EMBO/sd-character-level-ner huggingface dataset:
Loss of autophagy activity suppresses the retarded heterochronic defects in miRISC mutants.Figure 1A. Schematic illustration of seam cells in a wild‐type L1 larva. (B) Division pattern of seam cells at the postembryonic stage in hermaphrodites. Seam cells V1-V4 and V6 divide symmetrically at the L2 stage (highlighted in red), thus doubling in number. C-F Division pattern of seam cells at the postembryonic stage in hermaphrodites. Seam cells V1-V4 and V6 divide symmetrically at the L2 stage (highlighted in red), thus doubling in number. (G,H) dcr‐1 young adults have an increased number of seam cells (shown in brackets) and gaps in the alae (between arrows). (I,J) atg‐2 mutations suppress the retarded heterochronic defect in dcr‐1mutants. Complete alae (arrow) are formed in dcr‐1; atg‐2 double mutants. K-M Confocal images showing expression of hbl‐1::gfp::hbl‐1 reporter in VNC in wild type (K), atg‐2 (L) and epg‐6 (M) L3 larvae. N Distribution of relative fluorescence intensity of HBL‐1::GFP in 50 unit areas in VNC in the indicated strains. O-Q Confocal images showing the expression of the col‐10::gfp::lin‐41(3′UTR) reporter in wild type (O), atg‐2 (P) and atg‐3 (Q) late L4 larvae. R Distribution of relative fluorescence intensity of col‐10::gfp::lin‐41(3′UTR) in 50 unit areas of hypodermal cells in the indicated strains. S,T Distribution of relative fluorescence intensity of hbl‐1::gfp in VNC and col‐10::gfp::lin‐41(3′UTR) in hypodermal cells in 50 unit areas in wild type, let‐363(RNAi) or starvation‐treated animals. Black lines in (N,R-T) represent the average fluorescence intensity of the reporter in each strain. P values are determined by Student's two‐tailed unpaired t‐test in (N,R-T). (C,E,G,I) Scale bar, 20 μm; (D,F,H,J-M,O-Q) scale bar, 10  μm. GFP, green fluorescent protein; miRISC, miRNA‐induced silencing complex; RNAi, RNA‐mediated interference; UTR, untranslated region; VNC, ventral nerve cord.
NOD2 activation of M.Figure 5(A, B, D, E, G, H) The subcellular localization of autophagy proteins was ascertained in untreated and MDP‐treated cells using (A, B) anti‐IRGM, (D, E) anti‐LC3, and (G, H) anti‐ATG16L1 antibodies and detected with a secondary antibody coupled to 5‐nm gold particles indicated with arrowheads; bar represents 150 nm. (C, F, I) Gold particles co‐localizing with bacteria were manually counted in ten macrophages of each condition. Means ± SEM are depicted and differences between treatments are indicated, *p 0.05 using a two‐tailed paired t‐test. (B) Transmission electron microscopy (TEM) magnification ×50,000; the rest of the micrographs are TEM magnification ×80,000. Data were generated from one experiment.
Figure 4A. spMis18fl-Myc co-immunoprecipitates with both GFP-tagged spMis18fl and spMis18MeDiY, but shows reduced association when MeDiY-mediated dimerization is disrupted in spMis18flI31A. The asterisk (*) in the top panel denotes the IgG heavy chain. B C. Dimer-II interface mutations I31A and Y114A affect the ability of ectopically expressed spMis18fl to rescue the temperature sensitivity of mis18-818 and mis18-262 cells, while expression of spMis18MeDiY alone confers a dominant negative effect on growth in a MeDiY dimerization-dependent manner. Five-fold serial dilutions of cells expressing the indicated spMis18 constructs integrated at the leu1 locus in the genome, spotted on complete PMG + Phloxine B media supplemented with (repressed) or without (expressed) thiamine, and incubated at the indicated temperatures; dead cells stain dark pink. D. Mutations that disrupt MeDiY dimerization lead to reduced levels of spMis18fl association with centromeres. qChIP analyses of spMis18fl-GFP association with centromere 2 (cc2) in the indicated strains when grown in complete PMG media supplemented with (repressed) or without (expressed) thiamine. Error bars represent standard deviation between at least three biological replicates.
Figure 1A) SILAC screen design. B) Scatterplot with relative phosphopeptide enrichment in Ulk1/2 double knockout (DKO) vs wild-type (WT) MEFs in experiment 1. Log2 SILAC ratios from forward (fwd) and reverse (rev) conditions are plotted, with reciprocal ratios shown for the reverse condition. 16,308 phosphosites were detected in the forward and reverse experiments, and those with a log2 SILAC ratio of less than -1.5 in both (lower left quadrant) were considered to be depleted in DKOs. Nine phosphopeptides were assigned to the Fip200 (gene name Rb1cc1), shown in green, and 560 matched the ULK1 motif sequence shown in red. Two Fip200 phosphopeptides matched the ULK1 motif, indicated in red and green, 1 of which was significantly depleted in DKOs. See methods for use of ULK1 motif. C) Relative enrichment of amino acids centred on phosphosites depleted in DKOs depicted as consensus logos from experiments 1, 2 and 3. For experiment 2, average motifs for peptide subsets with and without proline at position +1 are shown in separate logos. D) TMT starvation time course protocol. E) Profile plots for the 3 control phosphopeptides. TMT enrichment (Y axis, log10Intensity) from the starvation time course (X axis, mins; RF=Refed) with WT enrichment values in green and DKO values in red. In the refed condition full media was added after 60 mins of starvation and cells harvested 60 mins later. F) Control and experimental peptides selected from peptide array-based in vitro kinase assay (see Fig EV2 for full details).
Figure 3Activated GFAP+ astrocytes in the spinal cord upregulate OTUB1 in EAE at maximal disease activity (day 15 p.i.) in an OTUB1fl/fl mouse (arrowheads), but not in a GFAP-Cre OTUB1fl/fl mouse. Note the OTUB1-expressing neuron in an OTUB1fl/fl mouse which is surrounded by GFAP-expressing processes of an activated astrocyte (asterisk). At day 22 p.i., astrocytes have downregulated OTUB1 expression while neurons express OTUB1 in an OTUB1fl/fl mouse (asterisk). Astrocytes in a GFAP-Cre OTUB1fl/fl mouse are OTUB1-negative while neurons express OTUB1 (asterisk). At day 22 p.i., demyelination in a GFAP-Cre OTUB1fl/fl mouse is much more severe and extended as compared to an OTUB1fl/fl mouse. Inflammation persists in the GFAP-Cre OTUB1fl/fl mouse, whereas it has resolved in an OTUB1fl/fl mouse. All photographs are representative of three mice per group; original magnification ×400; scale bars correspond to 50 µm. (A, C) Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC). (B, D) CV-LFB staining. EAE was induced in GFAP-Cre OTUB1fl/fl mice (n = 29) and OTUB1fl/fl control littermates (n = 29) by MOG35-55 peptide immunization with pertussis toxin. Graph represents data pooled from 4 experiments with 7-8 mice per group and shows the mean clinical scores + SEM. Statistical analysis performed using Mann-Whitney U test; * p < 0.05. EAE was induced in GFAP-Cre OTUB1fl/fl mice (n = 12) and OTUB1fl/fl control littermates (n = 12) by MOG35-55 peptide immunization without pertussis toxin. Graph represents the mean clinical scores + SEM. Statistical analysis performed using Mann-Whitney U test; * p < 0.05.
Figure 1(A) Morphological phenotypes of Wild type (WT), mkk1 mkk2 and summ3-1 mkk1 mkk2 plants. Photos were taken on three-week-old soil-grown plants. (B) Trypan Blue staining of cell death in the indicated genotypes. (C) DAB staining of H2O2 accumulation in the indicated genotypes. (D-E) Expression of PR1 (D) and PR2 (E) in the indicated genotypes as determined by quantitative RT-PCR. Values were normalized to the expression levels of ACTIN1. The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated three times with similar results.
Figure 9A) Confocal images at the lateral plane showing the immunostaining of the medium threshold Hh target Collier (Col) of wild type wing discs and discs after inhibition of vesicle fusion (α-Snap RNAi), vesicle trafficking (Hrs RNAi) or in over-expression of the Tsp96F-HA. Quantification of gradient lengths (μm) using Collier signal and normalized to the wing pouch size, showing the proportion of wing pouch covered by signal (1=total coverage). Quantification was performed for wild type condition as well as in temporally regulated RNAi expression for the ESCRT complex proteins Hrs, Tsg101, Shrub and Vps4 and for the SNARE proteins Syb, Snap24, α-Snap, as well as in transient over-expression of the Tsp96F-HA. Central horizontal lines show median values of N=7-22 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note a slight but significant increase in the length of Col signal for all treatment compared to the wild type condition. B) Lateral confocal images and quantification of the extension of immuno-labeled Engrailed as the high threshold Hh target. Quantification shows number of cell diameters covered by signal from the posterior/anterior border marked by Hh-GFP (Bac construct); central horizontal lines show median values of N=6-10 box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note a significant reduction after RNAi expression, suggesting Hh reception impairment and flattening of the signal gradient. C) Lateral confocal images and quantification of Ptc transcription as a Hh target through immunolabeling of the reporter β−galactosidase (Ptc-LacZ). Middle graph shows the extension covered by signal normalized to wing pouch size, in wild type condition (N=6) or after ectopic expression of the Tsp96F-HA construct (N=9). Green shaded areas show data variability and dotted line gives the average values. Note the greater distance covered after Tsp96F-HA transient expression. Boxplot (right) shows distance quantifications with central horizontal lines showing median values of N=6-9 , box showing lower and upper quartiles and whiskers showing the maximum and minimum excluding outliers. Note a significant extension under Tsp96F-HA expression, suggesting again flattening of the signal gradient.
Paused genes are more accessible than the TATA genes.Figure 4A) Average read-count normalized MNase signal (RPM) from 2-4 h and 14-17 h embryos are shown at the different effector gene promoter groups. The grey area highlights the changes in nucleosome occupancy, which are prominent at highly paused genes (arrow).  B) Chromatin accessibility is shown as the average read-count normalized ATAC-seq signal (RPM) from 14-17 h for each promoter group. Paused genes show higher accessibility at the promoter region than the TATA genes.  C, D) ATAC-seq chromatin accessibility was measured in different tissues isolated from 14-17 h embryos using INTACT. (C) Read-count normalized ATAC (RPM) signal at individual genes from the TATA group (Ccp84Aa) and highly paused gene group (ect) are shown. (D) Read-count normalized ATAC signals (RPM) from different tissues were calculated for each gene from 150 bp upstream of the TSS to the TSS. Paused genes show higher average accessibility across all tissues compared to the TATA genes (Wilcoxon two-sided test, *P <2.2*10-16).
Atg36 is required for pexophagy.Figure 1Atg36 is required for pexophagy. Validation of Pex11-GFP as a marker for pexophagy in WT, atg1, and atg36 cells by (A) microscopy and (B) western blot including pex14Δ cells. The fluorescence assay shows that like HcRed-PTS1, Pex11-GFP accumulates in the vacuole in starvation medium (A), which corresponds to the accumulation of GFP on a western blot (B). (B) Cells were grown 18 h in oleate medium and switched to glucose medium lacking nitrogen (SD‐N) for the times indicated. GFP* indicates the relatively protease‐resistant degradation product indicative of vacuolar breakdown. (C) Pexophagy assay by fluorescence of GFP-PTS1 expressed from the CTA1 promoter in WT, atg36Δ, atg1Δ, and pep4‐3, prb1‐1122 strains. Cells were grown as above for Pex11-GFP pexophagy assay and imaged after 22 h in SD‐N medium. Multiple fluorescent images were acquired in Z‐axis and flattened into a single image. Bright‐field image is a single plane. (D) FM4‐64 labelling of cells grown as in (C). The three central images of a Z‐stack were flattened into a single plane. Bright‐field image is a single plane. Bar, 5 μm. (E) Pexophagy as assayed by Pex11-GFP western blot of cells grown for up to 3 days in glucose, oleate or glycerol medium. Samples of WT cells (left panel) and atg36Δ cells (right panel) were taken from the cultures after 24, 48 and 72 h.
A type I IFN response is unleashed in ADAR1 knockout cells upon expression of LGP2.Figure 2A) HEK293 cells were genetically engineered to knockout ADAR1 using CRISPR/Cas9. Cells were treated for 24h with recombinant type I IFN to upregulate ADAR1 p150 and ISG60 to confirm correct gene editing and type I IFN responsiveness, respectively. Protein lysates were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=3). B) ADAR1-knockout HEK293 cells (clone 1) were co-transfected with an empty vector (EV) or a FLAG-LGP2-encoding vector (LGP2) combined with a vector encoding GFP-tagged ADAR1 p110 or p150. Cells were harvested 72h post-transfection and the type I IFN response was monitored by RT-qPCR analysis of IFN-β and IFIT1 expression, normalized to ACTB. Data are means ± s.d. from a representative of four biological replicate experiments. C) ADAR1-knockout cells (clone 1) were transfected as in (B). Protein lysates were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=4).
Figure 6(A-B) Representative phase contrast, red fluorescence (indicating geminin-mCherry levels), and yellow fluorescence (indicating p53-Venus levels) images of cells in response to a low (A) or high (B) frequency Nutlin-3 dosing regimen over 40 h. (C) Geminin-mCherry traces in single cells treated with the low, natural, or high frequency Nutlin-3 dosing regimens. N = at least 50 cells per condition. Percentage of cells undergoing cell division (D) within the 40 h imaging in response to the low, natural, and high frequency Nutlin-3 dosing regimens Percentage of cells positive for geminin-mCherry expression (E) within the 40 h imaging in response to the low, natural, and high frequency Nutlin-3 dosing regimens
Figure 2(A) Scheme of parallel analysis of codon effects (PACE). Codon tag reporter mRNAs were pooled as a library and coinjected into fertilized zebrafish eggs. Embryos were collected at 2 and 6 hpf and subjected to RNA-Seq analysis. Reads mapped on each codon tag region were quantified to calculate each codon's effect on mRNA stability. (B) Results of PACE in zebrafish embryos. Black circles show the relative stability of reporter mRNAs under normal translation (averages of three biological replicates). The stability of a codon-tag reporter with a UGA stop codon was set to one. Gray circles show the relative stability of reporter mRNAs under the condition in which translation initiation was inhibited by GFP MO (averages of two biological replicates). Error bars represent max and minimum data points. The relative effect of each codon on mRNA stability is shown as a color gradient from red (destabilizing) to blue (stabilizing). CSCs in Bazzini et al. 2016 are also indicated below as circles with the same color gradient. (C) PACE results shown in (B) are presented as a codon table. (D) Box plot of tRNA amounts for nonoptimal (red, 20 codons), neutral (light gray, 18 codons), and optimal (blue, 20 codons) codons based on CSCs Data information: the box represents the interquartile range (IQR), with the median indicated as a black horizontal line in the box. The whiskers represent the variation within 1.5 IQR outside the upper and lower quartiles. Outliers are shown as dots. (E) Box plot of tRNA levels for codons with strong (red, 20 codons), neutral (light gray, 18 codons), and weak (blue, 20 codons) destabilizing effects based on PACE. Data information: the box represents the interquartile range (IQR), with the median indicated as a black horizontal line in the box. The whiskers represent the variation within 1.5 IQR outside the upper and lower quartiles. Outliers are shown as dots.
miR-132 controls the balance between IL-10 and IFNγ production in CD4+ T cells.Figure 4A. Percentage of IFNγ+ live TCRβ+ CD4+ cells from L. donovani infected WT (blue) or miR-132-/- (red) mice, determined by intracellular cytokine staining. Data representative of 3 independent experiments with 3-5 mice per group. Data information: statistical significance was determined by unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001. NS: not significant. B. Percentage of IFNγ+/IL-10+ live TCRβ+ CD4+ cells from L. donovani infected WT (blue) or miR-132-/- (red) mice, determined by intracellular cytokine staining. Data representative of 3 independent experiments with 3-5 mice per group. Data information: statistical significance was determined by unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001. NS: not significant. C. IL-10 mRNA levels, determined by RNA-sequencing, in TCRβ+ CD4+ cells purified from spleens of L. donovani infected WT (blue) or miR-132-/- (red) mice (n=5 per group). D. Percentage of IFNγ+ WT (blue) or miR-132-/- (red) in vitro polarised Th1 cells (6 days) in the presence or absence of phenylephrine (PE), determined by intracellular cytokine staining. E. Percentage of IL10+ WT (blue) or miR-132-/- (red) in vitro polarised Th1 cells (6 days) in the presence or absence of phenylephrine (PE), determined by intracellular cytokine staining. F. Total cell counts following in vitro Th1 polarisation (6 days) in the presence or absence of phenylephrine (PE). For (D-E), cells were purified from 3 mice per group and 6 replicates performed. Data information: statistical significance was determined with 1-way ANOVA followed by Bonferroni's multiple comparison test. * p<0.05, ** p<0.01, *** p<0.001. NS: not significant.
LRRC8D but not LRRC8A correlates with shorter survival of high grade serous ovarian cancer patients treated with platinum‐based drugs A-D Differential survival based on LRRC8A (A, C) or LRRC8D (B, D) gene expression as extracted from the TCGA database (http://cancergenome.Figure 2Low expression of LRRC8D but not LRRC8A correlates with shorter survival of high grade serous ovarian cancer patients treated with platinum‐based drugsA-DDifferential survival based on LRRC8A (A, C) or LRRC8D (B, D) gene expression as extracted from the TCGA database (http://cancergenome.nih.gov/) (A, B) or using the data from Patch et al (2015) (C, D). As cutoff the lower tertile of LRRC8A or LRRC8D gene expression was used. P‐values were determined using the log‐rank test.
Figure 5A Promoter occupancy by MYC correlates with transcript levels in cells untreated with doxycycline (black line). In doxycycline treated cells promoter occupancy by MYC is lower, and no longer increases together with transcript levels. The scatter plot displays MYCChIP-seq reads at promoters (-1000, +100 regions with respect to TSS) in BT168FO cells - untreated (black) or upon 48 h Dox treatment (blue) - versus transcript levels (FPKM from RNA-seq data). B Distribution of transcript level differences (Δ FPKM) of single genes classified as promoter-targets (green), intragenic-targets (blue), intergenic-targets (red) in Dox treated (24 h) versus untreated cells, plotted against expression in untreated cells (n=3). Δ FPKM = FPKM_24h Dox - FPKM_no Dox. C The table first row shows the number of genes significantly up or down regulated at 24 h of Dox treatment, subdivided in promoter-, intragenic-, intergenic-targets (± 50 kbp from TSS), and the rest. The following rows depict - for each gene class - mean (average) values of FPKM in cells treated or not with Dox for 24 h and the corresponding Δ FPKM and Fold change (Fc). Data are from 3 independent biological replicates. D Transcript level distribution of MYC promoter-targets significantly down (red dots) or up (green dots) modulated in cells treated with Dox for 24 h vs. untreated cells. Transcript levels, expressed as FPKM, represent averages of three independent experiments.
Figure 4A Lipidomics-based quantification of indicated lipid species in mice that received tail-vein injection of adenovirus expressing PLTP or GFP (control). Blood and tissues were harvested after 10 weeks of HFD. n=4 for GFP and n=5 for PLTP in mice on HFD. LPC = (lyso) phosphatidylcholine, LPE = (lyso) phosphatidylethalonamine, PA = phosphatidic acid, SM = sphingomyelin, TAG = triglycerides, FFA = free fatty acid. Statistical analysis is demonstrated (P value) relative to GFP by Student's t-test. B Plasma total cholesterol levels in mice that received adenovirus GFP (control) or PLTP. Blood and tissues were harvested seven days after adenovirus infection. n=8 in mice on RD and n=7 in mice on HFD for 14 weeks. ***P<0.001 relative to GFP by Student's t-test. C Relative mRNA levels of indicated genes in the liver of mice on HFD. Tbp was used as an internal control. n=4 for GFP and n=6 for PLTP. *P<0.05, **P<0.01 relative to GFP by Student's t-test. D Bile acid levels in the liver of mice in (B). n=8 in mice on RD and n=6 in mice on HFD. *P<0.05, **P<0.01 relative to GFP by Student's t-test. E Bile acid levels in the plasma of mice in (B). n=8 in mice on RD and n=7 in mice on HFD. ***P<0.001 relative to GFP by Student's t-test. F Bile acid levels in the feces of mice in (B). Feces were collected from mice on RD and HFD after seven days of treatment. n=7 in mice on RD and HFD. **P<0.01, ***P<0.001 relative to GFP by Student's t-test. G Plasma bile acids composition of mice in (B). Pool of samples from mice on HFD that received GFP or PLTP. n=5 for both groups. H Relative expression of Ucp1 in differentiated brown adipocytes treated with cholic acid (CA). Cells were treated with CA at indicated concentrations for 24 h. 0.1% ETOH was used as vehicle control (Ctr). n=3. Ucp1 mRNA expression was normalized by Tbp as an internal control. **P<0.01 relative to Ctr by one-way ANOVA followed by Tukey's test. N.S., not significant.
Fig.Figure 7Both thermopriming/heat acclimation and SA187-induced thermotolerance are mediated by HSFA2 dependent H3K4 trimethylation of chromatin at HS memory gene loci. However, thermopriming is conveyed to HSFA2 via the family of HSFA1A, B, D and E transcription factors whereas SA187 regulates HSFA2 by ethylene signaling via the transcription factor EIN3.
Fig.Figure 6 A-B. Comparison between average enrichment values for Upf1-decapping and Upf1-23 components, Lsm proteins and CK subunits in Upf1 -TAP purified samples in the presence (grey bars) or absence (blue bars) of UPF2 (AError bars represent SD. White dots represent enrichment values for individual replicates, 6 for Upf1-TAP, 3 for the upf2Δ an conditions  A-B. Comparison between average enrichment values for Upf1-decapping and Upf1-23 components, Lsm proteins and CK subunits in Upf1-TAP purified samples in the presence (grey bars) or absence (blue bars) o UPF3 (B). Error bars represent SD. White dots represent enrichment values for individual replicates, 6 for Upf1-TAP, 3 for th upf3Δ conditions  C-D. Evaluation of the effects of UPF2 deletion on Upf3-TAP levels (C in total extracts, in comparison with a loading control (G6PDH) was done by immunoblot  C-D. Evaluation of the effect of UPF3 deletion on Upf2-TAP levels (D) in total extracts, in comparison with a loading control (G6PDH) was done by immunoblot  E. Comparison between average enrichment values for Upf1-decapping and Upf1-23 components, Lsm proteins and CK subunits in Upf2-TAP purified samples in the presence (grey bars, 6 experiments) or absence (blue bars, 2 experiments) of UPF1. Error bars represent SD. The levels of expression of Upf2-TAP and Upf3-TAP proteins have been verified in this condition (Appendix Fig. S2)