages as in f, cells were incubated with MitoSOX to measure mitochondrial ROS by fluorescence-activated cell sorting (FACS) analysis. (h) After the same treatment of peritoneal macrophages as in f, cells were stained with MitoTracker Red and MitoTracker Green for FACS analysis to determine mitochondrial potential. The numbers in g,h indicate the percentages of cells in the designated gate.
γ‐irradiation leads to preferential depletion of ISPCs with high Wnt signaling activity A–I Three‐month‐old mTerc+/+ mice were exposed to 12 Gy γ‐irradiation.Figure 5A-I Three‐month‐old mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR (n = 5 mice per group). (A-F) Representative pictures of PCNA staining. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. Note the depletion of PCNA+ CBC cells in position 1 and 2 in between the Paneth cells at 3-48 h after IR and the regeneration of these cells at 4-6 days after IR. (G) Quantification of PCNA+ ISPCs at the indicated positions in basal crypts at the indicated time points after IR. Mean values ± SEM are given. A-I Three‐month‐old mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR (n = 5 mice per group). (H, I) Representative pictures of Olfm4 in situ hybridization. Arrowheads point to positive cells. Dashed lines outline the crypts in irradiated samples. Scale bar: 20 μm. Note the selective survival of ISPCs above the Paneth cells at 24 h after IR. J-X Three‐month‐old LGR5‐GFPki, mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR. (J-N) Representative pictures of GFP staining at indicated time points after irradiation. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. J-X Three‐month‐old LGR5‐GFPki, mTerc+/+mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR. (O-X) Flow cytometry analysis of freshly isolated basal crypt cells at the indicated time points after IR (n = 4-10 mice per group). (O) The histogram depicts the number of LGR5+ cells in freshly isolated basal crypts at the indicated time point after IR. (P, Q) Flow cytometry analysis was used to determine the percentage of (P) LGR5hi cells and (Q) LGR5lo cells within the fraction of LGR5+ cells. (R‐W) Representative FACS plots of small intestinal crypt cells of non‐irradiated mice, 24 h after irradiation. Note the reduction in LGR5+ cells in irradiated mice with the remaining cells showing almost exclusively weak expression of GFP (LGR5lo). (X) Quantification of the survival rate of LGR5hi‐high cells and LGR5hi‐low cells within the fraction of LGR5hi cells comparing irradiated mice to non‐irradiated mice. Note the preferential depletion of LGR5hi‐high cells within LGR5hi cell population upon IR. Mean values ± SEM are given. NIR, non‐irradiated; IR, irradiated.
Figure 3(A) Venn diagram showing the overlapping subsets of modulated human genes (fold change | log2 | ≥ 1, P-value < 0.01) in the PLX4720, bevacizumab and COMBO treatments versus vehicle.(B) Venn diagram showing the overlapping subsets of modulated murine genes (fold change | log2 | ≥ 1, P-value < 0.01) in the PLX4720, bevacizumab and COMBO treatments versus vehicle. (C) Summary of the functional categories of mouse genes significantly enriched in response to PLX4720 and COMBO. GO analyses were performed individually on down- or up-regulated genes using DAVID tool (biological process). GO terms are ranked by p-value corrected by BH method, and the number of genes is indicated. For a complete list of significantly enriched GO groups see Appendix Tables SIII-SIV. (D) GSEA enrichment plots for "Reactome Cytokine Signaling in Immune System" and "Reactome Innate Immune System" (upper panels) and "Reactome Extracellular Matrix Organization" and "Reactome Collagen Formation" (lower panels) highlight significant enrichment of the pathways relative to the immune response and a decreased expression of the pathways relative to extracellular matrix remodeling in COMBO treated tumors as compared to the other treatments (vehicle, PLX4720, bevacizumab). (E) Heatmap representation of gene expression changes within the "Reactome Innate Immune System" (left panel) and "Reactome Extracellular matrix Organization" (right panel) gene set. Genes in heatmaps are shown in rows and samples are shown in columns. Expression level is represented as a gradient from high (red) to low (blue). V, P, B and C respectively indicate vehicle, PLX4720, bevacizumab and COMBO.
VEGF-A-induced AMD-like pathologies occur in an NLRP3 inflammasome- and IL-1β-mediated process, which is regulated by TLR2-signaling and autophagy pathways.Figure 8A. CNV lesion numbers per eye in 6-week old VEGF-Ahypermice compared with VEGF-Ahypermice lacking CASP1/CASP11, NLRP3, IL1R1, IL-18, TLR2, or those heterozygous for beclin-1 or homozygous for hypomorphic ATG16L1. CASP1/CASP11, NLRP3 or IL-1R1 deficiency results in significantly fewer CNV lesions. Each value represents the total CNV lesion number/eye for an individual mouse. Mean ± SEM is shown. P-values are based on comparison with the VEGF-Ahypermouse group. Absolute numbers of mice per group are indicated in ( ). P-values were calculated with a two-tailed unpaired Student's t-test. Separately, p-values are shown between VEGF-Ahyper/CASP1-/-/CASP11-/- and VEGF-Ahyper/NLRP3-/- or VEGF-Ahyper/IL1R1-/-mouse groups. WT mice developed no CNV lesions. B. Average CNV lesion area (in μm2/lesion) in 6-week old VEGF-Ahypermice compared with VEGF-Ahypermice lacking CASP1/CASP11, NLRP3, IL1R1, IL-18, TLR2, or those heterozygous for beclin-1 or homozygous for hypomorphic ATG16L1. Lack of TLR2 or CASP1/CASP11 significantly reduces CNV lesion size, while reduced autophagy moderately increases CNV size. Each value represents the average CNV lesion size/eye for an individual mouse. Mean ± SEM is shown. P-values are based on comparison with the VEGF-Ahypermouse group. Absolute numbers of mice per group are indicated in ( ). P-values were calculated with a two-tailed unpaired Student's t-test. Separately, p-values are shown between VEGF-Ahyper/CASP1-/-/CASP11-/- and VEGF-Ahyper/NLRP3-/- or VEGF-Ahyper/IL1R1-/-mouse groups. C.-E. Representative choroidal flat mount images show multifocal CNV lesions in VEGF-Ahyper mice and a significant reduction in CNV numbers in those VEGF-Ahyper mice that lack either CASP1/CASP11 or IL1R1 (and therefore IL-1β-signaling). Staining for phalloidin in green and for CD31 in red. Scale bars 200 μm. . Laser-induced CNV experiments demonstrate that treatment with a caspase-1 inhibitor (compared to DMSO treated controls) or a neutralizing TLR2 antibody (compared to isotype matched IgG treated controls) inhibits CNV lesion growth. P-values are shown. N=15 mice/group. Representative CNV lesions in choroidal flat mounts are shown. Scale bars 100 μm.
Knockdown of EXOSC10 increases repeat RNA and poly-GA expression A.Figure 1. Schematic representation of a plasmid expressing (G4C2) 80 repeats together with 5' flanking of the C9orf72 repeat (113 bp) and artificially introduced 3' 3xTAG. , C. siRNA-mediated knockdown of EXOSC10 (EXO10), but not of DIS3 and DIS3L, significantly increased generation of poly-GA in HeLa cells expressing (G4C2) 80 repeats. ctrl" indicates a control vector which lacks the G4C2 repeat but still contains the 5' flanking region and 3xTAG. . Increased G4C2 repeat RNA expression upon EXOSC10 knockdown on RT-qPCR. E, F. Knockdown of EXOSC10 increased RNA foci in HeLa cells expressing (G4C2) 80 repeats visualized by in situ hybridization targeting sense RNA foci. In total 25 (Ct) and 24 (EXO10#5) images were analyzed in 3 independent experiments. G. Knockdown of EXOSC10 increased RNA foci intensity in HeLa cells expressing (G4C2) 80 repeats. Each dot represents average cellular RNA foci intensity (Arbitrary unit) of RNA foci-positive cells in each randomly taken image. In total 25 (Ct) and 24 (EXO10#5) images were analyzed in 3 independent experiments.
Figure 3RNA-immunoprecipitation (RIP) analyses of EGFP-MIWI2 and EGFP. EGFP-MIWI2 and EGFP were immunoprecipitated from 16.5 dpc prospermatogonia, and long RNAs bound to these proteins and input RNAs were sequenced. RNAs expressed from HMR_-L1s and piRNA clusters, but not Ensembl and prospermatogonium-expressed mRNAs, are overall enriched by immunoprecipitation (IP) of EGFP-MIWI2 (left). This enrichment is not observed when EGFP is immunoprecipitated (right). The numbers of mRNAs, piRNA clusters, and HMR_-L1s analyzed were shown in parentheses. Prospermatogonium-expressed mRNAs are those that are highly and specifically expressed in prospermatogonia. The bottom and top of boxes represent 25% percentile and 75% percentile, respectively. The line in the box represents the median. Circles represent outliers, and the bottom (top) of whiskers represents the minimum (maximum) values except for outliers. RIP analyses of EGFP-MIWI2 in newborn testes from WT and Mili KO mice. RNA bound to EGFP-MIWI2 and input RNA were sequenced. All RNAs derived from each class was counted for this analysis. HMR_-L1s preferentially generate piRNAs. X- and Y-axes represent expression levels of long RNAs (16.5 dpc prospermatogonia) as determined by RNA-seq and MIWI2-bound piRNAs (18.5 dpc testes) as determined by small RNA-seq, respectively. Circles in the charts represent individual mRNAs, piRNA clusters and HMR_-L1s. More than half of the HMR_-L1s (right) are located in the area of y > x (upper part of the diagonal), whereas most mRNAs (left) are located in the area of y < x (lower part of the diagonal). RPKM, reads per kilobase per million mapped reads. Frequent 10-nt overlap between sense and antisense piRNAs derived from HMR_-L1s. Top 100 HMR_-L1s that generate the largest number of unique piRNAs were analyzed. Frequencies of 1−18-nt overlaps were analyzed for each HMR_-L1. piRNAs derived from HMR_-L1s are enriched in adenine at their 10th nucleotide. HMR_-L1s generating more than 40 unique piRNAs were analyzed (678 HMR_-L1s). The proportions of piRNAs with adenine (left) and uridine (right) at the indicated positions were analyzed for each HMR_-L1. See A for the explanation of box plots.
Phospholipase C gamma 1(PLCγ1) mediates netrin-1/DCC signaling.Figure 2(A) In primary mesencephalic neurons, the phosphorylation of PLCγ1 on Y783 was increased proportionally to the netrin-1 concentration. The lower graph presents quantification of the band intensities in (A) (n = 3) (B) Phosphorylation of PLCγ1 on Y783 was decreased after knockdown of the DCC receptor by siRNA (siDcc) (N = 3) (C) Western blot for the NTN1 secretion test. Lane #1: transfection-free lysate, #2: transfection-free supernatant, #3: NTN1-transfected cell lyasate, #3 and #4: NTN-1- transfected supernatant (D) Ventral mesencephalic (A9) cultures (daggers) derived from control or Plcg1f/f;Nes-Cre mice, were co-cultured with netrin-1-releasing 293T cell aggregates (asterisks; scale bar = 100 μm). The areas delineated by the white boxes between the mesencephalon tissue and 293T cell aggregates are magnified in the respective insets (n = 4) (E) Schematic diagrams of axon extensions The graphs were drawn according to the vector values between the borders of the cell aggregates and the mesencephalic tissues. (F) Mean angle of the axon extension (G) Quantification of the ventral mesencephalic axons. The bar graphs indicate the percentages of proper targeting of ventral mesencephalic axons correctly targeting the netrin-1-releasing cell aggregates (n = 200). Each experimental set was performed in quadruplicate (H) Anti-tyrosine hydroxylase (TH) immunostaining in ventral mesencephalic (VM) tissue. (scale bar = 200 μm) (I) Primary mesencephalic neuron culture in the absence or presence of NTN1-conditioned media (n = 100; scale bar: 500μm) (J) Schematic of the method for the measurement of neurite lengths (K) Mean length of neurites (F-value:1.82, t-test, P**<0.005)