EMBO/sd-character-level-ner huggingface dataset:
Figure 5A. DC domain binding footprints (residues within 4 Å distance) on MT lattice at GTP/GDP.Pi and GDP states. Interface areas were calculated using PDBePISA B. Differences between NDC binding sites in GTP/GDP.Pi- and GDP-MT lattices. Deviations in backbone (Cα) atom positions are illustrated with colour-coded spheres. C. Top, Binding of CDC and NDC at the vertex of 4 bent tubulin dimers of different MT nucleotide states with close-up cutaway views. Bottom, Superposition of CDC X-ray structure 5IP4 (darkened and transparent) with the GTP/GDP.Pi-MT-binding conformation (this study; bright yellow) and superposition of 5 NMR and X-ray NDC structures from Protein Data Bank (PDBs: 5IO9, 1MJD, 2BQQ, 5IKC, 5IN7) with the GDP-MT-binding conformation (this study).
Figure 4A) Interaction between Pvt1 and MYC was determined by immunoprecipitation followed with RT-PCR. Note significant decrease in Pvt1 and MYC interaction upon FTO treatment (left panel), calcium shift-induced differentiation (middle panel, Hi: high calcium), and Pvt1 methylation site mutations (right panel). n=3, P<0.01 (Student's t-test). Error bar represents S.D. B) Immunoblots show decreased MYC protein level in differentiated keratinocytes and Mettl14 KO cells. Undifferentiated WT and Mettl14 KO cells (C) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated. undifferentiated WT and Pvt1 KO cells (D) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated. WT cells before and after calcium shift-induced differentiation (E) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated. (F) Skin organoids derived from Mettl14 KO cells or KO cells rescued with exogenous expression of MYC were grafted to nude mice. The regenerated skin was analyzed by H/E staining. (G) Epidermal thickness of KO and KO skin rescued with MYC expression was quantified and presented as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=18, P<0.01 (Student's t-test). (H) Number of p63-positive cells in KO and KO skin rescued with MYC expression was quantified and shown as bar graph. n=5, P<0.01 (Student's t-test). Error bar represents S.D. (I) Fluorescence microscopy demonstrates different survival capability of WT and Mettl14 KO cells with inducible expression of MYC. (J) Ratio of WT and Mettl14 KO cells with inducible expression of MYC in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells without Dox treatment (no MYC expression) compared with WT cells or KO cells with Dox stimulation (exogenous MYC expression) at both Day 7 and 14. Error bar represents S.D.
Figure 4A, Intravitreal injection of LIF increases blood vessel density in the mouse eyes. Adult mice were intravitreally injected with VEGF (10 ng) or LIF (10 ng to 100 ng, Sigma). Seven days after injection, PFA-fixed choroid-sclera complexes and retina were subjected to CD31 IF. Representative images of CD31-positive vessels are shown in A. Scale bar = 100 µm.Vascular B) Intravitreal injection of LIF increases blood vessel density in the mouse eyes. Adult mice were intravitreally injected with VEGF (10 ng) or LIF (10 ng to 100 ng, Sigma). m.Vascular density, determined with ImageJ software, is shown in B), n=5-8. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. C, D) OCT-A imaging of LIF-treated mouse retina. Adult mice were intravitreally injected with 1 μl of LIF (50 ng, Sigma) or vehicle solution (PBS). Retinal OCT-A images were obtained 7 days after the injection and representative images are shown. Blood vessel density was determined as percentage of vessel-covered area/total area surface using ImageJ software and shown in D), n=7-8. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. E). STAT3 was phosphorylated in retinal ECs after LIF treatment. 50 ng of our in-house LIF with low-endotoxin levels was injected. Phospho-Stat3 was examined on cryosection of retina after 2 hour by IHC. Magnification in the dashed box is shown in the lower panel. n = 5. Scale bar = 100 µm. F). Five-day old neonatal mice were intravitreally injected with LIF (50 ng) or vehicle solution (PBS). After 3 days, retinas were subjected to IF staining with Dyight-488-labeled lectin. Representative images for similar ocular loci and quantification of lectin-labeled area using ImageJ software were shown, n=4. Scale bar = 100 µm. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.
Figure 7(A) Scheme with the rationale of the ChEC approach for non-DSB lesions. Rad6 fused to MNaseI will induce a detectable cut only if it is bound to a DNA lesion other than a DSB. (B) Rad6 binds to DNA in the absence and presence of MMS. ChEC analysis of G1-synchronyzed cells released in the absence or the presence of 0.033% MMS for the indicated times. Data information: DNA gel electrophoresis, DNA content (cell sorting) and quantification of DNA digestion profiles are shown. The experiments were repeated twice with similar results. (C-F) Rad6 binding to DNA depends on Rad18, Rad52 (C), Rad51 (D), Rad57 (E) but not Rad54 (F) as determined by ChEC analyses of asynchronous cell cultures without or with 0.05% MMS for 2 hours. Data information: DNA gel electrophoresis, DNA content (cell sorting) and quantification of DNA digestion profiles are shown. The experiments were repeated twice with similar results.
Figure 6B. Schematics depicting the rationale for designing a BRCT-Sgs1 chimera. The BRCT3/4-Sgs1 fusion protein is expected to engage at DNA lesions via the 9-1-1 clamp. For simplicity, the ssDNA-binding heterotrimer RPA and the obligatory Mec1 cofactor Ddc2 have been omitted from the diagram. Dilution assay of wild-type, ddc1Δ, or ddc1-T602A cells expressing BRCT3/4-SGS1 in the presence of 0.005% MMS. Immunoblot of Rad53 mobility shift assay in cells treated with 0.02% MMS expressing either DPB11BRCT3/4 alone or the BRCT3/4-SGS1 chimera. Dilution assay of wild-type, ddc1Δ, or ddc1-T602A cells expressing BRCT3/4-SGS1 in the presence of 0.005% MMS. Dilution assay of wild-type or rev3Δ cells expressing BRCT3/4-SGS1 in the presence of 0.0025% MMS.
LKB1 and CaMKKβ do not have an important function in TRAIL‐induced AMPK activation and autophagy.Figure 3(A) MCF10A-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs (top) and proteins (bottom by RT-PCR and immunoblotting, respectively. Similar results were obtained in three independent experiments. (B) MCF10A-eGFP-LC3 cells transfected with indicated siRNAs for 48 h, and left untreated or treated with 500 ng/ml TRAIL for 2 h, 10 μM ionomycin for 24 h or starved for amino acids and glucose for 24 h (starvation) were analysed for P‐ACC and tubulin (loading control) expression by immunoblotting. Similar results were obtained in two independent experiments. (C) Cells transfected as in (B) and left untreated or treated with 500 ng/ml TRAIL, 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation. The values represent mean±s.d. of three independent experiments. ***P‐value 0.001 as compared with cells treated in a same way, but transfected without siRNA.
Figure 5(A) Comparison of RNA isoforms and sRNA patterns generated by 35S:GFP-GUS and 35S:GFP-EVDint/ter-GUS. High and low molecular-weight RNA analysis using a GFP or GUS probe in two independent transgenic lines from each construct in the WT or rdr6 background. mRNA isoforms are indicated with arrows and correspond to the transcripts depicted in Fig.2A. EtBr staining of the agarose gel and miR171 probe serve as loading control for mRNAs and sRNAs, respectively. (B). Western blot analysis of the translation products from GFP and GFP-GUS transcripts. Coomassie (coom.) staining as a loading control. Black arrow: GFP-GUS fusion protein; white arrow: GFP protein. (C) Nucleo-cytosolic distribution of 35S:EVD and 35S:GFP-EVDint/ter-GUS RNA isoforms in rdr6 relative to that of ACT2 analyzed by qPCR. RNA extracted from Total, nuclear (Nucl) and cytoplasmic (Cyto) fractions was reverse transcribed with random hexamers and oligo(dT). snoRNA U5 is shown as a nuclear-only RNA control. (D) Same as in (C) but using exclusively oligo(dT) to reverse transcribe poly(A)+ RNAs.
Figure 1A-C. Immunodetection of BES1-CFP from heat-treated plants. Plants pre-grown at 21°C were (A) exposed for 60 minutes to the indicated temperatures, (B) to 45°C for the indicated period of time, or (C) to 45°C and, following sampling after 30 minutes (lane 2) and 60 minutes (lane 3), returned to 21°C for 3 hours (lane 4). CBB, Coomassie Brilliant Blue stained gel. D. Subcellular localization of BES1-CFP in roots of 7-day-old plants after heat shock. 35S:BES1-CFP seedlings were grown at 21°C and either left untreated or exposed for 60 minutes to 45°C. Left: representative photos. Right: quantification of the nuclear/cytoplasmic signal. Data show the mean ±SD. n=25. The asterisks indicate significant differences to the untreated plants by Student's t-test (***P≤0.001). E. Expression of the BES1-repressed BR biosynthetic genes ROT3, DWF4 and BR6ox2 (the basic regulatory pathway is illustrated) after a heat shock of 45°C. Plants were treated as in C, samples were harvested and qPCR analyses were performed. Data show the mean ±SD. n=4 biological repeats, each measured in 3 technical replicates, normalized to UBC. The asterisks indicate significant differences compared to the control condition at 21 °C by Student's t-test (*P≤0.05; **P≤0.01; ***P≤0.001). F. Heat stress resistance of bes1-1, bes1-2 and bes1-D as compared to wild-type. For basal resistance assays (top charts) plants were directly exposed to 45°C for the indicated time. For acquired resistance (bottom charts), plants were primed with the respective temperatures for 120 minutes before transfer to 45°C for 150 minutes. Data show the mean ±SE. n=7. The asterisks indicate significant differences compared to wild-type by Student's t-test (*P≤0.05; **P≤0.01; ***P≤0.001).
Figure 3A. Immunofluorescence of FLAG (red) and F-actin (gray) in oocytes after microinjection of negative control or mouse Mos siRNAs combined with or without wild-type human MOS or MOS variants mRNAs and culture in medium with 2.5 µM milrinone for 24 h, followed by release to maturation (n=15-20 oocytes each group). Scale bar= 10 µm. B. The relative intensities of F-actin were measured and compared among the indicated groups. The oocyte numbers for analyzed were labeled in each group. The box plot showing the F-actin intensities distribution in each group quantified using Image J (n=10-19/group). C. The immunofluorescence of F-actin (red) and TPX2 (green) in WT and Erk1/2oo-/- oocytes, using DAPI (blue) co-staining (n=15-20 oocytes for each group). The zoomed images of F-actin are displayed in gray. Scale bar=10 µm. D. The box plot summarizing the relative intensities of F-actin between control and ERK1/2oo-/- oocytes (n=7-10 oocytes for each group). E. Representative bright field images showing the morphology of oocytes and embryos from wildtype and ERK1/2oo-/- mice. The MII oocyte, zygote, 2-cell and blastocyst embryos were harvested in vivo at 14, 24, 48, and 96 h post-hCG, respectively (n=3 mice for each timepoints in two groups). Scale bar=100 µm. F, G. Bar graphs shows the blastocyst percentage (F) and fragment percentage (G) in wild-type (n=51) and maternal ERK1/2 deletion embryos (n=63).
Figure 4Workflow of the SILAC-based quantitative mass spectrometry method for identifying Dpb11 interactions. Identification of Dpb11 interacting proteins in wild-type cells using workflow shown in (A). DPB11-3xHA was overexpressed using the ADH1 promoter. P value calculated using Mann-Whitney u-test. C. Identification of Dpb11 interacting proteins in rad9∆ cells using workflow shown in a. DPB11-3xHA was overexpressed using the ADH1 promoter. P value calculated using Mann-Whitney u-test. Immunoblot showing co-immunoprecipitation between Dpb11 and Sgs1. DPB11-3xHA was overexpressed from an ADH1 promoter and SGS1-13xMYC was expressed from its endogenous locus. Schematic of the Sgs1 N-terminal truncation used in this study. Immunoblot showing co-immunoprecipitation of Dpb11 with Sgs1. Both DPB11-3xHA and SGS11-647-5xFLAG were expressed at their endogenous loci. Hypothetical depiction of the Dpb11-STR complex engaged at 5' recessed ends. See text for details. For simplicity, the ssDNA-binding heterotrimer RPA and the obligatory Mec1 cofactor Ddc2 have been omitted from the diagram.
mtON reverses mitochondrial dysfunction.Figure 3Day 1 adult animals expressing mtON were exposed to 50 µM rotenone (ETC complex I inhibitor) for 5 hours and survival was scored. Illumination was continuous throughout toxin exposure (see methods). ATR alone was protective (-ATR, -light vs. +ATR, -light p = 0.016.) The effect of mtON activation was greater than the ATR alone effect, one-way ANOVA with Tukey's post hoc test *p = 0.01. (-ATR, -light vs +ATR, +light p = 0.0002. -ATR, +light vs. +ATR, +light p = 0.0009. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 50 µM antimycin A (complex III inhibitor) and survival was scored 18 hours later. One-way ANOVA with Tukey's post hoc test *p = 0.02 (-ATR, -light vs +ATR, +light p = 0.03, -ATR, +light vs. +ATR, +light p = 0.01. n = 5 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 0.25 M azide (complex IV inhibitor) for 1 hour and scored for survival 1 hour after recovering, one-way ANOVA with Tukey's post hoc test *p = 0.0002. (-ATR, -light vs +ATR, +light p < 0.0001, -ATR, +light vs. +ATR, +light p <0.0001. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 31 µM oligomycin A (ATP synthase inhibitor) and survival was scored 18 hours later. No significant differences were found by one-way ANOVA (n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals in a complex I mutant background (gas-1) were scored for locomotion speed in the presence of food by counting body bends per minute. mtON activation rescued the decreased locomotion of the gas-1 mutant background, one-way ANOVA with Tukey's post hoc test, -ATR, +light vs. +ATR, +light *p = 0.0145, +ATR, +light vs. wildtype (WT) *p = 0.0008 (-ATR, -light vs +ATR, +light p = 0.0272, +ATR, -light vs. +ATR, +light p = 0.0332, all conditions vs WT, p < 0.001, n = 30, 30,30,41,41 animals each bar from left to right). Data show mean ± standard deviation.
Figure 4(A) Representation of the SNARE complex with Asp68 highlighted (top). BET1Asp68 is involved in a hydrogen bond with SEC22bLys169, with the most probable conformation of His68 in the mutant variant (bottom). (B) Stability of hydrogen bonds between BET1Asp68 and BET1Asp72 respectively was estimated using molecular dynamics simulation in three replicas. Parameter occupancy could be more than 100% if several stable hydrogen bonds could be established by residue. Bars represent average fraction of the simulated time the residue is involved in a hydrogen bond(s). Data represented are mean ± SD. (C) Sequence conservation of the corresponding regions of BET1 and SEC22b animal proteins. The relevant residues involved in hydrogen bond network are highlighted by color, hydrogen bonds are shown. (D) Representation of the SNARE complex with Ile51 (upper) and Ser51 (lower) residues highlighted. (E) Sequence conservation of the corresponding regions of BET1 and SEC22b and the relevant residues involved in the hydrophobic patch formed by Ile51 BET and SEC22bI148.
Figure 11At early night, wild-type (A) and 5X (B) TOC1 are equally present and recruit transcriptional repressors NF-YB/C and HDA15 with PRR5 (not shown) to inhibit expression of PHGs. As night progresses, PIF3 levels rise, and the advanced phase of 5X, more rapid degradation and poorer PIF3 interaction result in lesser presence of 5X at target promoters, relative to wild-type TOC1, leading to attenuated inhibition of PHGs and enhanced hypocotyl growth. At late night, low TOC1 and PRR5 levels reduce NF-YB/C and HDA15 recruitment, resulting in PIF3 release to activate downstream PHGs that leads to maximal pre-dawn hypocotyl growth.
Figure 4(A) Yeast two-hybrid mating screen showing interaction of BspF with a fragment of ACAP1 (amino-acid residues 460-740; ACAP1Lib) or full-length ACAP1 (ACAP1FL), compared to positive (p53/T antigen) and negative (empty vectors) control matings plated on permissive double dropout (DDO) or selective quadruple dropout (QDO) media. ACAP1 schematic indicates the region of interaction initially identified (ACAP1Lib). (B) Representative co-immunoprecipitations of HA-BspF and myc-ACAP1 in HeLa cells. HeLa cells were transfected to either co-produce or individually produce HA-BspF and myc-ACAP1 and either HA-BspF or myc-ACAP1 were immunoprecipitated using either anti-HA-conjugated (upper panel) or anti-myc-conjugated (lower panel) magnetic beads following cross-linking (+DSP) or not (-DSP) with dithiobis[succinimidylpropionate]. Input lysates (10% of the post-nuclear supernatant) and co-immunoprecipitates were separated by SDS-PAGE and probed for HA-BspF and myc-ACAP1 by Western blotting. (C) Representative confocal micrographs of HeLa cells transfected to produce mCherry-BspF and GFP-ACAP1 and treated with Cytochalasin D (200 nM) for 30 min and quantification of colocalization between mCherry-BspF and GFP-ACAP1. Arrows indicate areas of BspF and ACAP1 colocalization. Scale bars: 10 µm and 2 µm (insets). Data are means ± SD from n=3 independent experiments in which 10 cells were analyzed per experiment. Pearson's correlation coefficients were calculated from whole cells using NIH Fiji image analysis software and Coloc_2 plug-in.
Figure 4HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD44hiCD62Lhi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl or Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and monitored for tumour growth. TILs isolated from HER-2-E0771 mammary tumour versus lymphocytes isolated from the contralateral mammary fat pad were analysed for c) CD11b+ F4/80hiLy6ChiLy6Glo myeloid derived suppressor cells (MDSC) by flow cytometry. TILs isolated from HER-2-E0771 mammary tumour versus lymphocytes isolated from the contralateral mammary fat pad were analysed for CD4+CD25+FoxP3+ regulatory T cells (Tregs) by flow cytometry. Tgfb and Il10 mRNA levels in HER-2-E0771 tumours and mammary fat pads were assessed by quantitative real time PCR. HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. f) 13 or g) six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD44hiCD62Lhi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl splenocytes. Mice were injected with f) IL-2 (50,000 IU/day) or g) saline on days 0-4 after adoptive CAR-T cell transfer and monitored for tumour growth.
Figure 4(A) Volcano plots of common differentially expressed genes in shScr and Zfp207-depleted (sh1 and sh2) mouse ESCs. Up-regulated (Up) and down-regulated (Down) genes are indicated in red and blue, respectively (p < 0.05; > 1.5-fold). Zfp207 is depicted in black. Grey dots indicate non-significant (NS) and < 1.5-fold differential expressed genes. (B) Venn diagram depicting the overlap of down-regulated and up-regulated genes between sh1 and sh2 ESCs (FC >1.5 and p < 0.05). FDR value was calculated with the Benjamini-Hochberg correction Gene ontology (GO) analysis of biological processes associated with common (C) down-regulated genes in Zfp207-depleted ESCs (sh1 and sh2); NHEJ: Non-homologous end joining. Gene ontology (GO) analysis of biological processes associated with common (D) up-regulated genes in Zfp207-depleted ESCs (sh1 and sh2); NHEJ: Non-homologous end joining. E-H. Heatmap of column z-scores of log2 transformed values of genes related to (E) pluripotency and (G) ectoderm in shScr, sh1 and sh2 ESCs. GSEA plots depicting the expression of (F) ESC-related and (H) ectodermal genes in Zfp207-depleted ESCs compared to shScr. High and low expression of genes is represented in red and blue color, respectively. FDR, false discovery rate; NES, normalized enrichment score.
a) The CETN1–GFP signal is brighter in the upper centrosome/spindle pole of a mitotic spindle.figf2(a) The CETN1-GFP signal is brighter in the upper centrosome/spindle pole of a mitotic spindle. The merged differential interference contrast microscopy image with CETN1-GFP labelling at two centrosomes shows a metaphase chromosome. Insets: enlargements and semi-quantitative integrated intensity profiles of centrioles. Scale bar, 5 μm. (b) The brighter CETN1-GFP signal represents the older centrosome, as it co-stains more intensely for hCenexin1 and remains more intense throughout cell division (Supplementary Fig. S1a). Scale bar, 5 μm. Lower left, merge. (c,d) Time-lapse images show that the mitotic midbody is preferentially inherited by the daughter cell with the older centrosome in HeLa cells (c) and hESCs (d). Cells were imaged at the indicated times (h:min) from telophase by phase-contrast microscopy (c) and from metaphase by differential interference contrast microscopy (d). Middle panel of (c) and upper left panel of (d): CETN1-GFP at centrosomes; enlargements and integrated intensity profiles show that the daughter cell with the older centrosome (c, upper; d, lower) inherits the MBd (lower right images in c,d). Mitotic midbody and MBds (c,d; arrows). MKLP1, MBd marker (red); α-tubulin, mitotic midbody and cell-boundary marker (green); DAPI, DNA (blue). Scale bars, 10 μm (c,d).
Neutrophils induce paracrine senescence a) Schematic depicting experimental design.Figure 1Schematic depicting experimental design. effect of neutrophils and recombinant catalase on population doublings (data are from 3 independent cultures are shown). Blue arrow indicates time point in which neutrophils were added to culture for consecutive 3 days % ∆ Population Doublings (PD) following neutrophil co-culture d) representative images of Ki67 and p16Ink4a immunofluorescence and SA-β-Gal staining; e) % of Ki67 positive cells (28 days after co-culture); f) % of p16INK4A positive cells (28days after co-culture); g) % of SA-β-Gal positive cells (28 days after co-culture); h) Western blotting for p21 and quantification; i) MitoSOX fluorescence; j) Heat map depicting expression of 48 human cytokines in conditioned media at 8 and 28 days following co-culture.