EMBO/sd-character-level-ner huggingface dataset:
Figure 7A. Carfilzomib, Bortezomib and MLN9708 promote the expression of M1 macrophage markers (Il-1β, Il-6, Tnfα) and reduce the expression of M2 macrophage markers (Il-10, Tgfβ) in macrophages derived from PBMCs. After induced by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib (1 μM), Bortezomib (1 μM) or MLN9708 (2 μM). RNA was extracted from cells 6 hours after stimulation and the expression of mRNA were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test). B. Carfilzomib, Bortezomib and MLN9708 promote the secretion of inflammatory related cytokines in M2 macrophages derived from PBMCs. After induced by IL-4 (20 ng/ml) for 24hours to differentiate into M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib, Bortezomib (500 nM) or MLN9708 (500 nM). The secretion of IL-1β, IL-6 and TNFα were detected through ELISA experiments 24 hours after stimulation. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test). C-F. Carfilzomib, Bortezomib and MLN9708 increase the expression of CD80 (C) and reduce the expression of CD206 (E) in M2 macrophages derived from PBMCs. After activated by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM). The representative histogram of CD80 and CD206 expression was analyzed by flow cytometry 12 hours after stimulation. (D, F) Statistics represent the proportion of CD80 (D) or CD206 (F) positive cells in macrophages derived from PBMCs. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01 (student's t-test). G. Carfilzomib, Bortezomib and MLN9708 enhance the phagocytic ability of macrophages derived from PBMCs. After induced by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated with Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM) for 12 hours. Starved for 2 hours and incubated with L1210-GFP cells as targets in serum-free medium for another 2 hours. Macrophages were thoroughly washed and stained with fluorochrome-conjugated anti-CD11B antibody and analyzed through Flow cytometry. Phagocytosis efficiency was determined as the percentage of GFP positive cells in CD11B+ population. H. Statistics represent the percentage of phagocytes and data are means ± SD of three independent experiments. *p < 0.05 (student's t-test).
Schematic model for the role of NDST3 in the regulation of α-tubulin acetylation, V-ATPase assembly, and lysosomal functions.Figure 7Loss of NDST3 promotes α-tubulin acetylation and microtubule stability, increasing the level of the V-ATPase V1-V0 holoenzyme on lysosomal membranes and resulting in hyperacidification and dysfunction of the lysosomes.
Mutation in Cys81 reduces the redox sensitivity of HsAtg4A in vitro.Figure 7(A) Recombinant His6‐HsAtg4AWT, His6‐HsAtg4AC77A or His6‐HsAtg4AC81S (0.1 μg) was incubated with His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence or absence of 1 mM DTT. Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16. (B) His6‐HsAtg4AC81S (0.1 μg) was incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) following the same procedure as detailed in Figure 5C. Reaction mixtures were analyzed and are presented as explained in (A).
Synaptic DCVs localize to the border of the synaptic vesicle cluster.Figure 4Representative confocal image and zoom of a single isolated hippocampal neuron (DIV14) immunostained for the endogenous DCV marker ChgB (green), synaptic protein VGLUT1 (red) and dendritic marker MAP2 (blue). Area of zoom (B) is indicated in (A). Scale bars: 20 µm (A), 2 µm (B). Reconstructed ChgB localizations of dSTORM imaging of zoomed area in (B) (top). Middle panel shows overlay between dSTORM ChgB localizations and VGLUT1 immunofluorescence (confocal, 40x). Scale bar: 2 µm. Zooms of boxed areas containing synapses are visualized in lower panels (a-c). Arrows indicate extra-synaptic DCVs. Scale bar (a-c): 1 µm. Calibration bar represents n:o (0) to high (255) clustering of dSTORM localizations. Representative electron micrographs of synaptic sections containing DCVs. AZ indicates the active zone, dashed lines represent DCV diameter measurements. Arrow indicates extra-synaptic DCV. Scale bars: 100 nm. Histogram of number of DCVs in percentage of synapses. Inset: number of DCVs per synaptic section. Average diameter of DCVs (nm). Distance of DCVs to the active zone (nm), measured from center of DCV to the middle of the active zone. Histogram of distance of DCVs to the middle of the active zone (nm). Inset: shortest distance of DCVs to the active zone (nm). Histogram of normalized distance of percentage of synaptic vesicles (grey) to the active zone and corresponding distance of DCVs (black) to the active zone. Example electron micrograph zooms of synapses containing DCVs outside (top) or inside (bottom) the synaptic vesicle cluster. Scale bars: 100 nm. Percentage of DCVs outside or inside the synaptic vesicle cluster. Histogram of distance (nm) of DCVs to the border of the synaptic vesicle cluster, negative values are inside, positive values outside.
Figure 1(A-C) Bone marrow derived mouse macrophages (BMDMs) from wild type C57BL/6 mice were treated with 4µ8c (100 µM) or vehicle (dimethyl sulfoxide; DMSO) for 24 hours prior to sample processing for lipidomics analysis: (A) PI(3,4,5) P3 to PIP2; (B) PI(3,4,5) P3 to PI; (C) PI(3,4,5) P3 to PIP ratio are shown. Data represent mean values ± standard error of mean (SEM) of peak areas normalized to internal standards (n=3 biological replicates). (D) BMDMs were treated with 4µ8c (100 µM) or DMSO for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates; a representative blot is shown). (E) Wild type C57BL/6 mice were injected with tunicamycin (1 mg/kg) and 4µ8c (10 µg/kg) for 8 hours prior to sacrifice and collection of thiogylcolate-elicited peritoneal macrophages. Protein lysates were analyzed by western blotting using specific antibodies against, pIRE1S742, IRE1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin (n=3 biological replicates; a representative blot is shown). Western blot quantifications are shown next to the figure. (F) IRE1+/+ and IRE1-/- BMDMs were treated with PA (500 µM) for 6 hours and protein lysates were analyzed by western blotting using specific antibodies against pIRE1S742, IRE1, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates, a representative blot is shown).
Late compartment segregation is predicated on removal of Rab7 from SKIP-positive membranes.Figure 5A-C. Consequences of effector depletion on the endogenous Arl8b/Rab7 hybrid compartment. A. Representative confocal images of fixed HeLa cells harbouring GFP-tagged endogenous Arl8b (G-eArl8b, green), transfected with the indicated siRNAs and immunolabeled against endogenous Rab7 (eRab7, magenta). B. Colocalization (Mander's overlap) between endogenous Arl8b and Rab7 in response to effector depletion, n siC=10, n siRILP=9, n siSKIP=9 images (4≥ cells per image) analysed from 2 independent experiments. C. Immunoblot analysis for depletion efficiency of SKIP and RILP, with actin as loading control. D/E. Effect of Rab7 GTPase activity status on its association with the peripheral SKIP compartment. D. Left panels: Representative confocal images of fixed HeLa cells expressing GFP-Rab7 or its mutants Q67L or T22N (green) together with HA-RILP (red) and Myc-SKIP (blue), immunolabeled against the indicated epitope tags. Right panels: Schematic overview per condition. E. Colocalization (Mander's overlap) between the indicated protein pairs, n Rab7=5, n QL=5, n TN=7 images (2≥ cells per image) analysed from 2 independent experiments. F. Graphical summary of Rab7 removal from the SKIP compartment.
Figure 4(A) A strip plot summarizing differences in co-expression (x-axis) of pairwise combinations of 58 UPR genes (n= 3364) between low and high SCNA groups across 32 tumor types (y-axis) as quantified by the change in Spearman correlation coefficient. The side bar indicates the number of gene pairs with significant co-expression change relative to a null distribution obtained from 1000 permutations of SCNA status (B) A histogram showing the percentage of gene pairs (y-axis; n = 3364) that have significant co-expression change according to the number of types (x-axis) in which each gene pair was significant. Colored bars indicate the 2.68% of gene pairs (n = 45) that were significant in at least 9 tumor types, and that were selected for more in-depth analysis. (C) Change in Spearman correlation coefficient between SCNA high and low conditions for 45 gene pairs with significant co-expression changes across more than 9 tumor types (n = 45). Each point indicates the difference in correlation for one tumor type where the gene pair was significant. (D) Network plot showing top UPR gene pairs with reduced, preserved or augmented co-expression. Each node represents a UPR gene and each edge represents a co-expression relationship between a gene pair. Red double line edges depict increased co-expression in SCNAhigh tumors compared to SCNAlow. Solid lines depict preserved co-expression between gene pairs and blue dotted lines depict reduced co-expression between gene pairs. Node colors represent the UPR branch pathway membership of genes, with green representing the IRE1 pathway, blue representing the PERK pathway, orange representing the ATF6 pathway and purple representing membership in more than one branch pathway.
VAMP3 Is Required for Fusion of Vesicles Carrying mATG9 and ATG16L1.Figure 7(A) HeLa cells transfected with control, VAMP3 siRNA for 4 days were transfected during the last 20 hr with mStrawberry-ATG16L1 and labeled for mATG9. The correlation between mATG9 and ATG16L1 was quantified (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001. (B) HeLa cells were transfected with control or VAMP3 siRNA and then with mStrawberry-ATG16, and 5 min movies were recorded. Homotypic fusion events between mStrawberry-ATG16 vesicles were assessed in control and VAMP3 knockdown cells. Error bar, SEM. NS, not significant. (C) In vitro fusion assay of ATG16L1 and mATG9 vesicles. HeLa cells were transfected with control or VAMP3 siRNA for 4 days separately and, on the fourth day, either transfected with mStrawberry-ATG16L1 or ATG9L1-pEGFP. PNS of cells expressing the two constructs were mixed for 1 hr at 37C° and observed by confocal microscopy. Histogram shows the correlation of vesicles carrying ATG16L1 that fuse with vesicles carrying mATG9 (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001. The total number of structures per field (ATG16L1 and ATG9L1) is not significantly different in the control and KD samples (control, 37 ± 3; VAMP3 KD, 45 ± 3; ± SEM; p = 0.106). (D) Schematic diagram of mATG9 and ATG16L1 itineraries pertinent to their heterotypic fusion and autophagosome formation.
Figure 1A Expression patterns of pSEU::SEU-GFP and pWOX5::GFP in the embryos of the indicated genotypes at dermatogen, early globular, and heart stages. White arrows indicate the QC precursor cell, and white dashed lines indicate embryos. Scale bars: 10 µm. B Quantification of pWOX5::GFP GFP fluorescence in wild-type (WT) and seu-3 mutant embryos. Fluorescence intensity at the early globular stage of WT embryos was set to 1. Data Information: In (B) data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test). C, D Expression pattern of pWOX5::GFP in WT (C) and seu-3 (D) embryos at 5 days after germination (DAG). Scale bars: 20 µm. E Quantification of GFP fluorescence in the QC of pWOX5::GFP transgenic plants, as shown in (C) and (D). Fluorescence intensity was normalized to the WT. Data Information: (E), data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test). F RT-qPCR analysis of the relative expression levels of WOX5 in WT and seu-3 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Data Information: (F) data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test). G-J Modified pseudo-Schiff propidium iodide (mPS-PI) staining of stem cell niche areas in the indicated genotypes at 5 DAG. Blue arrows indicate the QC, and red arrows indicate the columella stem cells (CSCs). The numbers denote total number of scored samples, with similar phenotypes showing in (G-J). Scale bars: 20 μm. K Quantification of the CSC layer in the indicated genotypes at 5 DAG. Data Information (K), data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test). L, M Double staining of the QC184 β-glucuronidase (GUS) marker (light blue) and starch granules (dark brown) in WT (L) and seu-3 (M) seedlings at 5 DAG. In white arrows indicate the CSCs. Scale bars: 20 μm. N, O Expression pattern of J2341 in WT (N) and seu-3 (O) seedlings at 5 DAG. P, Q Expression pattern of CS9227 in WT (P) and seu-3 (Q) seedlings at 5 DAG. In white arrows indicate the CSCs. Scale bars: 20 μm.
Figure 1Primary hippocampal neurons were transduced with GFP or GA175-GFP (DIV4+4) and co-cultured with naïve primary neurons for 4 days. Endogenous TDP-43 and poly-GA aggregates in donor and receiver coverslips were analyzed by immunofluorescence. (A) Schematic representation of co-culture experiments. (B) Cytoplasmic TDP-43 immunostaining is elevated not only in poly-GA transduced neurons, but also in the non-transduced receiver cells. White and red arrows indicate cells with cytoplasmic TDP-43 in GFP positive and negative cells, respectively. (C) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced (donor), non-transduced (receiver) neurons. Cells with and without GFP signal were counted separately (indicated by +/­­−). Two groups (GFP negative donor, GFP positive receiver) were excluded due to very high GFP transduction rate and very low GFP transmission rate. n=4 biological replicates. In total 283 donor GFP, 273 donor GA175-GFP, 284 receiver GFP and 266 receiver GA175-GFP cells were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001. Co-culture model in HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and TDP-43ΔNLS-GFP in donor and receiver compartment. Immunofluorescence staining of TDP-43∆NLS aggregate number per cell Co-culture model in HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and TDP-43ΔNLS-GFP in donor and receiver compartment. automatic quantification of TDP-43∆NLS aggregate number per cell In (E) n=3 biological replicates with 368 donor iRFP, 251 donor GA175-iRFP, 430 receiver iRFP and 328 GA175-iRFP cells were analyzed. Cells with and without GFP signal were analyzed separately (indicated by +/­­−). White and red arrows indicate cells with cytoplasmic TDP-43 in GFP positive and negative cells, respectively. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. (F) in addition to filter trap assay of SDS-insoluble TDP-43ΔNLS-GFP aggregates compared in iRFP or GA175-iRFP transfected cells. (G) GFP mRNA expression levels were measured by qPCR. RNA levels were normalized to GAPDH, β-actin and β2-microglobulin mRNA. Bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction. * denotes p<0.05, and ** denotes p<0.01.
Figure 1A, Representative images of controls from the siRNA screen for HU-induced RPA accumulation. GFP-RPA1 and RFP-PCNA reporter cells were transfected with siRNAs and 48 hrs later treated with HU (3 mM) for 2 hrs prior to pre-extraction, fixation and imaging. B, Gene ranking based on siRNA scores from two independent screens. 219 genes were targeted by three individual siRNAs and ranked based on P-values derived from RSA and Fisher's analysis. Selected hits are highlighted. C, Validation of selected genes by single cell analysis of GFP-RPA1 intensity in pre-extracted cells. Only S-phase cells positive for RFP-PCNA were analysed. a, b, c denote independent siRNAs; Ctrl, Control. Median with interquartile range of relative intensity per cell is shown, n > 4000. Mann-Whitney: *** p < 10-4, n.s. non-significant. One representative experiment out of two biological replicas is shown. D, Analysis of DNA synthesis rate. Cells were pulsed 15 min with EdU and EdU intensity was analysed in S-phase cells positive for RFP-PCNA. EdU incorporation is shown relative to siCtrl.
Figure 3(A) SR protein specific temperature-dependent AS-decay. Sashimi plots are shown for heat-induced poison exon inclusion for Srsf2 (left) and cold-induced poison isoform formation for Srsf10 (right). Quantification of GE and AS are presented in the center (as in Figure 2B), lines represent mean. (B) Poison exon inclusion for Srsf2 (left) and Srsf10 (right) in a 24h temperature-rhythm in human cells. Hek293 cells were pre-entrained with square-wave temperature cycles (12h 34°C/ 12h 38°C) for 48h. For the last 24h, cells were treated with DMSO or CHX every 4h and harvested after 4h and analyzed by splicing sensitive RT-PCR (n=3, mean ± SD). White area: 34°C; Red area: 38°C. In Hek293 cells, inclusion of Srsf10 exon 3 is coupled to polyadenylation making it a weak decay target. (C) AS-decay of human targets at 34°C (blue) and 38°C (red) was investigated after a square-wave temperature regime (see B). RNAs were harvested after 56 and 68 hours, respectively (n=3, mean ± SD). Statistical significance was determined by 2-way ANOVA and is indicated by asterisks: adjusted p values: not significant (ns), **p<0.01, ***p<0.001, ****p<0.0001. Box plots show median PSI of validation PCRs, whiskers show min to max. Individual data points are shown as circles. (D) Intron conservation of alternative cassette exons. Shown are average placental conservation scores of introns surrounding all alternative exons (black, N=46,901), AS-decay exons (yellow, N=453) and temperature-regulated AS-decay exons (green, N=139). (E) Quantification of Srsf2 AS-decay in hamster, rabbit and chicken cells (n=2, mean ± SD). Statistical significance was determined by 2-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Box plots show median PSI of validation PCRs, whiskers show min to max. Individual data points are shown as circles.
Figure 1Schematic of MTA proteins with different protein domains indicated as coloured boxes, and the C-terminal epitope tags indicated. The model below shows the MTA- and HDAC-containing subunit joined to the CHD4-containing subunit through the MBD protein. Tagged MTA proteins in heterozygously targeted cell lines were immunoprecipitated and subsequent western blots probed with antibodies indicated at right. Solid black triangles indicate the locations of untagged proteins and open triangles show the position of epitope-tagged proteins in the anti-Mta1, Mta2 or Mta3 panels; and a grey triangle shows the location of IgG bands in the anti-Mbd3 panel. 10% of the input was loaded into "Input" lanes. Molecular weights in kDa are shown at left. Proteins co-purifying with Mta1, Mta2 or Mta3 in IP-mass spectrometry experiments in wild type cells (top) or Mbd3-null cells (bottom). Proteins showing significant enrichment with the bait protein are located outside the dotted lines. For all panels the protein being immunoprecipitated is indicated in red, the other MTA proteins in purple, and other NuRD components in blue. Each IP/Mass spec experiment set comprised three independent immunoprecipitations from one nuclear extract preparation. Relative enrichment of indicated proteins in MTA pulldowns from wild type (WT) or Mbd3-null (Mbd3KO) ES cells, normalised to 2x MTA proteins. NuRD components comprising the remodelling subunit are labelled in blue, those comprising the deacetylase subunit in red. As MTA proteins are the bait in these experiments, they may be isolated more efficiently than their interaction partners. Error bars represent standard deviation from three (Mta1 and Mta2) or six (Mta3) replicate pulldowns.
S-protein stimulated macrophages from convalescent individuals secrete IL-1β and show distinct gene expression signatures.Figure 3Subgroup analysis of IL-1β secretion of macrophages after stimulation with S-protein and nigericin from COVID-19 patients with severe (red/black; n=23) or mild (white/red; n=21) disease. Student's t-test with Welsh corrections was used to calculate statistical differences. Graph shows mean ± SEM. CD14+ cells of two convalescent individuals were isolated sequentially at d18, d26 and d56 after confirmation of SARS CoV2 infection by PCR. Following M-CSF differentiation, macrophages were stimulated with LPS (red triangle) or S-Protein (red circles) and IL-1β concentration (pg/ml) was quantified upon incubation with nigericin. Blue dashed line indicates mean IL-1β level of SC-naïve individuals stimulated with S-protein and nigericin. Quantification of IL-1β concentration (pg/ml) in the supernatant of primary macrophage cultures from convalescent COVID-19 patients (SC-conv) (n = 4; red bars) or SARS-CoV-2 naïve individuals (SC-naïve) (n = 4; blue bars) stimulated with S-protein (1 µg/ml) and nigericin. Students t-test with Welsh corrections was used to calculate statistical differences as indicated. Graph shows mean ± SEM. *p<0.05. Venn diagram showing number of differentially expressed genes (DEGs) in healthy/naïve (SC-naïve; blue) and convalescent COVID-19 patient-derived macrophages (SC-conv; red) upon stimulation with LPS (left) or S-Protein (right). Circle sizes of the venn diagram correspond to the number of genes. KEGG-gene enrichment analysis, based on RNA-Seq data, showing activated signaling pathways in S-protein stimulated macrophages compared to unstimulated in convalescent COVID-19 patient-derived macrophages (SC-conv). Padj values are indicated (color code) and size of the circles represent number of DEGs (count). P-values are calculated using Wald test and p-adjusted values using the FDR/Benjamini-Hochberg approach. Volcano plot showing DEGs in S-protein stimulated macrophages from convalescent COVID-19 patients (SC-conv) compared to unstimulated cells. Negative log10 adjusted p-values are plotted against the log2 fold-change. Select genes are labeled. P-values are calculated using Wald test and p-adjusted values using the FDR/Benjamini-Hochberg approach. Heat-map indicating DEG patterns comparing LPS or S-protein stimulation of SC-conv macrophages. Z-score is indicated in a color score.