EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A-C) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then with LLOMe (A and C) or starved in EBSS (B). (A-C) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then with LLOMe (A and C). (D) Secretory autophagy pathway. Upon lysosomal damage caused by inflammasome-agonists such as silica, alum, monosodium urate, and Leu-Leu-O-Me, inflammasome activation leads to caspase-1-dependent processing of cytosolic pro-IL1β into mature IL-1β. The autophagy regulatory systems (starvation/nutrition response systems: mTOR, LYNUS, TFEB) located at the lysosome transduce starvation signals (Napolitano Ballabio, 2016b). Upon lysosomal damage and stress, galectin-8 recognizes -galactosides on the exposed lumenal membrane and/or glycosylated lysosomal lumenal proteins released into the cytosol and activates the galectin-8-TRIM16 complex. TRIM16 binds secretory autophagy cargo (mIL-1β and potentially other cargo). TRIM16 forms complexes with Sec22b to transfer the cargo to the autophagy-induced LC3-II+ membrane 25k carriers (a direct interaction between Sec22b and TRIM16 remains to be determined). Depiction of mIL-1β between the two membranes or within the lumen of the double membrane-delimited autophagosomal intermediates reflects the sequestration processes that may be involved: IL-1β is sequestered either through the reported Hsp90-dependent import across the delimiting membrane into the lumen of carriers topologically corresponding to the intermembarne space in conventional autophagosomes (Zhang et al., 2015), or is sequestered into the inner lumen of conventional double membrane autophagosomal organelles, specializing in secretion, simultaneously observed in the same study (Zhang et al., 2015). Not depicted is the compatibility of TRIM16's receptor function with both of the above processes: i.e. either a hand-over of the cargo to Hsp90 or cargo sequestration via TRIM16 acting as a receptor/adaptor directly interacting with mAtg8s as previously reported (Mandell et al., 2014); the latter option is in keeping with the observations that TRIM16 is co-secreted with IL-1β (Munding et al., 2006). At this point, Sec22b molecules (acting as R-SNAREs) on the delimiting membrane facing the cytosol, carry out fusion at the plasma membrane in conjunction with the Qbc-SNAREs SNAP23 and SNAP29 and plasma membrane Qa-SNAREs syntaxins-3 and -4, thus delivering IL-1β to the extracellular milieu where it exerts its biological function. Data, means  SEM; n≥5. *P 0.05, **P 0.01, †P ≥ 0.05 (ANOVA or t-test).
Figure 6A series of major Rab family proteins were immunoprecipitated from HeLa cell extracts and then probed with an anti-BAG6 antibody. Co-precipitation of Rab8a (T22N) with BAG6 was used as a positive control for this experiment. Quantification of BAG6 co-precipitation with Rab family proteins. The graph shows the quantities of BAG6 protein in the respective anti-Flag precipitates that were normalized by the amount of Flag-Rab bait proteins. The value of BAG6 co-precipitation with Rab8a (T22N) was defined as 1.0. The quantities of the respective immunosignals were also normalized to the actin signal of each sample, and represent the median calculated from at least 3 independent biological replicates. The number of independent biological replicates was as follows: Rab1a, n = 4; Rab1b, n = 5; Rab2b, n = 3; Rab3a, n = 4; Rab4, n = 4; Rab5a, n = 3; Rab5b, n = 4; Rab6a, n = 5; Rab6b, n = 4; Rab7a, n = 3; Rab8a, n = 14; Rab9b, n = 4; Rab10, n = 6; Rab11a, n = 3; Rab11b, n = 3; Rab12, n = 2; Rab13, n = 6; Rab14, n = 3; Rab15, n = 5; Rab35, n = 3.
| DND1 is required for loading of the target RNAs to NANOS2-CNOT complex.Figure 5A-L, Sections of testes from Dnd1flox/flox (A, B, C, G, H, I, M, N, O) and Dnd1flox/flox_Tg(Oct4PE-CreERT2) (D, E, F, J, K, L, P, Q, R) embryos were prepared at E16.5 and then immunostained with antibodies against NANOS2 (A, D) (green), DCP1a (G, J) (green), DDX6 (M, P) (green) and TRA98 (B, E, H, K, N, Q). DNA was labeled with DAPI (blue). Tamoxifen was administered at E13.5. Scale bars: 50 m in A for A−F; 50 m in G for G-L; 50 m in M for M-R. Insets show an enlarged vision of each picture to better depict localization of NANOS2, DCP1a and DDX6. S-V, Immunoprecipitation with an anti-FLAG antibody from E15.5 male gonadal extracts of wild-type and the transgenic mouse line expressing FLAG-tagged DND1 (S, T), or with an anti-NANOS2 antibody from E15.5 male gonadal extracts of Dnd1flox/flox mice with or without Oct4PE-CreERT2 (U, V). Precipitates were analyzed by western blotting (S, U) or by RT-qPCR (T, V). The RT-qPCR data are shown as average relative mRNA levels ± SEs (n=3). W, X, Western blotting analyses of proteins in NANOS2-depleted (W) or DND1-depleted (X) testis extracts from E15.5 embryos. Proteins were analyzed with the indicated antibodies.
An S.Figure 1. An S. typhimurium strain devoid of its SPI-1 TTSS effector proteins is able to induce programmed cell death in macrophages. BMDPM from wild-type (A) or caspase-1−/− (B) mice (Kuida et al., 1995) were infected with wild-type S. typhimurium or mutant derivative either lacking all TTSS effector proteins (effectorless) or defective for TTSS secretion by virtue a having a mutation on an essential component of this system (invG). 6 h after infection, cytotoxicity was evaluated by lactate dehydrogenase release or by terminal deoxynucleatide transferase (TUNEL) staining. Values for the lactate dehydrogenase release represent the average of at least three independent measurements. Equivalent results were obtained in several repetitions of these experiments.
Figure 2A Western blot of lysed nematodes at the L4+48 hour stage, probed with antibodies recognizing, ubiquitin, GFP, RPN-11, multiple 20S proteasome subunits, or actin as a loading control. Animals express UbG76V-GFP protein in their hypodermis. The position of molecular weight markers is shown on the left of each blot. The position of UbG76V-GFP protein, as well as UbG76V-GFP with the indicated number of additional ubiquitin moieties, based on molecular weight, is indicated on the right of each blot. Twenty animals were loaded per lane for each indicated genotype (WT indicates wild type). B Western blot of lysed nematodes at the L4+48 hour stage, probed and annotated as in (A). Animals express UbG76V-GFP protein from the sur-5 promoter. Thirty animals were loaded per lane for each indicated genotype (WT indicates wild type). C Quantified fluorescence of UbG76V-GFP normalized to mRFP from lysates of transgenic nematodes at L4+48 hours that express these reporters in the hypodermis. Animals were exposed to bacteria containing the indicated RNAi clone or an empty RNAi vector as a control. *P<0.05, ANOVA with Dunnetts posthoc comparison to the empty RNAi vector control. N=3 trials. Error bars indicate SEM. D Quantified epoxomicin-sensitive proteasome activity (as measured through the turnover of fluorescent chymotrypsin substrate) from the same lysates and exposed to the same RNAi as in (C). *P<0.05, ANOVA with Dunnetts posthoc comparison to the empty RNAi vector control. N=3 trials. Error bars indicate SEM. E Quantified fluorescence of UbG76V-GFP normalized to mRFP from lysates of transgenic nematodes at L4+48 hours that express these reporters in the hypodermis. Animals were of the indicated genotype. ***P<0.001, **P<0.01, *P<0.05, ANOVA with Dunnetts posthoc comparison to wild type. N=4 trials. Error bars indicate SEM. F Quantified epoxomicin-sensitive proteasome activity (as measured through the turnover of fluorescent chymotrypsin substrate) from the same lysates as in (E). No statistical difference (P<0.05 cutoff) was detected by ANOVA. N=4 trials. G Percentage of live animals of the indicated genotype assayed after 6-hour exposure to heat shock. 100% of control animals of the same genotype but not given heat shock survived. *P<0.01, Student t-test. N=3 trials. Error bars indicate SEM. H Percent of animals of the indicated genotype containing aggregates of Poly(Q)44::YFP at the indicated time (in days) after L4 stage. *P<0.01, Student t-test. N=3 trials, 50-80 animals per trial per day. Error bars indicate SEM.
Figure 6A) Reporter cells expressing mFcγRIII, mFcγRIV or parental BW5147 cells were incubated with titrated amounts of synthetic sICs generated from rhTNF-α and mouse-anti-hTNF-α at a 1:1 ratio by mass. One experiment performed in technical duplicates. Error bars indicate reproducibility as range. B) Titrations of three mouse sera per group (C57BL/6, K/BxN or NZB/WF1) were incubated with mFcγR reporter cells and FcγR activation was assessed as described above. Sera from BL/6 mice served as negative control. Each symbol represents mean values of one mouse measured in technical duplicates. Error bars = Mean with SD.
Figure 7(A) A schematic representation of the heterogeneity within a neuronal population for HSV-1 reactivation following inhibition of the canonical AKT-mTORC1 signalling pathway. Dark blue neurons signifies cells undergoing "Successful" (productive) reactivation with Gadd45b localized in the cytoplasm (nuclear exclusion); light blue neurons shows cells remaining in the latent state with Gadd45b localized to both nuclear and cytoplasmic compartments; white-labelled cells with dark blue nuclear puncta signifies "Unsuccessful" (abortive) reactivation with Gadd45b localized to nuclear foci/puncta; and white-labelled neurons represent uninfected cells with low-level Gadd45b localized to both nuclear and cytoplasmic compartments. (B) Neurons that undergo "Unsuccessful" reactivation are conceived as those in which Gadd45b suppresses ICP4 mRNA transcription (and protein accumulation) and viral late gene expression. In contrast, neurons that can undergo "Successful" reactivation have relocalized or excluded Gadd45b protein from the nucleus to allow for ICP4 transcription and efficient viral late gene expression, and presumably culminating in synthesis of new viral progeny.
Overexpression of Rap1‐YFP in cay1∆ cellsRT–PCR analysis of pre‐mRNA splicing efficiencies of rap1+ and poz1+ pre‐mRNAs in cay1+ and cay1∆ cells expressing Cay1‐YFP or Rap1‐YFP.Figure 6RT-PCR analysis of pre‐mRNA splicing efficiencies of rap1+ and poz1+ pre‐mRNAs in cay1+ and cay1Δ cells expressing Cay1‐YFP or Rap1‐YFP. Amplification products corresponding to unspliced and spliced mRNAs are indicated by asterisks and gene names, respectively. rap1T indicates amplification products obtained with oligonucleotides spanning rap1 exon 3 and thereby amplifying both spliced and unspliced mRNA. Intronless act1+ was used as a loading control. Numbers at the bottom of each panel are ratios between spliced and unspliced forms (s/u) and spliced and act1+ (s/act1). Values are expressed as fold increase over wt. Western blot analysis of total proteins from early generation cay1+ and cay1Δ cells expressing Cay1‐YFP, Rap1‐YFP, and Taz1‐YFP using antibodies against Rap1, Taz1, and Act1 (loading control). Numbers at the bottom are ratios between Rap1 proteins (endogenous plus ectopic) and Act1 (Rap1/Act1). Values are expressed as fold increase over wt. Left: Representative YFPfluorescence microscopy and DIC images of cay1∆ strains expressing Cay1‐YFP. Scale bar, 10 μm. Right: Representative YFP and RFP fluorescence images of Cay1‐YFP‐ and Pot1‐RFP‐expressing cells. Arrows point to YFP and RFP foci within the same nucleus demonstrating lack of co‐localization. Scale bar, 5 μm. Representative YFPfluorescence microscopy and DIC images of cay1+ and cay1∆ strains expressing Rap1‐YFP. Scale bar, 5 μm.
sub-complex and the holoElongator complex.Figure 4A. The model of the Elp123 sub-complex. B. Crosslinks supporting the orientation of Elp3 (left) and Elp2 (right). The crosslinks are shown separately to highlight crosslinks linking from Elp3 and Elp2 to other subunits, respectively. C. The model of holoElongator. D. Crosslinks supporting the orientation of the Elp456 hexamer.
Figure 1(A) (B) CPT sensitivity assayed by 10-fold serial dilutions of different mutant combinations on YPAD plates. Four biological replicates have been performed. (C) Colony-forming unit assays to increasing CPT concentrations of the same mutant combinations as in (B). Mean values ± SD are plotted. Four biological replicates have been performed. (D) UV sensitivity assayed by 10-fold serial dilutions of different mutant combinations with rad3-102 allele on YPAD plates after exposure to the indicated UV-C doses. Five biological replicates have been performed. (E) Schematic representation of the BIR assay. Two fragments of the LYS2 gene sharing 2.1 kb of homology (lys and ys2) were integrated on chromosomes V and XI, respectively. Induction of HO endonuclease expression under control of the GAL1 promoter produces a DSB next to the lys fragment that can be repaired by BIR. BIR events can be scored by selecting survivor colonies, which harbor a functional LYS2 gene. (F) BIR frequencies (Lys+ survivors among total cells) for the WT and indicated mutant strains are plotted on a logarithmic scale. Data represent the mean ± SD from 3 to 6 independent experiments. ns, non-significant difference, * P-value = 0.0134, Mann-Whitney unpaired t test.
Figure 4(A) Schematic representation of the genomic locus of the chromosome 1 of V. cholerae with the three parS sites, named parS1-3. The rRNA operon (blue rectangle) spans the genomic coordinates 53823 to 59123 (B) ChIP-seq performed on strain N16961 is displayed as the number of nucleotide reads in function of the genomic coordinates. Correspondence to the parS1-3 location represented in (A) is indicated by grey dotted lines. The number of reads in the Input sample (grey line) is normalized to the total number of reads in the IP sample. (C) We modeled the ChIP-Seq data as in Fig.1C-E by means of MC simulations with a Freely jointed chain of size N=2000 monomers of size a=16-bp. Data are normalized after background subtraction to the read value at parS1 (genomic coordinate 62438). The best fit was achieved with σ=25nm and an amplitude κ=0.15 leading to Nt~50 ParB on the chromosome. In the MC simulation, we accounted for the finite width of the distribution around parS sites by including the average fragment size of the DNA librar
Figure 3A. HuH7 cells were mock treated, treated with 10,000 U/ml of IFNα for three days, infected with HCV for six days or infected with HCV for six days and treated with IFN during the last three days of infection. B. Control cells or cells collected 8h post-transfection with pIC. Then, the levels of CXCL10 mRNA and the indicated CSRs were quantified by qRT-PCR. GAPDH mRNA was also measured and used as a reference. Fold increase of each CSR in treated versus control cells is shown. Fold changes higher than 2 are indicated at the top of each bar. The experiment was performed at least twice in triplicate and each value shows the average (n=6). Error bars indicate standard deviations and asterisks mark significant differences (ns, non-significant; * denotes p<0.05, ** p<0.01) obtained with a two-tailed non-parametric Mann-Whitney U-test.
Figure 6Miniature inhibitory postsynaptic currents (mIPSCs) measured in CA1 pyramidal neurons in different mouse models at P14-P16. (A) example mIPSCs of WT (left) and Clcn3-/- neurons. Mean values ± s.e.m. and cumulative histogram of amplitude (top) and frequency/interevent-interval (bottom) of mIPSCs in different mouse-models as indicated . Several cells of 3 mice of each genotype were analyzed. Compared genotypes were siblings. No difference in amplitude or frequency/ interevent-interval distribution of mIPSCs was found in (B) Clcn3-/- and Clcn3+/+ (Clcn3+/+: 16 cells, Clcn3-/-: 15 cells; mean amplitude: p > 0.8, t test; mean frequency: p > 0.4, MW-test), in (C) Clcn3unc/unc and Clcn3+/+ (Clcn3+/+: 21 cells, Clcn3unc/unc: 13 cells; mean amplitude: p > 0.7; mean frequency: p = 0.3, t test), and in (D) Clcn4-/- and Clcn3+/+ (Clcn3+/+: 18 cells, Clcn4-/-: 18 cells; mean amplitude: p > 0.3, t-test; mean frequency: p = 0.6, MW-test). (E) No difference in mean amplitude (p > 0.5, t test), in cumulative amplitude and mean frequency of mIPSCs (p > 0.1, t test) was found in Clcn3unc/unc/Clcn4-/- and Clcn3+/+/Clcn4-/-. The interevent-interval distribution was shifted to the right, indicating a reduced frequency of mIPSCs in Clcn3unc/unc/Clcn4-/- (KS-test, p < 0,05) (Clcn3+/+/Clcn4-/-: 16 cells, Clcn3unc/unc/Clcn4-/-: 12 cells). (F,G) miniature excitatory postsynaptic currents (mEPSCs). (F) example traces of mEPSCs in WT and Clcn3-/- neurons (G) No significant difference between Clcn3unc/unc/Clcn4-/- and Clcn3+/+/Clcn4-/- mEPSCs in mean amplitudes (p > 0.9, t test) or in the distribution of amplitudes (Clcn4-/-: 12 cells, 2 mice; Clcn3unc/unc/Clcn4-/-: 8 cells, 2 mice). (H) Primary hippocampal neurons co-transfected with ClC-3 and synaptobrevin (syb2), both luminally tagged with pHluorin or the pH-sensitive mOrange Ramirez et al, 2012(), respectively, were stimulated by 200 action potentials (APs) at 20 Hz (indicated by bar). Vesicle exocytosis was monitored by fluorescence increase upon exposure to extracellular neutral pH. Note that only Syb2 and not ClC-3 shows signs of exocytosis. Traces were corrected for photobleaching. The initial decay of the pHluorin signal is probably due to the high light exposure, which forces the fluorophores into a transient dark-state Dean et al, 2011(). Averaged fluorescence traces (n=10), error bars, s.e.m.
Figure 7We hypothesize that the ncBAF complex is localized to the promoters of regulated genes by the interaction of the BRD9 bromodomain with histone acetylation marks. ncBAF activity may maintain the chromatin in an open state, thus enabling the efficient binding of ISGF3 complexes to ISRE sequences following IFN stimulation. Thus, maximal induction of ISG transcription may be 'primed' to enhance downstream antiviral activity. In the absence of BRD9 (through genetic loss or targeted degradation with small molecules), ncBAF is removed from promoter regions, which may increase chromatin compaction and result in less efficient transcriptional activation of ISGs following IFN stimulation.
Impact of the BH3‐like domain on Beclin‐1‐induced autophagy.Figure 5(A-D) Modulation of Beclin‐1‐induced autophagy by BH3 mutants and ABT737. Cells were transfected with GFP‐LC3 together with an empty control vector or Beclin‐1 (WT, L116A, F123A) and cultured for 48 h, followed by overnight culture in CM (B) or NF (A, B) in the absence or presence of 1 μM ABT737. Representative microphotographs are shown in (A) and the LC3‐GFP‐positive vacuoles per cell are quantified in (B) (means±s.d.; n=3 separate experiments). Alternatively, cells were not transfected and stained with CMFDA to detect vacuoles, as shown in (C) and (D), yielding similar results as for the LC3‐GFP method. (E) BH3‐dependent modulation of Beclin‐1‐induced autophagy by anti‐apoptotic Bcl‐2 proteins. Cells were transfected simultaneously with Beclin‐1 (or control vector) and equivalent amounts of plasmids coding for WT Bcl‐XL, Bcl‐XL G138A, Bcl‐2 or Mcl‐1 (or control vector), as well as GFP‐LC3. The culture in CM or NF in the absence or presence of ABT737 was performed during the last 12 h of the 60 h‐long experiment. The percentage of cells exhibiting the accumulation of LC3‐GFP in vacuoles (LC3‐GFPvac) is quantified as means±s.d. (n=3 separate experiments).
Figure 5A) Illustration of the experimental procedure. Cells were grown for two weeks in growth media containing either Heavy or Medium variants of lysine and arginine. At day 14, the media was replaced with normal growth media containing an excess of unlabeled (Light) lysine and arginine. Lactacystin or epoxomicin was added to one of the sets at this time. 4, 10 or 24 hours afterward, cells were harvested, mixed and separated by SDS-PAGE. Lanes were then cut into 5 slices, digested, and analyzed by MS analysis, resulting in a list of H/M ratios for each peptide. B) Illustration of H/M ratio meaning. For proteins whose degradation is retarded by proteasomal inhibitors, H-labeled copies are degraded at slower rates than M-labeled copies, resulting in H/M ratios greater than 1. For proteins whose degradation is not affected by proteasomal inhibitors, H-labeled and M-labeled proteins are degraded at similar rates, resulting in H/M ratios of ~1.
Figure 1A. Experimental design for the proteomic identification of PrimPol-interacting factors in S phase-synchronized cells. Immunoblots showing PrimPol levels in WT and KO cells. MEK2 is shown as loading control. B. Volcano plot showing statistical significance (-log p-value) vs enrichment ratio (log2) of identified proteins after PrimPol immunoprecipitation in WT and KO cells. The positions of PrimPol, RPA1-3, BLM, RMI1, RMI2, MHF1, SSBP1, RUNX1, HERC2 and BCCIP proteins are indicated. C. Volcano plot showing statistical significance (-log p-value) vs enrichment ratio (log2) of identified proteins after PrimPol immunoprecipitation in WT cells with or without MMC (2 µg/mL; 5 h). BLM, RMI1, RMI2, MHF1, SSBP1, RUNX1, HERC2 and BCCIP proteins are indicated. D. Immunoblot detection of the indicated proteins in whole cell extracts (WCE), PrimPol immunoprecipitates (IP), and non-precipitated flow-through fractions (FT). IgG shows the presence of anti-PrimPol antibody in the IP fractions (lanes 4-6).
Cardiomyocyte-specific p32cKO causes lysosomal enlargement in mice.Figure 2(A) Electron micrographs showing lysosomes in cardiac myofibrils of 2 and 6 months old mice. Lysosome size and density were different in WT (black dot) and p32cKO (red dot) heart tissues. Scale bars, 1 µm. The quantification of the average lysosomal length is presented in the plot in the right panel as mean ± SEM (two months old mice, n = 14-16; 6 months old mice, n = 35-60). Statistical significance was assessed by Student's t-test, ***p < 0.005. (B) Autofluorescence showing lipofuscin localization around the nucleus in the 6 months old p32cKO heart. Tissues were excited at a wavelength of 540 (upper panel) or 470 (lower panel) and emission spectra were collected with a confocal microscope at wavelengths (band path) of 580-630 nm (upper panel) or 510-560 nm (lower panel). Enlarged view of the white squares is shown right. Scale bars, 20 µm. The quantification of the ratio of autofluorescence of DAPI staining is presented in the plot in the right panel as mean ± SEM (three months old mice, n = 11; 6 months old mice, n = 9-14, 9 months old mice, n = 9-15, 12 months old mice, n = 11-21). Statistical significance was assessed by Student's t-test, ***p < 0.005. (C) Immunostaining of Lamp2 in the heart using TrueBlackTM to quench lipofuscin autofluorescence in the 6 months old WT and p32cKO heart. Enlarged view of the white squares is shown below. Scale bars, 20 µm. (D) Western blot analysis of Lamp2 in the 6 months old WT and p32cKO hearts. Quantification is shown in the lower panel (n = 4 mice per group). GAPDH was used as an internal control. Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, ***p < 0.005.
Residues that interact with E2~SUMO are mostly conserved between Siz2 and Siz1.Figure 5Structural comparison of Siz2 (green) and Siz1 (white; pdb 5JNE) highlighting the structural similarity of (A) the PINIT domain or (B) the SP-RING domain. Structure-based amino acid alignment of Siz1 and Siz2 PINIT and SP-RING domains. Residues in Siz1 that make contacts within 4 Å of SUMO and E2 in the PCNA/Siz1/Ubc9~SUMO complex (pdb 5JNE) are indicated by cyan circles and magenta triangles, respectively. Contacts determined using the CCP4 program CONTACT (Winn et al, 2011). Putative Siz2 E2~SUMO interaction interface modeled by aligning PINIT and SP-RING domains of Siz2 to those of Siz1 in the context of the PCNA/Siz1/Ubc9~SUMO complex (pdb 5JNE) (Streich & Lima, 2016) (D) and Siz1 E2~SUMO interactions present in the PCNA/Siz1/Ubc9~SUMO complex (E). Contact forming residues are shown in stick representation and Siz1/Siz2 residues that make contacts with SUMO and E2 are additionally colored in cyan and magenta, respectively.