EMBO/sd-character-level-ner huggingface dataset:
Figure 7(A) Effect of clodronate liposomes on tumor growth inhibition induced by COMBO treatment in in mice bearing A375 tumors. Tumor growth is expressed as % change of the initial tumor. Macrophage depletion by clodronate liposome enhanced the tumor growth of tumors treated with the combination of PLX4720 and bevacizumab (n=5 mice/group), *P < 0.05, ***P < 0.001 versus vehicle (vehicle Clodronate P = 0.049, COMBO P = 5.72E-06) ŦP < 0.05 compared to COMBO (P = 0.033). (B) Representative images of macrophage infiltration determined by F4/80 immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the F4/80+ area/tumor (n=3 tumors), ***P < 0.001 versus vehicle (P = 1.53E-25).(C) Representative images of collagen deposition determined by collagen I immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the collagen I+ area/tumor (n=3 tumors). (D) Waterfall plots showing the percent change in volume (relative to the initial tumor volume) for the individual A375 xenografts in each treatment group (vehicle, PLX4720, bevacizumab and COMBO) from week 1 to week 6. (E) Representative images of macrophage infiltration determined by F4/80 immunofluorescence staining in a responder and a relapsing A375 xenografts. Bar graphs indicate the F4/80+ area/tumor (n=3 mice), ***P < 0.001 versus vehicle (P = 1.25E-06).(F) Representative images of collagen deposition determined by collagen I immunofluorescence staining in a responder and a relapsing A375 xenografts. Bar graphs indicate the collagen I+ area/tumor (n=3 mice), ***P < 0.001 versus vehicle (P = 4.03E-05). (G) Real-time quantitative PCR of h-TGFB1 of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) of relapsing tumors compared to responder tumors after normalization for housekeeping gene TBP (n=3 tumors) ***P < 0.001 versus vehicle (P = 0.0006).
Figure 3HO-1-/- mice have more ECs and less CARs (p = 0.053) in BM, while frequency of PaS does not differ. Young HO-1-/- mice have lower frequency of Sca-1high fraction among BM ECs, what resembles phenotype of BM ECs in old mice. Intracellular flow cytometry reveals (C) lower levels of SDF-1α in HO-1-/- CARs and (D) lower levels of LAP protein in HO-1-/- ECs. Analysis of cell surface SCF revealed lower frequency of SCF+ ECs in HO-1-/- while no differences among CARs. The frequency of (F) CD163+ MQs and (G) CD163- MQs in BM of HO-1-/- mice is altered.
Changes in Ca2+ signaling are independent of autophagy stimulation and occur upstream of the Atg12-Atg5 complex.Figure 4A) Representative Western-blot analysis for Atg12 (showing the autophagic Atg12-Atg5 complex), GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with (+Dox) or without (-Dox) doxycycline and treated with DMSO or 0.1, 1 or 5 µM rapamycin (Rapa) for 5 h (n = 3). B-C) Representative measurements of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca2+ as indicated. B-C) Representative measurements of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca2+ as indicated. D) Quantification of the average amplitude of the response (F−F0) (n = 3, 4, 5 and 6 for ATP-Dox, ATP+Dox, Iono-Dox and Iono+Dox, resp.) * p<0.05; ** p<0.01; *** p<0.001, repeated measurements ANOVA.
Tumorigenic potential of late passage aneuploid cells(A) Tumor growth curve after later passage ICL cells were injected into flanks of athymic nude mice (n = 5 per group and error bars denote SD, p<0.Figure 5(A) Tumor growth curve after later passage ICL cells were injected into flanks of athymic nude mice (n = 5 per group and error bars denote SD, p<0.0005 for Ch9, p<0.05 for Ch10 and p<0.005 for Ch14). (B) Tumors sections were stained with H&E to assess tumor histology (scale bars denote 5mm for the inset and 200μm for the zoomed image) Arrows in the ICL tumors sections indicate regions where tumors have invaded into the adjacent tissue. (C) Tumor growth curve after later passage ICL cells were injected into flanks of NOD/SCID mice (n = 5 per group and error bars denote SD p<0.0005 for Ch10 and p<0.005 for Ch14).
USP42 inhibits paracrine Wnt signalling in colorectal cancer cells A, USP42 alterations in colorectal adenocarcinoma (Cancer Genome Atlas, 2012) (n = 524).Figure 4A, USP42 alterations in colorectal adenocarcinoma (Cancer Genome Atlas, 2012) (n = 524). Information was retrieved from the cBioPortal in 2017, and updated as displayed in 03/2020. B, FACS analyses of cell surface LRP6 protein levels in HCT116 cells upon knockdown of USP42. Cells were untreated (left panel) or treated with RSPO3 for 12 h (right panel). The grey dotted line in both panels shows background signal upon staining with control IgGs. Representative panels from one out of n = 4 independent experiments are shown. C, Western blots of lysates from HCT116 cells transfected with the indicated siRNAs. Where indicated, cells were treated for 6 h with RSPO3 conditioned medium. Representative blots from n = 3 independent experiments are shown. D, TOPflash reporter assays in HCT116 cells upon knockdown of the indicated genes using single siRNAs. Cells were stimulated with control or Wnt3a conditioned medium. Data is displayed as mean ± SD, and show one representative of n = 3 independent experiments with three biological replicates. E,F, qPCR analysis of USP42, LGR5 and AXIN2 expression levels in HCT116 cells. Data are displayed as mean ± SD and show n = 4 (E) or n = 3 (F) independent experiments.
Figure 1(A) Photos of forepaws from WT and RDEB mice of the indicated ages. In RDEB mice dermal fibrosis is apparent by fusion of digits and formation of mutilating deformities. (B) The stiffness of back skin and forepaw palms from 10-wk-old WT and RDEB mice was measured with a micro-indentation stiffness measurement tool. N = 3 mice per genotype and 5-7 measurements per mouse. Individual data points, mean ± S.E.M. are shown, P = 0.0348 (back skin) and P < 0.0001 (forepaws) tested with unpaired t-test. (C) Hierarchical clustering of forepaw samples of the indicated ages and genotypes based on protein abundances determined by label-free quantification MS analysis. Limited differences are seen between age-matched WT and RDEB mice, most notable differences are between ages. (D) Fuzzy c means clustering of significantly altered proteins in paws of RDEB compared to WT mice to visualize their dynamic regulation during disease progression. Average abundance differences of three biological replicates each (RDEB/WT) were log2 transformed and standardized. Shown are relative abundance changes over time (0, 4 and 10 weeks). Six clusters were generated and each cluster was analyzed for enrichment of pathways and processes using STRING DB Representative pathways are listed for each cluster. The number of proteins belonging to each pathway is indicated in brackets.
Klp5/Klp6 accumulates behind Tea2 at the plus end until just prior to microtubule catastrophe.Figure 4 (A) Kymograph (top left panels) showing fluorescently-tagged Klp5/Klp6 (Kinesin) co-imaged with fluorescently-tagged Tip1 (Tip1-Tdtom). Dashed yellow line indicates the cell end. Plots (bottom left panels) show both the fluorescence intensity and position of Klp5/Klp6 (green) and Tip1 (magenta) corresponding to the numbered sections of the kymograph. Scale bar, 2µm. Intensity of the maximal value pixel at the plus end (top right panel) and its position relative to the cell end (bottom right panel) for both Klp5/Klp6 (green) and Tip1 (magenta) plotted relative to MT shrinkage for this kymograph  (B) Data quantitated from multiple kymographs (n=7) of the type illustrated in (A) and Fig EV4 to display Klp5/Klp6 and Tip1 levels at the MT plus end (top plot) and the position of Klp5/Klp6 relative to Tip1 (bottom plot) before MT shrinkage  (C) Data quantitated from multiple kymographs (n=8) of the type illustrated in Fig EV5 where fluorescently-tagged Klp5/Klp6 is co-imaged with fluorescently-tagged Tip1 in the absence of Mcp1 and presented as in (B)
Model of lysosomal sensing and lysosome‐to‐nucleus signalling by TFEB and mTOR.Figure 7(A) (Left) Under full nutrients and in the absence of lysosomal stress, the complex formed by v‐ATPase, Ragulator, and Rag GTPases is in the active state and recruits mTORC1 to the lysosomal surface, where mTORC1 becomes activated. At the lysosome, mTORC1 binds and phosphorylates TFEB, which cycles between the cytoplasm and the lysosomal surface. Phosphorylation by mTORC1 maintains TFEB in the cytoplasm and prevents it from translocating to the nucleus. (Right) Starvation, v‐ATPase inhibition, or lysosomal stress switch the Rags off, leading to mTORC1 detachment from the lysosome and to its inactivation. TFEB can no longer be phosphorylated and translocates to the nucleus, where it activates gene expression programs that boost lysosomal function and autophagy. (B) Side‐by‐side diagrams of a healthy cell and a starved/stressed cell, showing the respective distribution of mTORC1 and TFEB in relationship to lysosomes, cytoplasm, and nucleus.
ATF7IP may have a role in SETDB1 import in HEK293T cells A ATF7IP KO HEK293T cells shows endogenous SETDB1 cytoplasmic localization phenotype.Figure 3A ATF7IP KO HEK293T cells shows endogenous SETDB1 cytoplasmic localization phenotype. Endogenous SETDB1 in HEK293T cells shows cytoplasm+nuclear localization profile. Scale bar: 10 µm. B Over-production of exogenous mouse SETDB1 induces endogenous SETDB1 nuclear localization in both WT and ATF7IP KO HEK293T cells. The representative data of two independent experiments is shown. Scale bar: 10 µm. C LMB treatment enhances SETDB1 nuclear accumulation in WT HEK293T cells, but no significant impact for ATF7IP KO HEK293T cells. Cells were treated with 10ng/ml of LMB for 5 h and then analysed with immunofluorescent staining. The representative data of three independent experiments is shown. Scale bar: 10 µm. D SETDB1 signal in the nucleus shown in C was calculated. The mean from three independent experiments is shown as a bar-graph ± SEM; n=3. Over 50 cells were analysed per sample per experiment. **P < 0.01 by Student's t-test (two-sided test). E 5 h of proteasome inhibitor bortezomib treatment do not have much impact on SETDB1 relative nuclear accumulation in ATF7IP KO HEK293T cells. The representative data of three independent experiments is shown. Scale bar: 10 µm. F SETDB1 signal in the nucleus shown in E was calculated. The mean from three independent experiments is shown as a bar-graph ± SEM; n=3. Over 50 cells were analysed per sample per experiment. Statistics comparison was only shown between without and with drug treatment in WT and ATF7IP KO cells. *P < 0.05 and **P < 0.01 by Dunnett's method.
Figure 4A. Diagram explaining how codon/amino acid expression bias was computed: correlation was calculated between the frequency of each codon/amino acid and mRNA levels per gene. Orange means correlation and purple anti-correlation. B. Heatmap showing the codon expression bias across the transcriptome for different developmental time points (2, 6, 12, 24 and 48 hpf) and adult organs in zebrafish, and Xenopus at 48h (right). The codon optimality, and CSC calculated during embryogenesis is shown to the right (red, optimal to blue, non-optimal). Heatmap shows the correlation of codon occurrence and Xenopus mRNA expression at 48 h. On the left a heat map (Red-Blue) shows the CSC values for zebrafish and Xenopus. The codons are sorted by their CSC values. For most tissues, optimal codons are correlated with highly expressed genes (orange), while non-optimal codons are anticorrelated with high levels of expression (purple). We also observe a correlation between mRNA expression and the CSC across different samples (2h P= 1e-07 R=0.6, 6h P= 2e-16 R=0.7, 12h P= 3e-09 R=0.6, 24h P= 2e-07 R=0.6, 48h P= 1e-07 R=0.6, Brain P= 1e-04 R=0.4, TestisP= 1e-07 R=0.6, Liver P= 1e-03 R=0.4, Spearman rank test). C. Heatmap of the codon composition bias showing the correlation between the CSC in zebrafish (left) with gene expression across 32 human tissues. The codon optimality is labeled as in (B). D. Biplots of the reporter amino acid stabilizing index (rASI) comparing 8h vs 8h+MO and amino acid stabilization coefficient (ASC) in Zebrafishembryos, P < 6e-3 R=0.6, Spearman rank correlation. Based on the consistent identification by both methods we defined: Optimal amino acids, highlighted in red and non-optimal amino acids in blue. E. Heatmap showing the amino bias across the transcriptome for different developmental time points (2, 6, 12, 24 and 48 hpf) and adult organs in zebrafish, and Xenopus at 48h (right). The optimal and non-optimal amino acid are shown to the right (red, optimal to blue, non-optimal).
Uncoupling of neurodegeneration and overgrowth.Figure 6(A) Regression analysis of the overgrowth versus the neurodegeneration observed in loss of function mutants (left) and overexpression of transgenes (right). Overgrowth quantification is shown in Supplementary Figure 14. For loss of function mutant, the neurodegeneration was quantified at 14 days in clones (as shown in Figures 4 and 5). For UAS‐overexpression transgenes neurodegeneration was quantified at 14 days when expressed with GMRGal4; UbiGal80ts. For CycD+Cdk4, these specific data are missing and have not been plotted; however, Supplementary Figure 13 shows that these mutants do not cause neurodegeneration in a different setup in which they are expressed during both development and adult life. In both cases the regression coefficient r2 is extremely low (0.21 and 0.03), indicating an absence of correlation between the two processes. (B) Tangential eye sections through the eyes of flies expressing with GMR-Gal4, Ubi-Gal80ts either UAS-EGFP or UAS-Yki or UAS-YkiS111A,S168A,S250A and aged 1 or 28 days. Arrows point at missing or degenerated photoreceptors. The UAS-YkiS111A,S168A,S250A is so effective that even the very low expression leaking out with this system is enough to affect development and generate mild eye roughness and overproliferation of lattice cells. Histograms showing the quantification of cell loss in eyes shown in A and also eyes from flies aged at the intermediate 14‐day stage. Mild but significant degeneration is observed for both Yki forms, with respect to the negative control; however, no statistical difference is observed between the two Yki proteins that have dramatically different effects on overgrowth. ***P0.001, **P0.01; *P0.05 in two‐tailed t‐test.
Figure 3A. Inhibition of p38 or JNK does not block osmotic stress-induced YAP nuclear localization. HEK293A cells were pretreated with 2 μM p38 inhibitor (SB203580) or 20 μM JNK inhibitor (SP600125) before treatment with 0.2 M sorbitol for 1 hr in the absence of serum. Quantification of YAP/TAZ subcellular staining is shown. Scale bar: 20 μm. A. Inhibition of p38 or JNK does not block osmotic stress-induced YAP nuclear localization. HEK293A cells were pretreated with 2 μM p38 inhibitor (SB203580) or 20 μM JNK inhibitor (SP600125) before treatment with 0.2 M sorbitol for 1 hr in the absence of serum. Cell lysates from identically treated samples were examined for phosphorylation of p38 substrate MK-2 and JNK substrate c-Jun. B. NLK knockout blocks osmotic stress-induced YAP nuclear localization. HEK293A cells were transiently transfected with CRISPR/Cas9 to knockout NLK. Wild type (WT) cells and the NLK KO cell pool were treated with 0.2 M sorbitol for 1 hr in the absence of serum. YAP/TAZ subcellular localization was determined by immunofluorescence staining. Scale bar: 20 μm. C. NLK induces YAP nuclear translocation. HEK293A cells were co-transfected with Flag-YAP together with vector control, Myc-NLK-WT (wild type NLK), or NLK-KN (kinase negative mutant). Cells were serum starved for 6 hr, and YAP localization and NLK expression were determined by Flag (green) and Myc (red) antibodies, respectively. Scale bar: 20 μm. D. NLK induces YAP phosphorylation. HA-YAP was co-transfected with NLK-WT or NLK-KN in HEK293A cells. The phos-tag gel showed NLK-WT but not NLK-KN caused a significant mobility shift of YAP. E. NLK phosphorylates YAP in vitro. NLK-WT and NLK-KN were immunoprecipitated from HEK293A cells and an in vitro kinase assay was performed using recombinant GST-YAP as a substrate in the presence of ATP-γ-S. Total phosphorylation of YAP was detected by immunoblotting with thiophosphate ester antibody, which identifies the alkylated thiophosphorylation on YAP.
Figure 7(A) Sequence similarities of MHR in HIV-1 (NL4-3), SIVmac239, and MuLV with the core SxIP-binding motif (bold text) within the EBH domain of EB1 from different species, adapted from (Honnappa et al., 2009). Residues identical to those in EB1 are highlighted in red, similar residues are printed in red. (B) Schematic representation of HA-tagged GAPDH constructs, with MHR from HIV-1 (GAPDH-MHRhiv), SIVmac239 (GAPDH-MHRsiv), or MuLV (GAPDH-MHRmulv). (C) Anti-HA co-IP in CHME3 expressing GAPDH-MHRhiv, GAPDH-MHRsiv or GAPDH-MHRmulv showing binding to endogenous CLIP170. Results are representative of three experimental replicates. Anti-HA co-IP in Rhesus macaque FRhK4 (D) cells expressing GAPDH alongside GAPDH-MHRsiv Anti-HA co-IP in rat fibroblast Rat2 (E) cells expressing GAPDH alongside GAPDH-MHRmulv Anti-EB1 co-IP was included as positive control to demonstrate CLIP170 binding by EB1 in Rat2 cells. Results are representative of three experimental replicates. FRhK4 cells infected with SIV-VSV-luc (F) stained for Ac-MTs, Tyr-MTs, and the nucleus (Hoechst) at 6 h.p.i Rat2 cells infected with MuLV-VSV-luc (G) stained for Ac-MTs, Tyr-MTs, and the nucleus (Hoechst) at 6 h.p.i, FRhK4 cells infected with SIV-VSV-luc analyzed by WB Rat2 cells infected with MuLV-VSV-luc analyzed by WB Measurements of SIV-VSV-luc infectivity in FRhK4 (J) cells depleted of CLIP170. Statistical significance was determined by t test. *** = p<0.001. Data are mean values from two independent experiments +/- SEM. Measurements of MuLV-VSV-luc infectivity in Rat2 (K) cells depleted of CLIP170. (L) 293T transfected with GAPDH-MHRhiv or R162Q mutant (GAPDH-MHRhivQ) were subjected to anti-HA co-IP. Representative WB analysis is shown (n = 2). Molecular weight markers (in kDa) are shown to the right of WBs.
Figure 7mESCs expressing arginine methylation-deficient mutant (RK) METTL14 are sensitive to ICL damage induced by MMC. WT, Mettl14 KO, KO+WT, and KO+RK mESCs were treated with various concentrations of MMC for 4 days before cell viability was measured.   Similar to (A), except that mESCs were treated with cisplatin, another ICL-inducing chemical, at various concentrations.   In both (A) and (B), each point represents the average of three independent replicates and error bars represent standard deviation (SD). Statistical analysis was performed using Student's t-test. *, p < 0.05. Proposed model for METTL14 C-terminal IDR arginine methylation-mediated regulation of m6A RNA modification and its effects on ICL DNA repair. PRMT1-mediated arginine methylation of the C-terminus IDR of METTL14 promotes its interactions with RNA substrates and RNAPII, which enables efficient m6A deposition on transcripts involved in ICL repair. The deposition of m6A enhances the translation efficiency of these DNA repair genes, promoting the recovery of mESCs from DNA damage. Inhibiting METTL14 arginine methylation using the type I PRMT inhibitor MS023 reduces m6A deposition and the protein synthesis of ICL repair genes, thus sensitizing mESCs to DNA damage-induced cell death.
Figure 2A. Immunofluorescence microscopy of control, parental and patient fibroblasts labeled for LAMP-1. Patient fibroblasts (VPS41S285P/R662* and VPS41c.1423-2A>G/R662*) show more LAMP-1 puncta, which are distributed throughout the cell. (A') Quantification shows a significant increase in the number of LAMP-1 positive compartments for both patients. >15 Cells per condition were quantified (n=3). Scale bars, 10µm. B. HeLa VPS41 knockout (HeLaVPS41KO) cells made using CRISPR/Cas9 methodology. Western blot analysis confirms a full knockout. The same HeLaWT and HeLaVPS41KO samples were analyzed in Appendix Figure S8C, showing the same actin control. C. LAMP-1 immunofluorescence of HeLaWT and HeLaVPS41KO cells. Similar to patient derived fibroblasts more LAMP-1 positive compartments are seen in HeLaVPS41KO cells (quantified in C'). >10 cells per cell line per experiment were quantified (n=3). Scale bars, 10µm. D. Immuno-electron microscopy of VPS41WT/WT and VPS41S285P/R662* fibroblasts incubated for 2 hours with BSA conjugated to 5 nm gold (BSA-Au5) and labeled for LAMP-1 (10 nm gold particles). Lysosomes are recognized by the presence of degraded, electron-dense material. LE = late endosome, LY= lysosome, PM = plasma membrane. Scale bar, 200nm. E. Morphometrical analysis showing that lysosomes in patient fibroblasts are significantly smaller than in control and parental cells. >100 Randomly selected lysosomes per condition were quantified. F. Relative labeling density of LAMP-1. Number of LAMP-1 gold particles per lysosome was divided by the number of grid intersections, representing lysosomal size. No significant difference was found between VPS41WT/WT, VPS41WT/S285P, VPS41WT/R662* or VPS41S285P/R662* fibroblasts. >53 Lysosomes per condition were quantified. G. Quantitation of LAMP-1 gold particles per lysosome. VPS41S285P/R662* lysosomes have significantly less LAMP-1 then control and parental cells. >53 Lysosomes per condition were quantified. Similar results were obtained for LAMP-2 (Appendix Fig S2).
CLRC promotes the shelterin-chromatin association.Figure 1A Scheme of the shelterin complex and CLRC at telomeres B Mass spectrometry analysis of proteins co-purified with Ccq1HA. Shown is a volcano plot for proteins significantly enriched in Ccq1HA relative to untagged Ccq1 (left panel: WT cells; right panel: rik1∆ cells). Members of the CLRC complex and shelterin are highlighted in red and blue, respectively. Bottom panel displays proteins enriched (log2 ≥ 5 or pval ≤ 0.01) ChIP-qPCR analysis of epitope-tagged shelterin components in WT and mutant strains as indicated (negative control: untagged strain). Positions on x-axis denote distances relative to telomeric repeats (telomere-proximal PCR primer) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEM ChIP-qPCR analysis of epitope-tagged shelterin components in WT and mutant strains as indicated (negative control: untagged strain). Positions on x-axis denote distances relative to telomeric repeats (telomere-proximal PCR primer) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEM ChIP-qPCR analysis of epitope-tagged shelterin components in WT and mutant strains as indicated (negative control: untagged strain). Positions on x-axis denote distances relative to telomeric repeats (telomere-proximal PCR primer) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEM F ChIP analysis of Ccq1HA and FLAG-TetR-Clr4* (see scheme). Expression and tethering of FLAG-TetR-Clr4* are controlled by thiamine/AHT addition (red dots indicate induced/tethered FLAG-TetR-Clr4*; empty circles indicate non-induced/non-bound controls) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEM