EMBO/sd-character-level-ner huggingface dataset:
Figure 3A Structure and N-terminal residues for potential acylation of ISP1 and ISP3. B Fluorescent microscopy of ISP and its N-terminal 20 residues (N20) fused with a BFP::3V5 peptide episomally expressed in zygotes. Scale bar = 5 μm. C Solubility assay detecting membrane association of ISP1_FL and ISP1_N20 using different detergents. Cytosolic soluble proteins are in hypotonic buffer (Hypo), peripheral membrane proteins in carbonate buffer (Carb)，integral membrane proteins in Triton X-100 buffer (Trx), and insoluble proteins in pellet (P). BFP::3V5 is in the soluble fraction, while ISP1_FL and ISP1_N20 are in the membrane-associated fractions. ER protein BiP is a loading control. D Acyl-RAC method detecting palmitoylation of ISP1 and ISP3 in zygotes of the DTS strain. Proteins from F (flow-through), W (wash), and E (elution) are analyzed. P28 as a loading control. E Substitutions of N-terminal cysteine or glycine residues to alanine (G2A or C7A/C8A) ablates palmitoylation of ISP1. ISP1 was fused with BFP::3V5 and episomally expressed in zygotes. Right panel shows quantification of palmitoylated protein band from two independent repeats, values are means ± SD. F G2A and C7AC8A mutations cause cytoplasmic distribution of ISP1 during zygote to ookinete differentiation. Scale bar = 5 μm. G Membrane association of ISP1 with either G2A or C7AC8A substitutions. H Expression and palmitoylation of ISP1 in ∆isp1/3 parasites complemented with the 3V5 tagged WT ISP1 or C7AC8A mutant. I IFA of ISP1 at zygote of the complemented parasites. Scale bar = 5 μm. In vitro differentiation to mature ookinete of the complemented parasites. Values are means ± SEM (n=4 replicates), two-tailed t test, ***, P<0.001, ns: not significant. K A model illustrating palmitoylation critical for apical localization of ISP1 and ISP3 at zygote.
Figure 2Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (A) Comparison of differentially expressed brain mRNAs from Tmem106b-/- and wild type mice. From 680 detected genes 21 are significantly upregulated, while 10 are significantly reduced. Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (B) Comparison of Grn-/- and wild type mice. From 680 detected genes 12 are significantly upregulated, while 6 are significantly reduced. Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (C) Volcano plot for Grn-/-/Tmem106b-/- and wild type mice. From 680 detected genes 79 are significantly upregulated, while 24 are significantly reduced. D. Display of differently expressed genes overlapping between the analyzed genotypes from (A-C). Overlap was considered if a gene was significantly changed in the respective mouse models with more than 20% change in at least one mouse model. Note that 9 genes are significantly altered in all 3 genotypes in comparison to the wild type. 7 genes are exclusively overlapping between the Grn-/- and the Grn-/-/Tmem106b-/- mice. Tmem106b-/- and Grn-/-/Tmem106b-/- mice share 15 significantly altered genes. Tmem106b-/- and Grn-/- mice share one significant altered gene. Data represent the mean ± SD. E. Gene set analysis based on NanoString advanced analysis R-script included in the Neuropathology panel.
Figure 7A Sensitivity of scc2-4 and scc2-4 irc5-Δ1 mutants to DNA damaging agents at permissive temperature 25°C. Logarithmically growing cultures of wild type (W303-1a), irc5-Δ1 (IL012), scc2-4 (TB069) and irc5-Δ1 scc2-4 (TB070) strains were 10-fold serially diluted and plated onto solid YPD containing indicated concentrations of DNA damaging agents B, C Levels of DNA repair foci in scc2-4 and scc2-4 irc5-Δ1 mutants. Logarithmically growing cultures of scc2-4 (TB071 or TB073) and scc2-4 irc5-Δ1 (TB072 or TB074) were treated with MMS for 1 hour at 25°C. Next, cells were washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=3) D Sensitivity of scc1-73 and scc1-73 irc5-Δ1 mutants to DNA damaging agents at permissive temperature 25°C or semi-permissive temperature 30°C. Logarithmically growing cultures of wild type (W303-1a), irc5-Δ1 (IL012), scc1-73 (JC1339) and irc5-Δ1 scc1-73 (TB075) strains were 10-fold serially diluted and plated onto solid YPD containing indicated concentrations of DNA damaging agents E, F Levels of Rfa1-YFP and Rad52-YFP foci in scc1-73 and irc5-Δ1 scc1-73 mutants. Logarithmically growing cultures of scc1-73 (TB076 or TB078) and irc5-Δ1 scc1-73 (TB077 or TB079) were treated with MMS for 1 hour at 25°C. Next, cells were washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=3) G Replication completion defect of scc1-73 is not exacerbated by IRC5 disruption. Intact chromosomes were isolated from scc1-73 (JC1339) and irc5-Δ1 scc1-73 cells (TB075), cultured at 25°C, and processed for PFGE. M, 0.03% MMS  H Quantification of data presented in G. Error bars represent mean value ± standard deviations of mean for the intensity values of two distinct chromosome bands representing chromosome XII or III (n=2). Values for G1 were set to 100
Survival of yw flies, PGRP-LE112 flies, flies treated with Atg5-directed RNAi (hml-Gal4>Atg5IR) and control flies carrying hml-Gal4 (hml-Gal4>GFP), after injection of wild-type L.figf3Survival of yw flies, PGRP-LE112 flies, flies treated with Atg5-directed RNAi (hml-Gal4>Atg5IR) and control flies carrying hml-Gal4 (hml-Gal4>GFP), after injection of wild-type L. monocytogenes (a), Δhly L. monocytogenes (b) or E. carotovora (c) into adult flies. *, P 0.01 (Wilcoxon-Mann-Whitney test): P = 0.0024, Atg5-directed RNAi versus control (a), P = 0.0007, PGRP-LE112 versus control (a); P = 0.0055, PGRP-LC7454 versus yw (c). Data represent the average of four independent experiments.
UV-induced decrease in acetylated histones.Figure 2(A) Identified acetylated histone peptides plotted against their log2 SILAC ratio (1h after 16 J/m2 UV/mock), ranked by SILAC ratio (B) Table listing the number of identified peptides per histone using MS following enrichment of acetylated peptides, and the number of peptides identified that are decreased (UV/mock log2<-0.5) or increased (UV/mock log2>0.5) one hour after UV irradiation (16 J/m2) (C) Western blot of acid extracted histones and WCE from HeLa cells, treated with HATi (CTK7A, 100 μM and CPTH2, 50 μM) or HDACi (TSA, 1 μM) for 4h or mock treated as indicated (D) A representative western blot of histone acetylation levels of HeLa cells lysed at indicated time points after UV-irradiation (16 J/m2). Blots were stained with α-acetyl-lysine (top panels, high and low exposure), α-histone H4 (middle panel) and α-tubulin (bottom panel) (E) Quantification of histone acetylation levels, normalized against either the histone H4 levels (red bars) or tubulin levels (blue bars). Error bars represent SEM. N=5 independent experiments. Significant differences between UV treated and mock treated conditions were calculated using a T-test and are indicated with * (p<0.1), ** (p<0.05) and *** (p<0.01) (F) Representative western blots of HeLa WCE from different time points after UV-C irradiation (16J/m2) using the indicated histone modification specific antibodies. Histone H4 and tubulin were used as loading controls (G) Quantification of specific histone acetylation marks normalized to tubulin levels. Significant differences were calculated using a T-test and are indicated with ** (p<0.05) and *** (p<0.01). N=3, error bars represent SEM
mediated degradation of SAMHD1.Figure 8Host restriction factor SAMHD1 but not phosphorylated SAMHD1 restricts EV71 replication. However, EV71 infection overcome the inhibitory of SAMHD1 by upregulating TRIM21, an E3 ubiquitin ligase, which induces the degradation of SAMHD1 via proteasome pathway. Moreover, the degradation of SAMHD1 induced by TRIM21 also affect its functions on HIV restriction and IFNs production.
AGO4 and AGO6 exhibit different co‐localization patterns with Pol VThe largest subunit of Pol V, NRPE1 (red), was visualized by immunofluorescence using its specific antibody in cells expressing FLAG‐tagged AGO4 (green).Figure 5A The largest subunit of Pol V, NRPE1 (red), was visualized by immunofluorescence microscopy using its specific antibody in cells expressing FLAG‐tagged AGO4 (green). AGO4‐NRPE1 co‐localized to nucleolar dot (yellow dot, marked with white arrow, upper panel) but not in nucleoplasmic foci (lower panel). B NRPE1 (red) was visualized by its specific antibody in cells expressing FLAG‐tagged AGO6 (green). The yellow signals due to the overlap of red and green channels in merged images indicate protein co‐localization. DNA (blue) was stained with DAPI. AGO6‐NRPE1 co‐localized to nucleoplasmic foci (yellow dots, marked with white arrow).
Figure 1Western blot analysis of BRD7 and γH2AX protein levels in breast cancer cell lines: MCF7 parental or doxorubicin-resistant MCF7/ADR (ADR, 0.5μg/ml) and irradiation-resistant MCF7/IR (IR, 10Gy), MDA-MB-231 parental or doxorubicin-resistant MDA-MB-231/ADR (ADR, 0.5μg/ml) or cisplatin-resistant MDA-MB-231/DDP (1μg/ml), non-small lung cancer cell lines: A549 parental or cisplatin-resistant A549/DDP (1μg/ml). MCF7, MCF7/ADR and MCF7/IR, MDA-MB-231 and MDA-MB-231/ADR cells were harvested and mRNA levels of BRD7 were determined by real-time PCR (n=3). Values are mean ± SEM and Error bars indicate SEM. Western blot analysis of BRD7 protein levels in MDA-MB-468 and MDA-MB-231 cell after treatment with ADR (5 μM) or camptothecin (CPT) (1 μM) for different intervals (n=3). Representative images of endogenous BRD7 (green) and γH2AX foci (red) in paraformaldehyde-fixed MDA-MB-468 cells after treatment with CPT (1 μM) for different intervals. Visualized by immunofluorescence using anti-BRD7 and Alexa Fluor 555 anti-γH2AX antibodies. DNA staining with DAPI; Scale bars, 2 μm. Quantification of average fluorescence intensity of BRD7 of cells in (E). Error bars indicate SEM; n>100.
Figure 3Figure 3. Helix-7 minimizes dwell time on off-targets. A) Cartoon schematic of our single-molecule FRET assay. B) Guide:off-target pairs examined listed with increasing values of N (the length of the matched region between miRNA and target RNA). N = 7 is the fully seed paired target. C) Representative fluorescence time traces (with a resolution of 100 ms for wt, 300 ms for MI-AA and Ηhelix-7) show docking and dissociation of wild type (top), MI-AA (middle), and Ηhelix-7 (bottom) Ago2-miRNA at single spots in the microfluidic chamber. D) Dwell time histograms obtained for N = 4, 5 and 6, with wild type Ago2; histograms were fit with single exponential decay. The first column of the data was not included in the analysis to avoid potential artifacts arising from the limit of the time resolution. The bin size is 0.5 sec for N =4 and 1 sec for N =5 and 6. E) Bar plot showing the dependence of dwell time on N for wild type Ago2. The dashed red line indicates the observation time limit (300 s), which is constrained by photobleaching.For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days. F) Dwell time histogram for different N's with the MI-AA helix-7 mutant. The dwell time histogram for N = 6 fit with double exponential decay (green; R2 = 0.98); The dwell time for N = 6 with single exponential fit was 15.2 ± 3.41 sec, R2 = 0.96). G) Bar plot for dwell times of MI-AA Ago2 as a function of N. For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days. H) Dwell time histogram for different N's with the ∆helix-7 mutant. The dwell time histograms for N = 5 and N = 6 were fit with double exponential decays (green; R2 = 0.97 for N = 5 and R2 = 0.96 for N = 6); The dwell time for N = 5 and N = 6 with single exponential fit was 9.1 ± 0.91 sec (R2 = 0.86) and 22.3 ± 5.08 sec (R2 = 0.90) respectively). I) Bar plot for dwell times of ∆helix-7 Ago2 as a function of N. For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days.
Figure 1A) ESCs were grown in either self-renewal conditions (serum plus LIF) or differentiation conditions (knock-out serum replacement, KSR, plus Retinoic Acid, RA). Extracts were subjected to immunoprecipitation with an antibody against either ZRF1 or ID1. Levels of co-immunoprecipitation of ID1 with ZRF1 (left), or of ZRF1 with ID1 (right), were assessed by Western blot. B) ZRF1 ChIPs were performed in ESCs transfected with an empty vector (Ctrl) or in Id1-Flag over-expressing ESCs. Zrf1-depleted ESCs were used as a control. Values are reported as percentage of input. Standard deviation (SD) is representative of three independent experiments. *P < 0.05 and ** P < 0.01 (paired t-test) represent comparison with Ctrl ESCs at the corresponding time point. C) mRNA fold-change of Zrf1 targets under differentiation conditions (24hrs RA). SD is representative of three independent experiments. *P < 0.05; ** P < 0.01 (paired t-test).
Figure 2(A) HOIL-1 activity towards purified bovine liver glycogen was measured using Cy5-labelled ubiquitin. Control assays lacking ATP, UBE2L3, HOIL-1 and glycogen are indicated, as are subsequent treatments with 50 μg/mL human salivary α‑amylase and 1.5 M hydroxylamine (NH2OH). The gel has been stained using the periodic acid-Schiff (PAS) method to visualise glycogen (purple-magenta) and Coomassie Blue stain to visualise protein. (B) Prior to PAS and Coomassie staining, the gel shown in (A) was stained with Flamingo fluorescent protein stain (shown in red) and glycogen ubiquitylation observed by visualising the fluorescent Cy5-ubiquitin signal (shown in green). (C) Chemical structure of the α-1,4-linked heptasaccharide maltoheptaose. (D) In vitro ubiquitylation of maltoheptaose by HOIL-1. Assays were treated with α‑amylase or hydroxylamine as indicated. Reaction products were detected by Coomassie staining. (E) Ubiquitylated maltoheptaose was purified from reaction components and analysed by MALDI-TOF, revealing an additional 1135 Da attached to the ubiquitin. This is consistent with addition of maltoheptaose to ubiquitin via an oxyester linkage (expected additional mass is 1153 for maltoheptaose minus 18 to account for the loss of a water molecule during ester bond formation).
Loss of POMC neuronal atg7 impairs glucose tolerance.Figure 4(A) Blood glucose levels in 6 mo (n=7-9) and (B) 12 mo Con and KO mice on RD (n=6), and in (C) 12‐mo‐old Con and KO mice on HFD (n=4). (D) Serum insulin levels in 6 mo, (n=7-9) and (E) 12‐mo‐old RD‐fed Con and KO mice that were fed or 6 h fasted (n=6). (F) HOMA values from 6‐h‐fasted 6‐mo‐old (n=7-9) and (G) 12‐mo‐old Con and KO mice (n=6) on RD. (H) Glucose tolerance tests in 6‐h‐fasted 6‐mo‐old mice on RD (n=4) and in (I) 6‐h‐fasted 12‐mo‐old Con and KO mice fed HFD for 10 mo (n=4). (J) Insulin tolerance tests in RD‐fed 6‐mo‐old (n=4) and (K) HFD‐fed 12‐mo‐old Con and KO mice (n=4). (L) Insulin tolerance tests and (M) body weights of 1‐mo‐old Con and KO mice on RD (n=3-5). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, **P0.01, ***P0.001. Con, control; HDF, high‐fat diet; HOMA, homeostasis model of insulin resistance; KO, knockout; mo, month; POMC, proopiomelanocortin; RD, regular chow; Stv, fasted.
Figure 6A-B. Involvement of filamentous cytoskeletal structures in BiFC foci polarization. Time-lapse micrographs of agarose-embedded cells expressing the Dsl1pVN•ε-COPVC BiFC combination at RT with fresh PM Glc+ura medium supply. (A) Upon disruption of actin filaments with 1,25 μM latrunculin A, COPI•Dsl BiFC signals dispersed. Arrowheads: polarized signal, asterisks: unpolarized localization. Fluorescent images are displayed with inverted brightness values. Scale bar 10 μm. A-B. Involvement of filamentous cytoskeletal structures in BiFC foci polarization. Time-lapse micrographs of agarose-embedded cells expressing the Dsl1pVN•ε-COPVC BiFC combination at RT with fresh PM Glc+ura medium supply. (B) Microtubuli disruption through 24 μg/ml nocodazole did not affect polarization of the COPI•Dsl BiFC signals. Arrowheads: polarized signal, asterisks: unpolarized localization. Fluorescent images are displayed with inverted brightness values. Scale bar 10 μm. C-E. Involvement of myosin motors in BiFC foci polarization. Time-lapse micrographs of agarose-embedded cells carrying the COPI•Dsl BiFC pairs Dsl1pVN•ε-COPVC or β'COPVN•Dsl3pVC were taken. Either wild type cells (E) or mutants carrying the myosin V mutations myo2-66 (C) or myo4∆ (D) were analyzed. Cells were grown at RT with fresh PM Glc+ura medium supply. In the myo2-66 cells, the BiFC signal polarization was hardly detectable even at permissive temperature, while it was unaffected in myo4∆ compared to the control cells. F. Fluorescence micrographs of β'-COPVN•Dsl3pVCcells carrying the myo2-66 defect. COPI•Dsl BiFC, as well as ER or Golgi markers were analyzed. While the BiFC spots were dispersed in these mutants, they retained their association with ER and Golgi. Scale bar 5 μm.
Figure 5FRET labels are positioned at the N-terminus of the nascent chain (red star) and at the cytoplasmic face of the translocon (yellow star). NC, nascent chain.
Activation of p38 inhibits autophagy and mAtg9 trafficking.Figure 4(A) 293/GFP-LC3 cells were treated with 10 μM anisomycin for 30 min, or exposed to UV irradiation for 3 min followed by a 40‐min recovery and incubation in full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin for 2 h. Cells were then fixed and visualized by confocal microscopy. GFP-LC3‐positive structures per cell were quantified as in Figure 3A. Bars=5 μm. Data are represented as mean±s.e.m. n=60 cells, mock versus anisomycin EBSS (***P=0.0001); mock versus UV EBSS (***P=0.0001); mock versus anisomycin EBSS with leupeptin (***P=0.0001); mock versus EBSS with leupeptin UV (***P=0.0001). All analysed using Student's t-test. (B) HEK293A cells were pretreated with 10 μM anisomycin for 30 min, or UV irradiation for 3 min followed by a 40‐min recovery, before incubation in EBSS for 2 h. Images were analysed as in Figure 2E. Bars=5 μm. Data are represented as mean±s.e.m. of 60 cells, EBSS mock versus EBSS anisomycin (*P=0.0142); EBSS mock versus EBSS UV (*P=0.0150); Student's t‐test.
Figure 3(A-B) Volcano plots obtained by DESeq2 analysis showing the effect on the transcriptome of ALSC9orf72 MN exerted by Apamin and XE991 in the ALSC9orf72 II and ALSC9orf72 III lines. (C) PASTAA ranking of the top transcription factors activated by Apamin and XE991. Significance was set with the algorithm published in Roider et al. (2009). (D) Venn diagram and showing the shared 263 CREB-controlled genes significantly altered by treatment with K+ channel blockers; the bar plot represents the significantly altered genes involved in neuronal activity and autophagy obtained by DESeq2 analysis. (E) Apamin and XE991 increase the levels of pCREBS133 in all the ALSC9orf72 lines (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments with each hiPSC line. Scale bar: 10 µm. Data information: *p<0.05; **p<0.01. Error bars represent SEM. Exact p-values are reported in Appendix Table S1. (F) ALSC9orf72 MN showed an increased number of Homer1b/c+ synaptic contacts after treatment with the K+ channel blockers, which in contrast had no significant effect on Healthy MN. n=3 independent treatments with each hiPSC line (two-way ANOVA). Scale bar: 5 µm Data information: *p<0.05; **p<0.01. Error bars represent SEM. Exact p-values are reported in Appendix Table S1. (G) Venn diagram showing the number of proteins significantly altered upon Apamin and XE991 treatment in ALSC9orf72 MN. The lines ALSC9orf72 II and ALSC9orf72 III were used as representative of the ALS genotype. (H-I) Volcano plot displaying the up- and down-regulation of the proteins altered by Apamin and XE991 (t-test). The blue dots indicate proteins involved in synaptic structure and function. (J) Up-regulated GO (biological processes) terms in ALSC9orf72 MN treated with either K+ channel blockers (Fisher enrichment test). (K) Synapse- and neuronal processes-related proteins significantly up-regulated by both treatments.
Figure.Figure 7A. Examination of mRNA levels of genes involved in innate immunity in two constitutively active atfs-1 mutants (et15 and et17) revealed that innate immunity genes are upregulated when ATFS-1 is activated. This indicates that activation of the mitoUPR can cause activation of genes that are regulated by the p38-mediated innate immune pathway. Gene expression changes were determined by RNA sequencing of six biological replicates of each genotype. Results represent counts per million (CPM) expressed as a percentage of wild-type. Data information: Error bars indicate SEM. *p<0.05 **p<0.01, ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison test. B,C. Constitutive activation of ATFS-1 does not increase activation of PMK-1/p38 as measured by the ratio of phosphorylated-PMK-1/p38 to total PMK-1/p38 using Western blotting. Representative blots are shown in panel (B). There is also no significant difference in the levels of PMK-1/p38 or phosphorylated-PMK-1/p38 in atfs-1(gof) and atfs-1(lof) mutants compared to wild-type (C). atfs-1(gof) is the et17 allele. atfs-1(lof) is the gk3094 allele. Eight biological replicates per strain were quantified. Data information: Error bars indicate SEM. *p<0.05 **p<0.01, ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison test.
Figure 4A-C) Analysis of RNA Pol II binding (RPB-1/AMA-1 ChIP-seq) at upregulated genes in rpb-9 (mj261) mutants, as defined in Figure 3C. Class I: upregulated genes with increased RNA Pol II binding (A); Class II: upregulated genes with invariant RNA Pol II binding (B); Class III: upregulated genes with reduced RNA Pol II binding (C). n=3 biological replicates are shown per each genotype. (PolyA-selected RNA-seq libraries). Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers. D) RNA Pol II binding profile over the piRNA sensor locus. E) RNA Pol II binding profile over the Chapaev-2 and CEMUDR1 DNA transposons, scaled to 1kb.