EMBO/sd-character-level-ner huggingface dataset:
Figure 3(A) Expression levels of our hits in M229 melanoma cells during the BRAFi-resistance acquisition. P: parental cells, 2D: two days of treatment, DTP: drug tolerant persister cells, DTPP: drug tolerant proliferating persister cells, SDR: single-drug resistant cells (BRAFi) (B) Expression levels of our hits in M238 melanoma cells during the BRAFi-resistance acquisition. (C) Expression levels of our hits in parental, single drug resistant (SDR, BRAFi) or dual drug resistant (DDR, BRAFi+MEKi) SKMel28 melanoma cell lines (D) T-distributed Stochastic Neighbor Embedding (t-SNE) plot showing the 4 drug tolerant states (NCSC (neural crest stem cells), invasive, SMC (starved-like melanoma cells) and pigmented cells) according to single cell RNA-seq (scRNA-Seq) performed in PDX MEL006 model exposed to BRAFi+MEKi Our BRAFi-resistance genes are mainly expressed in NCSC and invasive cells. (E) AUCell score for each drug tolerant states. (F) Expression level of our genes (median) in cutaneous melanoma biopsies before the BRAFi-treatment (baseline) and during the relapse. The expression level of our BRAFi-resistance genes is increased in relapse samples (48%, n=21). (G) Expression level of our genes (median) in cutaneous melanoma biopsies before the BRAFi-treatment and during the relapse. The expression level of our BRAFi-resistance genes is increased in relapse samples (31%, n=16).
Monitoring Syx1 transport in HEK293T cell.Figure 3A Representative images of HEK293T cells transfected with mCherry2-Syx1 or EGFP-Munc18-1. Lipophilic dye DiD (1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine) was used as the plasma membrane marker. B Representative images of HEK293T cells co-transfected with mCherry2-Syx1 and EGFP labeled WT or mutants of Munc18-1. C,D Summary graphs of the membrane distribution of Syx1 (C) and Munc18-1 WT or mutants (D) quantified by the ratio of fluorescence at the plasma membrane (PM) to the total fluorescence in the whole cell (total). DiD is the PM marker. Numbers of pictures analyzed are shown as follows: no Syx1, n = 14; no M18, n = 15; WT, n = 24; QKAA, n = 23; KKEE, n = 12; L348R, n = 22. E Munc18-1 mutants retain interaction with Syx1 shown by GST pull-down experiment. A representative gel displayed is from one of three independent replicates.
Associations between components of the retinoid signaling pathway and ATRA sensitivity.Figure 6The gene-expression microarray and RNA-seq data associated with 40 of the breast cancer cell lines were used for the analyses. The panels illustrate the associations of RARα and PPARβ/δ with ATRA sensitivity. The left panels show the basal average levels of the indicated transcript in the cell lines belonging to the T1 and T3 groups (13 cell lines in each of the T1 and T3 groups) defined by ascending ATRA scores. The intermediate and right panels indicate the same results after stratification for the Luminal (microarray and RNA-seq data = 7 cell lines in each of the T1 and T3 groups) and the Basal (microarray and RNA-seq data = 7 cell lines in each of the T1 and T3 groups) phenotype, respectively. fpkm = fragments per kilobase of exon per million fragments mapped. The basal expression levels of the indicated RAR-isoform variants were determined with the use of specific Taqman assays. The results are associated with ATRA sensitivity before (TOTAL) and after stratification of the cell lines for the Luminal and Basal phenotype as in (A).Data information: Significant P-values (Student's t-test) are indicated in red.
Figure 5A) Representation of the differential glutamylation of α- and β-tubulin, and which epitopes can be detected by the glutamylation-specific antibodies used in this study. The most prominent tubulin isotypes in brain, α1- and β2-tubulin are shown with their C-terminal tails represented as amino-acid sequences. The main modification sites, glutamates (E) 445 on α1-tubulin (Eddé et al, 1990) and E435 on β2-tubulin (Rüdiger et al, 1992) are indicated. However, all glutamate residues in these C-terminal tails (light grey circles) could potentially be glutamylated. Glutamylation generated by TTLL1 is colour-coded red, while TTLL7-catalysed glutamylation is orange. Note that under wild-type conditions, TTLL1 and TTLL7 exclusively modify α- and β-tubulin, respectively in brain tissue. B) Representation of glutamylation patterns found in the knockout mouse models studied here, and interpretation of the participation of the TTLL1, TTLL7 and CCP1 in their generation. Based on the immunoblot analysis shown in Fig. 2C, it can be deduced that TTLL1 is able to hyperglutamylate both, α- and β-tubulin in the absence of CCP1. By contrast, loss of CCP1 in the absence of TTLL1 does not lead to hyperglutamylation, suggesting that TTLL7 is not sufficient to generate this phenotype. In this context, our observation that hyperglutamylation of β-tubulin is abolished with TTLL7 indicates that while TTLL1 can hyperglutamylated β-tubulin, it requires prior glutamylation by TTLL7 to do so. We thus conclude that TTLL1 specifically elongates nascent glutamate chains generated by TTLL7.
Figure 1A. Localization of TRAPP complex components after sodium arsenite (SA) treatment in HeLa cells treated with 300 µM SA for 30 min. Fluorescence microscopy of fixed cells using antibodies against TRAPPC2 and eIF3 (to label SGs) at steady state and after SA treatment (top two rows). Other panels show localization of GFP-TRAPPC3 and endogenous TRAPPC1 to eIF3-labeled SGs and of endogenous TRAPPC9 and TRAPPC8 to G3BP-labeled SGs. DAPI (blue). Scale bar, 10 µm. B. Representative images of a time course analysis of TRAPPC2 redistribution to SGs. Cells were treated The graph (B') shows quantification of TRAPPC2 localization at SGs over time as the ratio between TRAPPC2 (mean fluorescence intensity) in SG puncta and cytosolic TRAPPC2. Mean ± s.e.m. n = 50 cells per experiment, N = 3. Scale bar, 10 µm. C. Analysis of TRAPPC2 (green), TRAPPC8 (blue) and TRAPPC9 (orange) localization at SGs upon TRAPPC2, TRAPPC4 TRAPPC8 and TRAPPC9 depletion. Mean ± s.e.m. n>100. *p<0.05; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test. D. Schematic representation of MS/MS analysis of TRAPPC2 interactors. In addition to the TRAPP components and known TRAPPC2 interactors, 53 RNA binding proteins known to associate with SGs were co-IP with TRAPPC2. Proteins that associate with TRAPPC2 only upon SA-treatment are highlighted in red. E. Analysis of TRAPPC2 localization at SGs upon CLIC1, CRYAB and ENO1 depletion. Mean ± s.e.m. n>100. ****p<0.0001; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.
Figure 6(A) Effect of the rad54∆ and rad52∆ mutations in spontaneous and UV-induced uSCE. The frequency of spontaneous and UV-induced recombinants was determined by irradiating or not cells plated onto solid medium after the corresponding dilutions (SMM and SMM without histidine to calculate total and recombinant cells, respectively). The mean and SEM of 3-5 independent fluctuation tests are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. Two and three asterisks represent p-values <0.01 and <0.001, respectively. ssDNA repair efficiency (B) of the indicated mutants. ssDNA repair efficiency was determined from the maximal and final percentages of cells with RPA foci during the time course (B, right panel). Cells were synchronized in G1, irradiated with 20 J/m2 and released into S phase for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (B, middle panel). Cell cycle progression was determined by cell sorting (B, left panel). Counting and quantification were done with the MetaMorph software. The mean and SEM of 4 independent experiments are shown. Statistically significant differences according to a paired two-tailed Student's t-test are shown, where two and three asterisks represent p-values <0.01 and <0.001, respectively. fluorescence signal at RPA foci per cell (C) of the indicated mutants. ssDNA repair efficiency was determined from the maximal and final percentages of cells with RPA foci during the time course (B, right panel). Cells were synchronized in G1, irradiated with 20 J/m2 and released into S phase for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (B, middle panel). Cell cycle progression was determined by cell sorting (B, left panel). Counting and quantification were done with the MetaMorph software. The mean and SEM of 4 independent experiments are shown. Statistically significant differences according to a paired two-tailed Student's t-test are shown, where two and three asterisks represent p-values <0.01 and <0.001, respectively. (D) Effect of the rad54∆ and rad52∆ mutations in spontaneous and UV-induced mutagenesis. The frequency of spontaneous and UV-induced forward mutagenesis at the CAN1 locus was determined by irradiating or not cells plated onto solid medium after the corresponding dilutions (SMM and SMM with canavanine to calculate total and mutant cells, respectively). Cell viability (right panel) was calculated from total irradiated cells relative to non-irradiated cells. The mean and SEM of 6 independent fluctuation tests are shown. Two asterisks represent a p-value <0.01, according to a two-way ANOVA followed by a Bonferroni test.
Transcriptome analysis of lymphatic vessels identifies FOXP2 as a collecting vessel-specific transcription factor.Figure 1Isolation of dermal EC subtypes from the ear skin of 5 weeks old Prox1-GFP mice using multicolor flow cytometry. Whole-mount immunofluorescence for LYVE1 (on the left) defines three Prox1-GFP+ lymphatic vessels subtypes that are sorted using the indicated gating scheme (on the right): LYVE1low/- collecting vessel (green arrow, col); LYVE1intermed pre-collecting vessel (grey arrow, pre-col); LYVE1high lymphatic capillary (magenta arrow, cap). Top 100 genes up-regulated in collecting in comparison to lymphatic capillary LECs. Expression in BECs is shown for comparison. Heatmap color coding shows log2 fold change. Red box indicates LEC-specific/enriched collecting vessel signature genes that include Foxp2 (red arrow). Whole-mount immunofluorescence of ear skin, mesentery and flank, showing nuclear FOXP2 staining in collecting lymphatic vessels (arrows, col), but not in LYVE1+ lymphatic capillaries (cap) or blood vessels (bv). qRT-PCR analysis of Foxp1-4 in murine ECs freshly isolated from P11 mesentery (n = 6 mice, individual data points shown). Data are presented as mean relative expression (normalized to Gapdh) ± SD. Transcript levels for each transcript are presented relative to levels in LECs. P, Student's t-test. nd, not detected.
LDM chemotherapy selectively offsets HIF-1α levels in experimental colon cancers A: HIF-1α levels in HT29 primary tumors.Figure 1A: HIF-1α levels in HT29 primary tumors. Left: vehicle-treated controls Middle/left: Doublet LDM cyclophosphamide + capecitabine (LDMCTX + LDMCPB) Middle/right: LDM cyclophosphamide + MTD capecitabine (LDMCTX + MTDCPB). Inset, high-magnification image of the region marked with a green asterisk (*). Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on HIF-1α+ area. F(5,12) = 8.791 and P = 0.001 for overall treatment by Brown-Forsythe ANOVA; *, P= 0.046 vs vehicle; #, P< 0.0001 vs LDMCTX + MTDCPB by Benjamini, Krieger and Yekuteli post hoc test. B: HIF-2α levels in HT29 primary tumors. Left: Example of immunostaining in vehicle-treated controls. Inset, high-magnification image of the region marked with a green asterisk (*). Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on HIF-2α+ area. F(5,18) = 1.215 and P = 0.3424 (not significant) for overall treatment by Brown-Forsythe ANOVA. C: CA9 levels in HT29 primary tumors. Left: Example of immunostaining in vehicle-treated controls. Inset, high-magnification image of the region marked with a green asterisk (*). Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on CA9+ area. F(5,11) = 2.466 and P = 0.0961 (not significant) for overall treatment by Brown-Forsythe ANOVA. D: Correlation between HIF-1α levels and proliferation indexes in HT29 primary tumors. Left and middle/left: HIF-1α and Ki67 expression in vehicle-treated controls; consecutive sections are shown. Middle/right: HIF-1α+ vs Ki67+ proliferation index scatterplot. Each point represents median values for each tumor. Regression line (red) and 95% CI (shaded blue area) are indicated. F(1,27) = 34.45 and P = 10-4; slope ≠ 0 by F-test Right: Machine-learning quantification of the effect of monotherapies or doublet LDM/MTD regimens upon Ki67+ proliferative index. Median indexes per tumor are indicated. F(5,12) = 1.609 and P = 0.2312 (not significant) for overall treatment by Brown-Forsythe ANOVA.
p62-dependent aggrephagy compensates for proteasomal deficiency or overload.Figure 7 Model for p62-dependent cargo nucleation and autophagic degradation of poly-ubiquitinated proteins, and its crosstalk with the UPS
Ectopic 5hmC retention is incompatible with POU5F1 chromatin association a.Figure 6Heatmap of POU5F1 bound peaks in both mESC and pre-IPSC (Cluster 1) and mESC alone (Cluster 2) with 5hmC levels during Day 0, 2 and 10 of AA+2i treatment. Metaplots of POU5F1 binding for Cluster 1 (Left) and Cluster 2 (Right) regions in pre-iPSCs and mESCs. Metaplots of 5hmC Enrichment for Cluster 1 (Left) and Cluster 2 (Right) regions for Day 0, 2 and 10 of AA+2i treatment. Left - Scheme for identifying sites that lose 5hmC and their association with POU5F1 binding. Right - Heatmap of 5hmC profile during AA+2i treatment of WT pre-iPSC and POU5F1 binding in pre-iPSCs or mESCs at the loci identified in the left panel. Representative 5hmC profiles during AA+2i conversion of WT and Kdm3b-KO pre-iPSC with POU5F1 binding in pre-iPSCs and mESCs. Loci were assigned to the nearest gene. ChIP-PCR for POU5F1 during AA+2i reprogramming of WT and Kdm3b-KO pre-iPSCs. Values are fold change of enrichment between WT and Kdm3b KO (WT/Kdm3bKO).
The limiting, degrading entity hypothesis.Figure 6The dependence of lag time on glucose pulse frequency can be explained with constitutive degradation of the limiting entity. In the model shown, the entity abundance is synthesized with each glucose pulse and depletes constitutively. Three example frequencies (f1 > f2 > f3) are shown where slight changes in period time dramatically changes the time for the entity to reach the threshold needed to engender division. When synthesis and degradation of the entity are equal, the TI feedrate is at the critical rate (f3). Arrows indicate the glucose pulse frequency
Figure 5A. Volcano plots of differentially expressed genes in pair-wise comparison of 40-day Drosophila treated without ALA administration to 40-day Drosophila treated with 0.5 mM ALA administration. Blue symbols indicate significantly downregulated mass bins (Log2 FC < -0.6 and p < 0.05), red symbols indicate significantly upregulated mass bins (Log2 FC > 0.6 and p < 0.05), and gray symbols indicate mass bins that were not significantly changed. B. KEGG pathway enrichment analysis of up- or down-regulated genes in pair-wise comparison of 40-day Drosophila treated without ALA administration to 40-day Drosophila treated with 0.5 mM ALA administration. Both adjusted p-value and gene ratio denote the significance of the respective pathway. C. Gene list of endocytosis from KEEG analysis in a pair-wise comparison of 40-day Drosophila treated without ALA administration to 40-day Drosophila treated with 0.5 mM ALA administration. D. Relative mRNA fold changes of endocytosis-related and autophagy-related genes with sorted esg-GFP+ cells of 40-day Drosophila treated without ALA administration and 40-day Drosophila treated with 0.5 mM ALA administration. Error bars indicate the SD of three independent experiments. Data information: Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.
Organization of LAMP‐2A in different regions of the lysosomal membrane.Figure 9(A) The Triton X‐114‐resistant and soluble regions of the lysosomalmembrane were subjected to centrifugation through a continuous (10-80%) sucrose density gradient, SDS-PAGE and immunoblot for LAMP‐2A. The molecular weight range of each fraction is as follows: fraction 1 (100 kDa); fraction 2 (100-200 kDa); fraction 3 (200-400 kDa); fraction 4 (400-600 kDa); fraction 5 (>600 kDa). (B) Fed rat liver lysosomes were incubated with 1 mM CaCl2 (+Ca2+) in an isotonic buffer in the absence or presence of a cocktail of protease inhibitors (PI). Samples were subjected to Triton X‐114 extraction, sucrose density gradients centrifugation and LAMP‐2A immunoblot. (C) Fed rat liver lysosomes treated or not with 25 mM MBCD were incubated in an isotonic buffer for 30 min and then subjected to SDS-PAGE and immunobloted with antibodies against the cytosolic (Top; L‐2A) and luminal (Bottom; L‐2) regions of LAMP‐2A. Arrow: LAMP‐2A proteolytic fragment (Cuervo and Dice, 2000a). (D, E) Rat liver lysosomes incubated or not with 1 mM CaCl2 (+Ca2+) were subjected to Triton X‐114 extraction and sucrose density gradients centrifugation. Levels of cathepsin A (D) and carboxypeptidase A activity (E) were measured in each fraction by immunoblot or using a standardized colorimetric assay, respectively. Values in (E) are expressed as times the activity in untreated membranes and are mean+s.e. of two experiments. (F) Hypothetical model for the distribution of LAMP‐2A in microdomains at the lysosomal membrane and its changes with CMA activity (see text for details).
Dexamethasone accelerates maturation of fetal organoids.Figure 4Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ), as revealed by real-time qPCR. Decrease in relative expression of neonatal marker A Blimp-1 and increase in expression of adult marker B Sis occur earlier in dexamethasone treated organoids Dexamethasone treatment accelerates increase in activity of sucrase maltase reaching adult organoid level ( ) at day 13 of the culture. Activity is given in µM glucose/µg protein·min-1 (n = 3 independent organoid cultures). Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ), as revealed by real-time qPCR. expression of adult marker E Treh and F Arg2 remains unchanged (n=3 individual wells from single organoid culture Dexamethasone treatment accelerates increase in activity of G trehalase and H arginase reaching adult organoid level ( ) at day 13 of the culture. Activity is given in µM glucose/µg protein·min-1 (n = 3 independent organoid cultures).
a) Vps34 overall structure with its helical domain (blue, residues 286–532) and catalytic domain (green, residues 533–873).figf2(a) Vps34 overall structure with its helical domain (blue, residues 286-532) and catalytic domain (green, residues 533-873). Regions are colored as follows: gatekeeper (red), P-loop (cyan), hinge (blue), DFG (orange), activation loop (magenta) and catalytic loop (gray). (b) Surface representation of the ATP binding site of Vps34, showing the shape complementarity between SAR405 (in sticks) and the site. (c) Description of the binding mode of SAR405, showing the main nonbonding interactions as black dashes (numbers represent distances in Å).
Figure 5(a) The workflow used to analyse ERV expression in single nuclei RNA-seq data. (b) Mean plots show the changes of TE subfamily expression in each cell cluster upon the Emx1-Cre/Trim28-KO. A differential expression analysis performed with DESeq2 (described in material and methods) showed upregulated TEs in cell types in which Trim28 was deleted (excitatory neurons, astrocytes, oligodendrocytes and oligodendrocyte precursors), (indicated with red dots: p-adj < 0.01, log2FC > 3). The specific upregulated elements and their fold changes are listed in bar graphs under each mean plot. In cell types in which Trim28 was not deleted, TE expression remained unaffected (inhibitory neurons and microglia).
Figure 1(a) Representative 2D μ-CT scans of RasV12; ctrl at day 6, RasV12; scrib-/- larvae at day 6 and day 10. Muscle (green), fat body (blue) and eye-antennal discs/tumor (red) are outlined.Scale bar: 1mm. Anterior (A), Right (R). (b) Representative 3D rendering of genotypes indicated in (a). Anterior (A), Dorsal (D), Left (L). (c) Representative 3D rendering of RasV12; scrib-/- tumors (red) and central nervous system (yellow), over time.
Figure 7In the proposed roadmap of HSPC development, Stage I refers to the differentiation of ESCs into HM/E, which required Group XII genes. The transition from HM/E, which is dependent on Group IV genes, is denoted as Stage II. Group X genes are specifically required for HSPC specification, indicated as Stage III. See text for details.
Figure 3A. Human GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase-1. The protein was cleaved in a time-dependent manner into two bands of 31 kDa (GSDMDNterm) and 22 kDa (GSDMDCterm). B. Cross-linking experiment of full-length and cleaved GSDMD. GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase-1. After enzymatic cleavage, GSDMDNterm is highly cross-linked by DSS, resulting in the gel-impenetrating species highlighted by the arrow. GSDMDCterm is not cross-linked. C. GSDMD at a concentration of 1 μM was incubated at room temperature with 5 nM caspase-1 and liposomes composed of 4 mM DMPC or polar lipid extract derived from E. coli. After 2 h, the lipid fraction (L) was separated from the supernatant (S) by ultracentrifugation at 4C for 1 h at 120,000g.
Figure 1The expression heatmap shows that all the somatic cct subunits from TRiC are up-regulated in X1 (stem cells) compared to Xins (differentiated cells) at 30dS. The colour-coded scale indicates Row Z-score of normalized TPMs (transcripts per million) values per replicate. The asterisks indicate that these genes are significant differentially expressed (q-value (FDR) < 0.01). RNAi injections schedule in starved conditions. Planarians are at 14dS when the amputation is performed (indicated by the grey cross at day 14). Live images show that cct3A(RNAi) planarians form a minimal blastema compared to controls at the time points shown. At the bottom are the number of planarians with the phenotype shown. The remaining planarians are dead by the time point of regeneration shown. dR indicates days of regeneration. RNAi injections schedule in feeding conditions. One extra feeding in respect to (B) is introduced one day prior to injections. Planarians are at 8dS when the amputation is performed. The live images show that most of the cct3A(RNAi) planarians regenerate like controls. At the bottom are the number of planarians with the phenotype shown. At 8-10dR, the remaining planarians are either dead (4/124) or have a tiny blastema (23/124) and either died or regenerated later. At 51-68dR, the remaining planarians are either dead (10/93) or have a tiny blastema and died later (5/93).
IL‐1β activates AMPK and autophagy.Figure 5(A) Protein lysates from MCF10A cells left untreated (0) or treated with 50 ng/ml TNF, 500 ng/ml TRAIL (positive control) or 10 ng/ml IL‐1β for indicated times were analysed by immunoblotting for phosphorylated ACC (P‐ACC), phosphorylated IκB (P‐IκB) and GAPDH (loading control). The values show the P‐ACC/GAPDH and P‐IkB/GAPDH ratios as percentages of the ratios in untreated cells (left lanes) and are representative of two independent experiments. (B, C) MCF10A-eGFP-LC3 cells left untreated or treated with 500 ng/ml TRAIL, 50 ng/ml TNF or 10 ng/ml IL‐1β for 24 h were analysed for LC3 translocation (B) and the DNA content (C). The values represent mean±s.d. for two independent experiments. **P‐value 0.01 and ***P‐value 0.001 as compared with untreated cells.