EMBO/sd-character-level-ner huggingface dataset:
Figure 2A) Schematic of the ub-HS-motif mapping strategy. A 1038 bp stretch of ub-HS4 (blue line) was cloned and integrated at the LEU2 locus (grey) using the integrative YI128-vector (green). Initial mapping led to the identification of fragment F1 (Appendix Fig. S2A), which was further truncated for fine-mapping ((B), F5-F8). qPCR primers were designed to bind within the YI128 backbone. B) A 39 bp fragment of ub-HS4 is sufficient to drive ectopic ub-HS formation. Fragments of ub-HS4 were integrated ectopically and ub-K48 ChIP-qPCR was performed for the endogenous ub-HS5 and the ectopic ub-HS4 fragments as depicted in (A). contr.: control, empty YI128-vector was integrated at LEU2. Experiments were performed in cdc48‑6 background. Data represent means ± SD (n = 2). Experimental mapping and bioinformatic prediction identify a similar ub-HS-motif. Comparison between the experimentally mapped ub-HS4 F7 (B) and the consensus motif of all ub-HSs identified by the MEME software. D) A yeast one-hybrid screening strategy to identify proteins binding to the ub-HS-motif. Three copies of the ub-HS4-motif (F7) were cloned upstream of a minimal promoter followed by a HIS3 reporter-gene and integrated at the URA3 locus. A yeast cDNA library with N-terminally fused Gal4-AD was used to screen for survival on media lacking histidine and multiple plasmids coding for 4 different genes were recovered (bottom). E) Euc1 binds to the ub-HS-motif in a Y1H assay. Gal4-AD- or Gal4-AD-Euc1-encoding plasmids were transformed into a Y1H reporter strain as described in (D). Serial dilutions were spotted on control plates and plates lacking histidine with 20mM 3-amino-triazole (3AT). Cells were grown at 30°C for 3 days. F) Euc1 is required for formation of ub-HSs. ChIP with ub-K48 specific antibodies was performed for the indicated strains and enriched DNA was analyzed by qPCR. Data represent means ± SD (n = 4). Euc1 binds to endogenous ub-HSs. Genome-wide binding profiles of K48-linked ubiquitin chains were obtained in ChIP-chip experiments as described in Fig. 1A. Euc1-ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292-462. Data represent means from two independent replicates. Euc1 binds to endogenous ub-HSs. Genome-wide binding profiles of Euc1 were obtained in ChIP-chip experiments as described in Fig. 1A. Euc1-ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292-462. Data represent means from two independent replicates.
ALKAL stimulates ALK signaling and transcriptional responses in NB cells.Figure 1A. RNA-Seq based differential gene expression (DE) was measured in NB1 and IMR32 NB cell lines in response to ALKAL2 stimulation. See Table S1 for detailed results. B. Volcano plot showing DE 1hr after NB1 (top) and IMR32 (bottom) cell treatment with ALKAL2. Dashed lines show DE thresholds. Up-/downregulated genes indicated in blue. Six genes that are DE in both cell lines and sensitive to the ALK inhibitor lorlatinib are indicated and labelled in red. C. Venn diagram indicating the number of DE genes between different conditions as indicated. Outer circles (labels below diagram) indicate the number of DE genes after ALKAL2 addition for NB1 cells (34 genes) and IMR32 cells (13 genes). Inner circles (labels on top) correspond to the number of DE genes after addition of lorlatinib. Six genes that are DE in both cell lines and sensitive to lorlatinib are indicated. D. Temporal dynamics of ALKAL2-induced transcription of ARC, EGR1-3, FOS and FOSB in NB1 and IMR32 cells in the presence and absence of the lorlatinib, as indicated. E. Immunoblot validation of ALKAL2 induction of EGR1 and FOS at the protein level in NB1 cells. Cells were treated for 0, 1 and 6h in the presence and absence of lorlatinib as indicated. F. Transcription factor prediction based on a gene set enrichment analysis (GSEA) of the identified 6-gene set. Bar plot shows the log10(q) values of all enriched transcription factors at 10% FDR. G. Immunoblot analysis of ALKAL2 induction of pSRF in NB1 cells. Cells were treated for 0 and 1h in the presence and absence of lorlatinib as indicated. RNA-Seq analysis was performed using 3 biological repeats. Immunoblots are representative of at least 3 independent experiments.
Figure 3Distribution of SiE5′NT orthologues across higher fungi. End nodes are color-coded based on the presence (blue) or absence (red) of 5′NT genes in a particular fungal taxa. Numbers in parentheses besides the nodes specify the number of species that have 5′NT genes with respect to the total number of genomes analyzed. Left tree: distribution of 5′NT without signal peptide (SP) and GPI anchor. Right tree: E5′NT with SP and GPI anchor. The distribution shows that E5′NT genes are mostly present in Ascomycota such as Sordariomycetes (107/121), followed by Dothideomycetes (24/37) and finally Leotiomycetes (6/18). Few species of Eurotiomycetes (6/118) possess an E5′NT orthologue. In Basidiomycota, E5′NT members are only found in the class of Agaromycotina (12/85). Comparison of the SiE5′NT structural homology model (green) with the crystal structure of human E5′NT (PDB id 4H2I, red) with 32% sequence identity. C. Comparison of the SiE5′NT structural homology model (green) with the crystal structure of Thermus thermophiles 5′NT (2Z1A, blue) with 36% sequence identity. *: position of the loop involved in dimerization of human E5′NT.
Figure 8LD biogenesis is initiated in the ER. Nascent LDs harboring seipin at their ER contact recruit proteins and lipids from the ER to promote LDgrowth. LDs remain associated with the ER during maturation, allowing continuous cargo exchange between these compartments. In seipin deficient cells, ER-LD contacts are morphologically abnormal, either missing, rudimentary or aberrantly extensive. Most newly formed LDs lack functional contacts with the ER. HPos, a model ER-to-LD targeting peptide can initially reach LDs but its access to LDs is later perturbed. ACSL3, a lipid biosynthetic enzyme normally targeted to the LD surface remains in the ER or at ER-LD junctions. Oleic acid flux into neutral lipids is initially intact but subsequently impaired. In contrast, ADRP, a cytoplasmic coat protein, reaches LDs independently of seipin. Defective ER-LD contacts result in discontinued cargo exchange and arrested LD maturation, where the majority of LDs fail to grow beyond the diameter of ~200 nm.
a, Immunoblot analysis of Atg5 and LC3.figf1a, Immunoblot analysis of Atg5 and LC3. Brain homogenates were prepared from six-week-old control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice. Immunoblot analysis was performed using antibodies against Atg5 and LC3. GSK-3β was used as a loading control. The positions of the Atg12-Atg5 conjugate, LC3-I and LC3-II (LC3-PE conjugate) are indicated. Atg5 monomer was not detected in either lane (data not shown). b, A representative male control (Atg5flox/+; nestin-Cre) and an Atg5flox/flox; nestin-Cre (Atg5flox/flox) littermate at three weeks of age. c, Left, paw placement records of eight-week-old mice. Right, stride lengths corrected for paw base widths (stride/width ratio) of Atg5flox/flox; nestin-Cre and control (Atg5flox/+; nestin-Cre) littermate mice. Values represent means ± s.d. of four mice. Asterisk, P 0.01 (Students t-test). d, Abnormal limb-clasping of an Atg5flox/flox; nestin-Cre mouse compared with a control mouse (Atg5flox/+; nestin-Cre) when suspended by its tail. e, Rotarod testing of Atg5flox/+; nestin-Cre (open symbols) and Atg5flox/flox; nestin-Cre (closed symbols) mice. One male and one female mouse were analysed for each genotype. The time until drop from the rod (rotating at 20 r.p.m.) is shown.
PfCDPK7 is involved in PC biosynthesis in P.Figure 2A. Total lipids were extracted from PfCDPK7-KO or 3D7 parasites and separated by HPTLC. After quantification by GC-MS content relative to total fatty acids was determined. Relative abundance of individual phospholipids was quantified. Disruption of PfCDPK7 caused a significant decrease in PC (mean± SEM, n=3; biological replicates, ***p<0.001, t-test). B. Total fatty acids were extracted and separated before quantification by GC-MS analysis. The analysis shows an increase of C18:1 as well as the general decrease of PUFAs (inset) and more particularly C20:4 (mean ±SEM, n=3; biological replicates, ***p<0.001, t-test). C. Metabolic pathways depicting the synthesis of PC and PE in P. falciparum. PMT and EK, which are putative targets of PfCDPK7, are indicated in red D. Metabolic labeling to monitor PC and PE synthesis. Equal number of WT (3D7) and PfCDPK7-KO (KO) parasites were incubated with 14C-Eth or 14C-Cho (in RPMI) for 12h. Subsequently, parasites were harvested and lipids were extracted and separated by thin layer chromatography (TLC). The radiolabeled lipids were detected by phosphorimaging (upper panel). Bottom Panel, Radiolabelled PC and PE were quantitated by performing densitometry on phosphorimage of TLC plates in above panel and % change in PC or PE formation in PfCDPK7-KO parasites with respect to 3D7 (100%) was calculated (mean ± SEM, n=4; biological replicates, **p<0.01, ANOVA; ns, nonsignificant). E. 3D7 or PfCDPK7-KO parasites were cultured in the presence or absence of 200 μM choline in culture medium. After 3 days, parasitemia was determined by counting parasites on thin blood smears (mean± SEM, n=6; biological replicates, *p< 0.05, ***p<0.001, ANOVA).
Figure 3Prevalence of individual RKPT residues and prevalence of carbonylated form in experimentally measured peptides combining all 3 biological replicates of both conditions for each organism. Ratios are given above each pair of bars. All proportions are significantly different between each RKPT and their respective carbonylation state by two-tailed z-test of two proportions (p-values < 0.01; see Materials and Methods), meaning carbonylated proportions are not determined simply by relative prevalence of RKPT. See also Appendix Fig S1. Prevalence of all canonical amino acids before irradiation of E. coli and D. radiodurans, combining all 3 biological replicates for each condition. Ratios are given above each pair of bars. All proportions are significantly different between species by two-tailed z-test of two proportions (p-values < 0.01). See also Fig EV1 and EV2.
Figure 6Primary ILC3 cells were sorted from colonic LP cells and were stimulated with indicated cytokines. Productions of IL-22 were detected by ELISA. mRNA expressions of Il-22 were detected by qPCR. BMDMs were stimulated with C.albicans for 24h and the supernatant was isolated and added to primary ILC3 cells. (1) Supernatant from BMDMs without stimulation. (2) Supernatant from BMDMs with stimulation of C.albicans. (3) Supernatant from BMDMs with stimulation of C.albicans and IL-7 antibody. Productions of IL-22 by ILC3 cells were detected by ELISA. mRNA expressions of Il-22 in ILC3 cells were detected by qPCR. Primary ILC3 cells were sorted from LPs and were stimulated with (IL-7 group) or without IL-7 (None group). mRNA expressions of relative genes were detected by qPCR. Primary ILC3 cells were sorted in LPs from STAT3fl/fl and STAT3fl/fl VillinCre mice and were stimulated with IL-7. Productions of IL-22 were detected by ELISA. mRNA expressions of Il-22 in ILC3 cells were detected by qPCR. Primary ILC3 cells were sorted from LPs and were stimulated with IL-7 in combined with FIZC and AhR Inh. Cells without stimulation were used as control (None). Production of IL-22 was detected by ELISA. AhR, STAT3, ROR binding sites in the il-22 promoter. HEK293T cells were transiently transfected with the indicated expression constructs (Ctl or AhR) and were stimulated with (IL-7 group) or without IL-7 (none group). ChIP analysis of the interaction of AhR and STAT3 to binding sites in the il22 promoter with or without IL-7, respectively. HEK293T cells were transiently transfected with the indicated expression constructs. Whole cell extracts were subjected to anti-HA immunoprecipitation, and were then immunoblotted with HA antibody and anti-STAT3 antibody. EL4 cells were transduced with retroviruses encoding AhR and STAT3. IL-22 mRNA expression was analyzed by qPCR.
Fig.Figure 5A) Comparison of USP1WT and deletion mutants (+UAF1) for activity on DNA-loaded PCNA-Ub (solid lines) and free PCNA-Ub (dashed lines) shows no increase in USP1ΔL1 activity on the loaded substrate. Left panel: Quantification of gel-based activity assays showing percentage of cleaved PCNA-Ub at the mentioned time points (n=2, biological replicates), Right panel: Quantification of the activation fold observed in USP1WT and mutants on DNA-loaded PCNA-Ub versus free PCNA-Ub (n=2, biological replicates). B) Multiple sequence alignment of USP1 insert L1 which highlights the DNA binding region and the conservation of the positively charged residues across species. Three groups of positively charged residues (***) are mutated separately and tested for binding to double stranded DNA (65bp) using SPR which shows the importance of this region for DNA binding of USP1. C) Comparison of USP1DM1, USP1PIP1 and USP1PIP1+DM1 (+UAF1) activity on DNA-loaded PCNA-Ub (solid lines) and free PCNA-Ub (dashed lines) shows reduced increase in activity of these mutants compared to USP1WT. Left panel: Quantification of gel based activity assays showing percentage of cleaved PCNA-Ub at the mentioned time points (n=2, technical replicates), Right panel: Quantification of the activation fold observed in USP1WT, USP1PIP1, USP1DM1 and USP1PIP1+DM1 on DNA-loaded PCNA-Ub versus free PCNA-Ub (n=2, technical replicates). D) EMSA based DNA binding experiment shows that USP1PIP1 and USP1WT has similar DNA binding capability while USP1DM1 has lost its ability to bind DNA. E) Schematic model for the role of Insert L1 in USP1 activity regulation by UAF1 and DNA loaded PCNA-Ub.
Figure 8A-E. Expressions of Rab5-GFP reporter in ISCs of 14-day WT Drosophila (A), 40-day WT Drosophila (B), 40-day Drosophila with ALA administration (C), 14-day Drosophila carrying esgts-GAL4>UAS-lacZ (D), and 14-day Drosophila carrying esgts-GAL4>Las RNAi (E). GFP (green). F. Quantification of fluorescence intensity of RAB5-GFP in experiments (A-E). Each dot corresponds to one cell. n is indicated. The numbers of quantified guts from left to right are 13, 11, 12, 10 and 13. G Data information: DAPI stained nuclei are shown in blue. Scale bars represent 2 μm Error bars represent SDs. Student's t-tests, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. G-K. Expressions of Rab7-GFP reporter in ISCs of 14-day WT Drosophila (G), 40-day WT Drosophila (H), 40-day Drosophila with ALA administration (I), 14-day Drosophila carrying esgts-GAL4>UAS-lacZ (J), and 14-day Drosophila carrying esgts-GAL4>Las RNAi (K). GFP (green). L. Quantification of fluorescence intensity of RAB7-GFP in experiments (G-K). Each dot corresponds to one cell. n is indicated. The numbers of quantified guts from left to right are 11, 10, 11, 13 and 12. M Data information: DAPI stained nuclei are shown in blue. Scale bars represent 2 μm Error bars represent SDs. Student's t-tests, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. M-Q. Immunofluorescence images of midgut section from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (M, control), constitutively active form of RAB5 (N), constitutively active form of RAB5 with ALA administration (O), constitutively active form of RAB7 (P), or constitutively active form of RAB7 with ALA administration (Q). GFP (green) and Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm R. Quantification of the number of esg-GFP+ cells, Dl+ cells, and pH3+ cells in experiments (M-Q). n is indicated. The numbers of quantified guts from left to right are 17, 18, 18, 17, 19, 17, 18, 18, 17, 19, 17, 18, 18, 17, and 19. Data information: Error bars represent SDs. Student's t-tests, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. S. Quantification of luciferase activity in Drosophila with indicated genotypes and applied manipulations. Error bars show the SD of six independent experiments. Data information: Error bars represent SDs. Student's t-tests, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.
Fig.Figure 1(A) Images of host armyworm Mythimna separata larvae. Upper: emergence of Cotesia kariyai parasitoid wasps. Lower: inside of host body extensively occupied by parasitoid larvae before their emergence. (B) Survival of parasitized and unparasitized host larvae 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=10). **P<0.01 vs. unparasitized hosts. Day-4 of last instar larvae were used for this assay. Test larvae were parasitized by the wasp on day 0 of last instar. (C) Survival of armyworm larvae injected with PBS, unparasitized, and parasitized larval hemolymph 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=12). **P<0.01 vs. PBS. Test larvae were exposed to the heat stress 4 h after injection of each sample. (D) Time-dependent survival of armyworm larvae injected with 0.8 µmol/larva N-acetyl-L-tyrosine (NAT): post NAT-injection 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=12). **P<0.01 vs. zero time. (E) Dose-dependent survival of armyworm larvae injected with NAT after heat stress at 44oC for 50 min (data are means ± SEM; n=12). Injection of more than 10 nmol NAT/larva caused a significant difference compared with zero (log rank test using R version 3.2.2). (F) Survival of armyworm larvae injected with indicated chemical solution 1 day after heat stress at 44oC for 50 min (data are means ± SEM; n=10). **P<0.01 vs. PBS. Each chemical (0.5 µmol/larva) was dissolved in PBS; a minimal volume of dimethyl sulfoxide was used for poorly soluble chemicals such as tyrosine.
Figure 2(A) Representative immunofluorescence confocal microscopy images of young, aged and aged + CASIN (5µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of Cdc42 and borg4. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar= 5µm. (B) Quantification of the level of fluorescent signal in young, aged and aged + CASIN (5µM) treated LT-HSCs to quantify the Cdc42-borg4 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n=at least 17 cells per condition, error bars indicate mean±SEM, unpaired t-test, ****P<0.0001. (C) Representative immunofluorescence confocal microscopy images of young, aged and aged + CASIN (5µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of borg4 and septin 7. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar= 5µm. (D) Quantification of the level of fluorescent signal in young, aged and aged + CASIN (5µM) treated LT-HSCs to quantify the borg4-septin 7 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n=at least 17 cells per condition, error bars indicate mean±SEM, unpaired t-test, ***P<0.001, ****P<0.0001.
Figure 9(A) Schematic model of the molecular interactions between dynein/p150Glued/STIM1 and p150Glued/KIFC1 complexes at the MT plus ends. Black arrows point at already known binding motifs, whereas red parts show interaction domains found in this manuscript. TM = transmembrane domain. (B) Schematic model of the distinct motor sets regulating EE (above) and LE (below) retrograde transport, along MTs. Stars indicate EB1-binding SxIP motifs.
Figure 2A Schematic representation of the pTY-LAI-luciferase reporter virus used in secondary screening assays, where luciferase expression is under the control of the 5' LTR, in cloned lines derived from JurkatTAT cells bearing single copy integrants. B-F Luciferase activity was measured from reporter cells treated for 24 hours with the indicated compound concentrations. Results are presented as percent activation relative to PMA treated controls. The EC50 for each compound is indicated. Mean and standard error (SE) for the results are determined from three biological replicates (n=3) and technical duplicates. G Illustration of chemical structures of the PH compounds.
Figure 3(A) Confocal microscopy images of ECs silenced with a control siRNA (siCTL) or one targeting STIM1 (siSTIM1) and stained for endogenous Rab5 (in green) and EEA-1 (in red) to visualize EEs and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm. (B) Average size, normalized on cell size, of EEA-1+ endosomes as in a. Results are the average ± SEM of three independent experiments for a total 4483 early endosomes in 18 siCTL cells (299 ± 33 endosomes per cell) and 2454 early endosomes in 13 siSTIM1 cells (189 ± 17 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***. (C) Average number, normalized on cell size, of EEA-1+ endosomes as in a. Results are the average ± SEM of three independent experiments for a total 4483 early endosomes in 18 siCTL cells (299 ± 33 endosomes per cell) and 2454 early endosomes in 13 siSTIM1 cells (189 ± 17 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,01 **. (D) Distribution of distance to nucleus overtime, normalized on cell size, quantified by image (data correspond to movies EV1 and EV2) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 60 seconds. Results are from three independent experiments for a total of 903 early endosomes in 18 siCTL cells (50 ± 5 endosomes per cell) and 935 early endosomes in 20 siSTIM1 cells (47 ± 9 endosomes per cell). Data are analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,01 **.
Figure 7(A) Deletion of EUC1 does not lead to ub-HS adjacent gene deregulation, but rather widespread transcriptome changes. Total RNA isolated from WT and euc1∆ cells was polyA-enriched and sequenced (RNAseq, triplicates). Significance testing was based on the Wald test, see Materials and Methods for details. See also Appendix Fig. S8A for quantification of selected transcripts by qPCR. (B) RNAseq transcriptome analysis of EUC1 overexpression as in (A). ∆euc1 cells with pGAL-EUC1 or pEUC1-EUC1 (control) integrated at the URA3-locus (YIplac211) were grown to mid-log phase and 2% galactose was added for 3h. See also Appendix Fig. S8E. (C) EUC1 overexpression is toxic at elevated temperatures and upon exposure to the membrane fluidizing drug benzylalcohol. For (C, D, F), serial dilutions of the indicated strains were spotted and grown on YPD control (or selective media) plates or under conditions as indicated. See also Fig. EV4A-B. (D) EUC1 overexpression toxicity depends on DNA binding and transactivation. The indicated EUC1 alleles with endogenous or galactose-inducible promoters were integrated at the URA3 locus (YIplac211, euc1∆ background) and spotted on glucose control or galactose plates to induce EUC1 overexpression. See also Appendix Fig. S9A-C. (E) Aberrant binding of overexpressed EUC1 partially depends on Euc1 DNA binding. ChIP-qPCR of Euc1 after 3 hours galactose induction. Note that IP/input ratios of Euc1 signals are shown, also for the control locus (contr., TOS1 promoter) to highlight Euc1 binding at non-ub-HS sites. STI1-CIN5: intergenic region. TEC1-us: upstream (promoter) region. Data represent means ± SD (n = 3). See also Fig. EV4C-D. (F) Simultaneous overexpression of SLX5 and SLX8 rescue EUC1 toxicity. Experiment as in (D), but with concomitant plasmid-borne overexpression (ADH promoter) of SLX5 and SLX8 (bottom panels) and on media selecting for SLX5/SLX8 plasmids. See also Fig. EV4E and Appendix Fig. S9D.