EMBO/sd-character-level-ner huggingface dataset:
Figure 7Experimental scheme to determine whether JNK signaling promotes resistance to chemotherapy. MDA231-LM2 or SUM159-LM1 cells were implanted bilaterally into the fourth mammary fat pads of NSG mice. Treatment with JNKi was initiated at day 5 post implantation and repeated every three days; paclitaxel chemotherapy was started at day 10 post implantation and repeated every five days. Mammary tumor growth curves in NSG mice implanted with MDA231-LM2 or SUM159-LM1 cells and treated with paclitaxel as single agent or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2: n = 12 tumors per group. SUM159-LM1: vehicle, JNKi and PAX n = 12 tumors per group; PAX + JNKi n = 10 tumors. **P < 0.01, ***P < 0.001. Metastatic burden in lungs of MDA231-LM2 and SUM159-LM1 tumor-bearing mice treated with paclitaxel as single therapy or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2 tumor-bearing mice: n = 6 mice per group. SUM159-LM1 tumor-bearing mice: vehicle, JNKi and PAX n = 6 mice per group; PAX+ JNKi n = 5. Ten fields were quantified per lung section. FOV, field of view, LS, lung section. PAX, paclitaxel. Representative histological images of metastatic foci (arrows) in lungs of the MDA231-LM2-implanted mice described in panel C, stained for human vimentin expression in differentially treated groups. Scale bar, 100 μm. Apoptosis was determined by quantification of TUNEL-positive cells per field of view (FOV) in MDA231-LM2 tumors (from the animals described in (C)) treated with JNKi and/or PAX. Values are means from 6 mice per condition ± SEM. GSEA showing enrichment of the JNK signature in tumor samples from breast cancer patients from the TOP trial (GSE16446), stratified according to pathological response to neoadjuvant chemotherapy. NES, normalized enrichment score. pCR, pathological complete response. Kaplan-Meier analyses of chemotherapy-treated breast cancer patients associating JNK signature (JNK-S) with metastasis-free survival (TOP trial data set, JNK-S low n = 54 patients; JNK-S high n = 53 patients (G)) or overall survival (METABRIC data set, JNK-S low n = 135 patients; JNK-S high n = 135 patients (H)). HR, hazard ratio. P values were determined by log-rank test. Model illustrating the link between stress-induced ECM, stem cell and wound healing network and metastatic progression and therapy resistance in breast cancer.
Altered metabolism accompanies decreased fission and autophagy.Figure 8(A) FIS1 and DRP1 knockdown reduced maximal respiratory chain capacity in FIS1 RNAi and DRP1‐DN compared with their control (n=4 in each group; glucose=11 mM). Inhibition of autophagy by 3‐MA in INS1 cells (2 mM, 5 days, n=4) and C2C12 cells (1 mM, 5 days, n=4) showed significant decrease in respiratory capacity. ATG5‐deficient MEF cells (n=4) had significantly decreased respiratory capacity compared with control MEF cells. Maximal respiration was tested using 5 μM FCCP or 100 μM DNP (*P0.001, **P0.04). (B) Expression of mtDNA‐encoded subunit I of cytochrome oxidase (COX I) in INS1 cells infected with FIS1 RNAi and control RNAi lentiviruses and tested after 1 week. (C) GSIS in FIS1 RNAi and control RNAi cells (n=5 for each column, *P0.05). Values are normalized to number of cells in each well and represent 30 min stimulation.
a) LRO (lysosome-related organelle) live Nile red-stained images of hlh-30(tm1978) and wild-type (WT) L3 larvae starved for 60 h show that hlh-30 is required for the expansion of the lysosomal compartment following fasting.figf5(a) LRO (lysosome-related organelle) live Nile red-stained images of hlh-30(tm1978) and wild-type (WT) L3 larvae starved for 60 h show that hlh-30 is required for the expansion of the lysosomal compartment following fasting. Quantification as mean±s.e.m. relative to fasted wild-type worms is also presented; n = 3 independent experiments. (b) Transcriptional levels of the autophagy genes lgg-1, lgg-2 and atg-16.2 in fasted wild-type and hlh-30(tm1978) young-adult worms show that transcriptional activation of autophagy following fasting is hlh-30 dependent (mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms). n = 3 independent experiments (c) The mean percentage (+ s.d.) of L1 larvae alive after 48 h fasting in minimum media shows that hlh-30 (tm1978) mutant animals are sensitive to starvation. n = 3 independent experiments. For L4 survival see Supplementary Fig. S6g.
Figure 8(1) Iron (red circle) enters the chloroplast and is then captured by the conserved cysteine residues in the CR domain of DJA6/5 proteins. (2) During ATP binding, dimerization of SUFC occurs, which induces a large conformational change of the SUFBC2D complex and exposes the buried assembly sites to the SUFB-SUFD surface. (3) This conformational change facilitates the recruitment of the NFS2-SUFE1 complex. Then the sulfur (yellow circle) is extracted from the substrate L-cysteine by the action of NFS2, and further delivered via SUFE1 to SUFB. (4) Iron-loaded DJA6/5 proteins directly interact with SUFE1 and SUFC via their J domain and deliver iron to proposed iron recipient SUFD. (5) Arrival of the electrons allows the building of Fe-S cluster at the SUFB-SUFD interface. After assembly, DJA6/5 protein and NFS2-SUFE1 complex are released and ready for a new cycle. (6-7) The nascent Fe-S cluster (red and yellow circle) is transferred by carrier proteins (NFUs, SUFA, GRXs and HCF101) to client proteins (such as PetC, PsaA/B, PsaC, Fd, PAO).
Genes linked to growth phenotypes via GWAS analysis in 925 natural yeast isolates.Figure 4S-score heatmap of the yeast natural isolates across 34 conditions that were also used in the KO screening. B) Number of variants significantly associated (p-value < 1E-6) with phenotypic variation in each growth condition. C) Number of associated variants that overlap (i.e. are in a 3kbp window) with a conditionally essential gene in the same condition, in any of the four KO libraries. Manhattan plots showing examples of overlaps between associated variants and the KO screening. The top plot shows the associations between variants and growth in the natural isolates as a function of the -log10 of the association p-value, while the four bottom plots show the strength of the KO phenotypes across the four yeast strains, as a function of the -log10 of the corrected S-score p-value. Sections shaded in grey indicate the overlap between associations and KO data. Position in the yeast chromosome is reported in kilobase units.
Figure 2A Primary mousecardiomyocytes were treated with H2O2 (1 mM) for 3 hours and cardiomyocyteviability was determined by flow cytometry. Representative results of flow cytometry analysis. B Quantitative analysis of PI-positive cells treated with H2O2 was shown (42.9 ± 3.1% in wild-type group vs. 10.1 ± 4.4% in Cuedc2-/- group, * p = 0.0004, n = 5 wells per group and repeated for three times). C Mouse neonatal cardiomyocytes were subjected to hypoxia for 6 hours and then reoxygenation for 30 minutes. Cardiomyocytes were then stained with 5 μM CellROX® Deep Red Reagent and analyzed by flow cytometry. Representative results of flow cytometry analysis are shown, and all experiments were repeated for three times.D ROS levels were quantitated and summarized. * p = 0.0108, n = 3 wells per group and repeated for three times. E HPLC detection of a superoxide probe oxidized dihydroethidium (DHE) product in sham and I/R injury heart tissue (30-min ischemia followed by 30-min reperfusion), 2-hydroxyethidium (EOH), a specific product for superoxide anion radical. *p < 0.0001, n = 5 in each group.F In the same samples of (E), HPLC detection of ethidium (E), oxidized by other reactive oxygen species such as H2O2 (mainly) and ONOO. *p = 0.0001, n = 5 in each group.
Figure 6A The data were analyzed by categorizing the splice events into seven types: exon skip (ES), alternative 5' donor (AD), alternative 3' acceptor (AA), retained intron (RI), mutually exclusive exons (ME), alternative first exon (AP) and alternative last exon (AT). The analysis of splice events was performed using the following filter criteria: Min Gene RPKM>2, |dPSI|>0.1, p-value≤0.02, Min Group Obs % >.85; RPKM-reads per kilobase of transcript per million aligned reads; and |dPSI|-absolute changes in percent splicing (dPSI, ∆Ψ). The events are presented in Fig 6 Source data file 1 as differential expression of splice events between ESRP1_Low and ESRP1_High cases. B TCGA Splicegraphs for the EMT signature genes in ESRP1high versus ESRP1low tumors. A splice graph of the gene's exons is shaded based on expression level and shows the selected splice event outlined in red.
Arrayed CRISPR screens using solid phase transfection A) Viability of NCI-N87 and NCI-H358 cells upon transfection of 45 different gRNAs by solid phase transfection.Figure 4A) Viability of NCI-N87 and NCI-H358 cells upon transfection of 45 different gRNAs by solid phase transfection. Cas9 expressing NCI-N87 and NCI-H358 cell were seeded on pre-coated plates. 5 days post transfection, cell viability in each well were measured by CellTiter-Glo. Values were background subtracted, normalized to scrambled controls and plotted against each other. Blue dots represent scrambled controls, whereas the yellow dots represent POLR2A gRNAs. Green dots represent the genes that affect the viability in a cell line dependent manner. Results are representative of 3 independent experiments. B, C) Logarithm of the odds (LOD) scores of cancer associated gene KOs in NCI-N87 and NCI-H358 cells derived from screens with gRNAs (B) and RNPs (C). Bottom panels show distribution of positive (POLR2A) and negative (scrambled) controls for each experiment. LOD score of 3 is highlighted with a dashed line, indicating statistical significance.
Figure 2A. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 ama1∆ control cells and spo13 mutants. Top, time-lapse series. Bottom, meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. Data information: data are representative of two independent experiments. B. TEM analysis of SPBs. Left, SPBs in SPO13 and spo13∆ cells lacking all APC/C activators. TEM samples were collected at 12 h in SPM. The spo13∆ mutation causes MP formation at metaphase I (P < 0.0001, Fisher's exact test). Right, SPBs at metaphase II. CDC20-mAR and CDC20-mAR mpc54∆ mpc70∆ cells were synchronized by arrest/release at metaphase I, and TEM samples were collected at metaphase II (80 min after release). MP formation is blocked in the absence of Mpc54 and Mpc70 (P < 0.0001, Fisher's exact test). Scale bar, 0.2 μm. C. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 ama1∆ control cells and cells containing clb1∆ or cdc28-as1, which have been treated with 1NM-PP1 at metaphase I (8 h in SPM). Meiotic events (bottom) were analyzed as in (A). Data information: data are representative of two independent experiments.
Figure 4pAla computed FLR estimate for each type of canonical and non-canonical phosphorylated residue at ptmRS values ≥0.99, ≥0.90 or ≥0.75. Total number of phosphorylation sites estimated to be 'true positives' based on the pAla-determined residue specific FLR, for each phosphorylated residue according to ptmRS score. pLys* and pArg* represent non-C-terminal pLys or pArg residues respectively.
Fig.Figure 4(a-h) The migration of HEK293 cells expressing mGPR15, qGPR15, tGPR15, cGPR15, aGPR15, and their mutants induced by ligand C10ORF99. Data information: All data shown are generated from at least three technical replicates. Error bars indicate SD (n=3). ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05 (t-test). (i-p) The migration of CD4+ cells from GPR15-knockout mice, engineered to express mGPR15, qGPR15, tGPR15, cGPR15, aGPR15, and their mutants induced by ligand C10ORF99. Data information: All data shown are generated from at least three technical replicates. Error bars indicate SD (n=3). ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05 (t-test). Ad-NC represents adeno-virus-based gene-delivery vector as negative control.
Figure.Figure 2(A) Heatmap showing the differential expression of a selection of miRNAs known as "FibromiRs" in human BRAFV600E mutant melanoma cells sensitive to MAPK-targeted therapies (M229, M238, M249) and the corresponding BRAFi-resistant cells. Expression of indicated FibromiRs was evaluated by RT-qPCR; log2 (Resistant vs Parental). Expression level of AXL and MITF phenotypic markers for each resistant cell line is indicated. (B) Relative miRNA expression levels were quantified in M238P cells treated for 72 h with BIBF1120 (BIBF, 2 μM), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) (1µM), or with MAPKi (1 μM) plus BIBF (2 µM) by RT-qPCR and normalized to miR-16-5p. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. One-way ANOVA was used for statistical analysis. **P≤0.01, ***P≤0.001. Significance was calculated against the control group. Statistical significance of BRAFi/MEKi vs BRAFi/MEKi + BIBF was also calculated. (C) Expression of miR-143-3p and miR-145-5p in control mice and mice treated with the indicated therapies was quantified by RT-qPCR. Data is represented as mean ± SEM from 2 independent experiments performed on 6 mice, with 2 sites of injections. One-way ANOVA was used for statistical analysis. *P≤0.05, ****P≤0.0001. Significance was calculated against the control group. Statistical significance of BRAFi/MEKi vs BRAFi/MEKi + BIBF was also calculated. (D) Relative miRNA expression levels was quantified in M238P cells stimulated for 48 h with TGF-β (10 ng/mL) or PDGF-BB (20 ng/mL) by RT-qPCR and normalized to miR-16-5p. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. P-values were calculated using Paired Student t-test. *P≤0.05, ***P≤0.001. (E) Relative miRNA expression levels was quantified in M238R cells treated for 48 h with the triple kinase inhibitor Nintedanib/BIBF1120 (BIBF, 2 μM), the TGF-β receptor kinase inhibitor SB431542 (SB, 10 µM), and the pan-AKT inhibitor GSK690693 (GSK, 10 µM) by RT-qPCR. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. P-values were calculated using Paired Student t-test. *P≤0.05, **P≤0.01, ***P≤0.001. (F, Phenotype switch/invasive/ECM markers were quantified in therapy-naïve (N) and resistant (R) PDX samples. The log2 fold change or the ratio of the fold change R vs N is shown for each couple of samples. G) relative miRNAs expression levels (G) were quantified in therapy-naïve (N) and resistant (R) PDX samples. The log2 fold change or the ratio of the fold change R vs N is shown for each couple of samples.
Figure 5A. xiSEARCH is an open source search engine that takes a peak list as input. Users can define any type of crosslinker, modification, digestion, and fragmentation method. The output is a list of matches in .csv format. We use xiFDR to filter results to the desired confidence level. B. xiSEARCH+xiFDR(Xi), pLink 2 and Kojak(+PeptideProphet) comparison at 5% residue-pair FDR. The same trypsin dataset of the 26S proteasome was searched with all three software packages. xiSEARCH was run twice - once giving same likelihood for matching Lysine, Serine, Threonine and Tyrosine (xiSEARCH), as is the case for Kojak and pLink 2, and once giving priority to Lysine (Xi*).
Figure 3Hypocotyl lengths in SD condition of Col-0, toc1-101 and two independent native or 35S promoter lines of wild-type TOC1 and each phosphosite mutants. The middle line of the box represents the median, the x in the box represents the mean. The bottom line and the top line of the box represent 1st and 3rd quartile, respectively. The whiskers extend from the ends of the box to the minimum value and maximum value. Data information: 18 to 27 seedlings per line were entrained in 12h/12h light/dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2-h intervals for 1 week. B, Hypocotyl lengths in free-running period of Col-0, toc1-101 and two independent native or 35S promoter lines of wild-type TOC1 and each phosphosite mutants. The middle line of the box represents the median, the x in the box represents the mean. The bottom line and the top line of the box represent 1st and 3rd quartile, respectively. The whiskers extend from the ends of the box to the minimum value and maximum value. C-E, Average CCA1:LUC bioluminescence traces of indicated plant lines. Data information: 18 to 27 seedlings per line were entrained in 12h/12h light/dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2-h intervals for 1 week. White and gray regions indicate subjective light and dark period. Data are representative of at least two independent trials with similar results. Different letters indicate statistically significant differences in period between genotypes (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test).
Figure 1(A) Scheme for the two major mechanisms of DDT: HR and TLS. An asterisk represents a blocking DNA lesion for the replicative polymerase. (B) Representative image of wild-type cells with MMS-induced Rad52-YFP foci (bright field and fluorescence signal) (C-G) ssDNA repair efficiency of the indicated TS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (right panels). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour (60+), treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. They were also released into medium without MMS for 1 hour to control the formation of spontaneous Rad52-YFP foci (60-). The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels). Cell cycle progression was determined by cell sorting. The mean and SEM of 3 (wild type, rad18∆, mms2∆, rad51∆, rad57∆, rad54∆), 4 (pol30-K164R) and 5 (rad5∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where three asterisks represent p-values <0.001.
Figure 3A. UMAP of scRNA-Seq data from human ileum-derived organoids (n=16,682 cells); dots corresponding to cells are colored by the cell type. B. Dot plot of the top-three marker genes for each cell type. The dot size represents the percentage of cells expressing the gene; the color represents average expression across the cell type. Fig.S3B shows an extended list of the marker genes. C. Representative images showing multiplex in situ RNA hybridization of marker genes in an organoid. DAPI in blue. D-I. Visualizations for cell-type markers for the major intestinal cell types, showing single-cell expression intensities of the cell-type markers on the UMAP (left) and multiplex in situ RNA hybridization; red corresponds to viral RNA and a green corresponds to cell type markers (right). J-M. Percentage of stem cells (OLFM4) goblet cell (FGCBP), mature enterocytes (APOA4) and cells of the enterocyte lineage (FGCBP) from the total organoid cells population (black bar) in mock-treated organoids and after HAstV1 infection. The percentage of infected cells (red bars) and bystander cells (blue bars) within each cell type is shown. The data was obtained from multiplex in situ RNA hybridization in 2D organoids. n=10, mean ± SEM. Statistics show comparison of infected cells between mock-treated, 4h pi and 16h pi organoids. An Ordinary one-way ANOVA and Tukey's multiple comparisons test was used. n.s non significant, * p<0.05, ** p<0.01, *** p<0.001.
TP53INP1-deficient MEFi contain a dysfunctional mitochondrial pool.Figure 6A-D High-resolution respirometry was performed on permeabilized MEFi TP53INP1 deficient (−/−, black bars) or not (+/+, white bars) using glucose (A), lipid-related substrates (C) or complex IV substrate (D). High-resolution respirometry was also performed on intact MEFi (B). For different respiratory state details (routine, 3, leak, and ETS capacity), please refer to the Materials and Methods section. Results are expressed as the mean ± SEM and are representative of three independent experiments. In (A): P (−/− versus +/+; State 3) = 0.0079; P (−/− versus +/+; ETS) = 0.0079; n = 5 in each group. In (B): P (−/− versus +/+; routine) = 0.0159; P (−/− versus +/+; leak) = 0.0159; P (−/− versus +/+; ETS) = 0.0159; n = 5 in each group. In (C): P (−/− versus +/+; State 3) = 0.029; P (−/− versus +/+; ETS) = 0.0079; n = 5 in each group. In (D): P (−/− versus +/+; ETS) = 0.0259; n = 5 in each group. *TP53INP1−/− versus TP53INP1 +/+; 1 character: P < 0.05; 2 characters: P < 0.01. E TCLs from MEFi were analyzed by immunoblotting for CPT1A, CPT1B and β-tubulin. Results are representative of three independent experiments.
Figure 7PKA activities in testicular sperm from WT (+/+) and KO (-/-) mice. Graph shows average PKA activity in testicular sperm from KO mice (black bar) relative to that from WT mice (white bar), expressed as a percentage. Representative ProQ Diamond-stained 2D gel images of isolated proteins from testes in WT (left) and KO (right) mice. Testes lysates were separated by 2DE, and phosphoproteins were stained with ProQ diamond. Images in the boxes are enlarged in insets shown below. Circles with numbers indicate spots with significantly changed volume relative to those on WT gels. Comparative phosphorylation intensities of selected spots in 2D gels. The intensities of selected spots in KO gels were compared with intensities in WT gels. Average intensities of the indicated spot numbers (#) in WT (white bar) and KO (black bar): #1205, 1092.2 (WT) and 244.98 (KO); #1207, 729.9 (WT) and 140.43 (KO); #3303, 1165.5 (WT) and 279.5 (KO); #4302, 2725.8 (WT) and 1 (KO); #4405, 984.5 (WT) and 1 (KO). An intensity value of 1 indicates no spot, but is provided for calculation of fold change. Identification of proteins with altered phosphorylation patterns in Spatc1l-KO testis by PMF. SS, standard spot; Seq. cov., sequence coverage calculated based on the amino acid count; MW, molecular weight; pI, isoelectric point. Immunoblot analyses of five candidate proteins in testis (E) and sperm (F) of WT and KO mice. Anti-GAPDH, anti-ADAM2, and anti-α-tubulin antibodies were used as controls. Loss of SPATC1L in KO testis was confirmed by immunoblotting with anti-SPATC1L antibody. Localization of SPATC1L (red) and CAPZB (green) in testicular sperm. Overlay with bright field image is shown at right. The white arrow indicates the spatial overlap between SPATC1L and CAPZB. Scale bar = 5 μm.
Figure 1(a) Reaction scheme for Sld3/7/Cdc45 recruitment experiments. DNA substrate was bound to beads and reaction mixtures were removed after each step. (b, c) Immunoblots of recruitment reactions performed as described in (a), with the indicated proteins omitted. In (c), a mid reaction wash in high salt (0.5 M NaCl) buffer (HSW) was included following DDK phosphorylation as indicated. (d) Recruitment reaction performed as in (a), using wild type (wt) or a Cdc45-binding mutant (3E1) of Sld3. (e) Schematic showing position of Cdc45-interacting region in Sld3. Alignments were generated in Jalview using Clustal. Sld3 from various fungal species is included (S.c., Saccharomyces cerevisiae, S.m., Saccharomyces mikatae, S.b., Saccharomyces bayanus, S.ca., Saccharomyces castellii, S.ku., Saccharomyces kudriavzevii). Residue numbers correspond to S. cerevisiae Sld3. Residues were substituted for Glu. as indicated. (f) Wild type or mutant FLAG-Sld3 was added to an S-phase protein extract and tested for interaction with Cdc45. Immunoprecipitated proteins were analysed by immunoblot. (g) Reaction scheme for in vitro replication reactions. Reaction mixtures were removed after each step. (h) Reaction performed as in (g). Nascent DNA was separated in a 0.7% alkaline agarose gel in this and all subsequent replication reactions.