EMBO/sd-character-level-ner huggingface dataset:
Figure 1A. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing ER marker GFP-TMD (green). The cells were loaded with SiR-lysosome to visualize LE/Lys (orange). Zoom insets show a merge of the ER (GFP-TMD) (green) and SiR-lysosome labelled LE/Lys (orange). Zoom insets show a region of interest containing a mobile endosome as denoted by the white arrow. Scale bars: full size 10µm, zoom inset 3µm. Images related to movie EV1. B. Kymograph of the representative image in panel (A). C. Schematic flow of membrane displacement analysis method. D. Representative time-lapse spinning disk confocal images of n = 3 of COS7 cells stably expressing GFP-TMD (ER) (green) and SiR-lysosome (orange). Zoom insets highlight regions of interest containing a static (upper panels) or mobile endosome (lower panels) as denoted by the white arrows. First row shows ER (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Scale bars: full size 10µm, zoom inset 3µm. Images relate to movies EV2 and 3. E. Kymograph of the mobile vesicle in panel (D). F. MDA quantification of (D). Mobility of static and mobile endosomes was quantified and normalized to the static endosomes. Graph represents mean ± s.d. of analyses at multiple locations within the same cell(n=20 cells per condition from 3 independent experiments). All images acquired for 90 frames at 4Hz. Significance two-tailed student t-test, **** P<0.0001.
Figure 6A Depletion of METTL3 in Hepa1-6 cells by CRISPR-Cas9 knockout enhanced sorafenib-resistance in vivo. Growth curve (B) of Hepa1-6 cells derived xenografts in the subcutaneous implantation mice model in six treatment groups for 12 days (n=5). tumor weight (C) of Hepa1-6 cells derived xenografts in the subcutaneous implantation mice model in six treatment groups for 12 days (n=5). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD. D Knockdown of METTL3 slightly increased liver tumor growth treated with sorafenib in orthotopic xenograft mouse model. Stable METTL3-knockdown Bel-7402 cells and control cells were injected into the liver of each NOD/SCID mouse. 3 weeks after injection, livers were separated for pathological analysis. Red circle indicates tumors in the livers. E Liver tumors in orthotopic xenograft mouse model were confirmed by hematoxylin and eosin staining. F Overexpression of FOXO3 rescued sorafenib-resistance mediated by METTL3-knockdown in xenograft liver tumors. Representative xenograft tumors at endpoint, containing 6 groups. Growth curve (G) of Hepa1-6 cells derived xenografts in the subcutaneous implantation mice model in 6 treatment groups for 10 days (n=5). tumor weight (H) of Hepa1-6 cells derived xenografts in the subcutaneous implantation mice model in 6 treatment groups for 10 days (n=5). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD. I H&E and immunohistochemical images of Ki67, LC3-B, VEGF-A in Hepa1-6 cells derived xenografts in 6 treatment groups. J-L Quantification of Ki67 (K), LC3-B (K), and VEGF-A (L) expression in immunohistochemical images by Image Pro Plus (IPP) analysis. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD.
Figure 6(A and B) Lactate production assay in BT549 (A) and MDA-MB-231 (B) cells with Luciferase or GLUT1 knockdown. (C) Cell proliferation assay in BT549 cells with Luciferase or GLUT1 knockdown. (D) Cell proliferation assay in MDA-MB-231 cells with GFP or GLUT1 knockdown. (E and F) Immunoblotting analysis for GLUT1 protein expression in control and MIG-6 knockdown BT549 (E) and MDA-MB-231 (F) cells with or without GLUT1 overexpression. (G Lactate production assay in control and MIG-6 knockdown BT549 cells with or without GLUT1 overexpression. H) Lactate production assay in control and MIG-6 knockdown MDA-MB-231 cells with or without GLUT1 overexpression. (I and J) Cell proliferation assay in BT549 (I) and MDA-MB-231 (J) cells with control knockdown, MIG-6 knockdown, or MIG-6 knockdown plus GLUT1 overexpression.
The Hippo/YAP and the ERBB pathways interact with each other to regulate cervical cancer cell growth.Figure 8Representative images showing the morphology of spheroids derived from ME180-siCtrl and ME180-siLATS1/2 cells growing in a 3D hanging-drop culture system for 10 days. Scale bar: 1.0 mm. Quantitative data showing changes in the volume of spheroids derived from ME180-siCTRL and ME180-siLATS1/2 cells growing in a 3D hanging-drop culture system. Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.0004). Quantitative data showing changes in the volume of spheroids derived from ME180 cells growing in a 3D hanging-drop culture system in the absence or presence of AREG (20 ng/ml, 8 days). Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.0030). Soft agar assay showing the effect of AG1478 and VTPF on colony formation in ME180-MXIV, ME180-YAP and ME180-YAPS127A cells. Scale bar: 500 μm. Representative images showing the effect of AREG, EGFR inhibitor (AG1478), and YAP antagonist verteporfin (VTPF) on the growth of ME180 cell in a 3D hanging-drop culture system. ME180 cells were incubated in the 3D hanging-drop culture system for 10 days in the absence or presence of AREG, AG1478 or verteporfin for 8 days. Scale bar: 1.0 mm.
Parkin ubiquitinates BAK on a conserved lysine in the hydrophobic groove.Figure 2(A) Subcellular fractionation of cytosol and membrane fractions of HeLa cells expressing HA-Parkin following 2 h of antimycin A and oligomycin (AO) treatment. (B) Immunoblotting of whole cell lysates immunoblotted following treatment with antimycin A and oligomycin (AO) for 3 h with MG132 (20 µM). Graph shows densitometric analysis of non-ubiquitinated proteins relative to untreated control from three independent experiments. Error bars represent mean +/- SD. (C) UBA pull-down of ubiquitinated proteins following antimycin A and oligomycin (AO) treatment for 3 h and treatment with the non-specific DUB USP2 for 30 min at 37°C, representative immunoblot from two independent experiments. (D) Structure of human BAK (PDB 2IMS) with the hydrophobic binding groove comprising α-helices 3-5 highlighted in blue. The ubiquitination site, K113, (red) resides in the binding groove. A modelled transmembrane anchor (α9) and the C-terminal K210 localised to the inter-membrane space is based on evidence that the BAK transmembrane anchor spans the mitochondrial outer membrane (Iyer et al CDD, 2014). The orientation of the soluble portion (α-helices 1-8) of BAK with respect to the mitochondrial outer membrane is hypothetical. (E) Whole cell lysates of HA-Parkin and BAK variant-expressing HeLa cells following 3 h antimycin A and oligomycin (AO) treatment with 30 min pre-treatment with MG132 (20 µM), representative immunoblots from three independent experiments.
Figure 6A, B. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP in ndt80∆ cells expressing PEST-IME2-∆C and/or PEST-CDC5 upon addition of estradiol (EST) at 4 h in SPM (t = 0). Top, time-lapse series. Bottom, quantification of cells with Spc72 at SPBs. (A) ndt80∆ cells expressing Ime2-∆C or Cdc5. (B) Expression of Ime2-∆C and Cdc5 in ndt80∆ cells containing wild-type Spo13 or the phospho-site mutants Spo13-10A and Spo13-10D. While Spo13-10D delays Spc72 removal by 127 min (95% CI, 105-149; P < 0.0001), Spo13-10A has little effect (95% CI, -24-0.8; P = 0.07; Welch's t-test). Data information: data are representative of two (A) or 2-4 (B) independent experiments. C, D. Immunoblot analysis of whole-cell extracts. C, sample from proliferating cells. (C) Protein levels in the cultures shown in (A). (D) Protein levels in the cultures shown in (B).
Impaired Mitophagy in gba−/− Neurons and Astrocytes.Figure 6(A) Midbrain astrocytes were transfected with GFP-LC3, treated with 100 nM bafilomycin A1, and stained with MitoTracker Red and imaged using confocal microscopy. (B) Midbrain gba+/+ and gba−/− neurons and astrocytes were transfected with YFP-Parkin. Half the transfected cells were treated for 1 hr with 10 μM FCCP and all bathed in TMRM containing recording solution and imaged using confocal microscopy. Scale bar, 20 μm.(C) TMRM fluorescence intensity of cells from (B) was analyzed via confocal microscopy. Data represent the mean ± SEM, (n = 3, >4 cells analyzed per experiment). (D) Mitochondria within midbrain neurons were uncoupled by the addition of 10 μM FCCP for 1 and 6 hr. PINK1 expression analyzed via immunoblotting on isolated mitochondria from gba+/+ and gba−/− neurons. Mitochondrial CI subunit NDUFS4 was used as a loading control.
Figure 6Sequence alignment of SUD between SARS-CoV (Genbank: AY291315) and SARS-CoV-2 (GenBank: MN908947). Domains Mac2 (SUD-N), Mac3 (SUD-M) and DPUP (SUD-C) range from K389 to L526, G527 to S652 and K653 to S720, respectively. The first 16 residues of Mac2 are boxed in blue. This figure was generated by using the ESPript server (http://espript.ibcp.fr/ESPript/ESPript/). SARS-CoV-2 SUD (SARS2-SUD) interacts with Paip1 in HEK-293 cells as demonstrated by the split-YFP assay. SARS2-Mac2 binds to Paip1. SARS2-Mac2 but not Δ16-SARS2-Mac2 interacts with Paip1M. MERS-CoV SUD-MC (Mac3−DPUP) or XMC (Mac1−Mac3−DPUP) do not bind to Paip1. Scale bars represent 50 µM.
Figure 6Study design. Two fin clips were taken at 10 and 20 weeks post hatching (wph) from 159 killifish and individual lifespans monitored. RNA sequencing was employed to compare transcriptome changes between 10 and 20 wph for each individual fish. A Cox Hazard model was used to correlate lifespan to gene expression changes. Two groups of genes were identified: (i) genes whose increased expression between 20 and 10 wph is a risk factors (i.e., associated to increased mortality risk) (red), and (ii) genes whose decreased expression is a risk factors (blue). KEGG pathways enriched among genes whose regulation is associated to mortality. Only pathways with FDR<0.05 are shown. Distribution of change in expression for proteasome transcripts across the entire cohort of 159 fish. Lifespan was compared among fish that showed extreme changes in proteasome levels between 10 and 20 wph (32 fish showing the most pronounced decreases, shown in black vs. 32 fish showing the most pronounce increases). *** p<0.001 Log-rank test.
Figure 2Tumor number, tumor size, and tumor load in colons were measured. Tumors were microscopically analyzed and classified as low or high grade. Histological score was assessed by a pathologist. Tumor tissues were stained for Ki67. The percentages of Ki67-positive tumor cells were quantified. Scale bars, 25 µm. Mice were treated as described in Fig 1A and Fig 2A. Feces were collected from colons in each mouse. Total fungal burden in feces were quantified using qPCR. Mice were treated as described in Fig 1A. Fungal ITS2 rDNA gene sequence was performed in each group. Fungal-taxon-based analysis at the phylum level in feces of mice. Color from blue to red indicates enrichment of fungus. Fungal-taxon-based analysis at the genus level in feces of mice. 1 Candidan 2 Fusarium 3 Kazachstania 4 Thermomyces 5 Phaeococcomyces 6 Mycosphaerella 7 Penicillium 8 Aspergillus 9 Coprinellus 10 Cryptococcus 11Mortierella 12 Guehomyces 13 Preussia 14 Chaetomium 15 others Three-dimensional principal component analysis (PCA) based on fungal ITS2 rDNA gene sequence abundance in the feces. WT: samples are from WT mice, De: samples are from Dectin-3-/- mice. Specific fungal burden of C.albicans in the feces were quantified using qPCR. GF mice were orally gavaged with C.albicans (twice a week, 1×107) during administration with AOM-DSS (n=8, each group). Mice were euthanized on Day 100, tumor load in each mouse were measured. ELISA results for binding assays of Dectin-3 with C. albicans. Plates were coated with C.albicans and then were added with 100 ml/well recombinant Dectin-3 at indicated concentrations. GFP-C. albicans (5×106) were added onto 1×106 BMDMs, and incubated at 37°C for 1h. Wells were washed and fresh media containing fluconazole (300 μg/ml) was added. At 6 hours and 18 hours, C. albicans CFU inside BMDMs were photographed and calculated by plating on YPD agar. Scale bars, 25 µm.
Mitochondrial defect in atg mutants.Figure 3(A) WT and atg1Δ cells expressing mitochondria targeted mCherry cultured in SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours were observed by fluorescent microscopy. Mitochondrial DNA was stained with SYBR green I, and mitochondria were visualized by mitochondria targeted mCherry. Scale bar, 2 µm. (B-C) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. ROS accumulation was detected by DHE staining (B). Each photo contains about 200 cells. Scale bar, 20 µm. (C) shows quantification of ROS accumulated cells (n>200 cells). (D) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) with 10 mM NAC for the indicated time. Cell viability was determined by phloxine B staining. Cells from these cultures were plated on YEPD agar and overlaid with TTC agar to examine respiratory competency. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. These data represent the average of three independent experiments and bars indicate standard deviations
Figure 4A. Schematic drawing of 2-step QTIP (improved version of the quantitative telomeric chromatin isolation protocol (QTIP)). B. Quantification of precipitated telomeric DNA in 2-step QTIP (% of input) and fold enrichment of precipitated telomeric DNA compared with precipitated Alu repeat DNA (based on dot blot analyses). Bars represent data from three independent experiments ± SD. C. Scatter plots representing immunoprecipitation specificity (2-step QTIP/IgG ratio) versus difference between non treated cells (NT) and cells at day 4 upon 4-OHT induction (4d 4OHT/NT ratio) or difference between non treated cells (NT) and cells at day 7 upon 4-OHT induction (7d 4OHT/NT ratio) respectively (data from Table EV1). MRN complex in red, 9-1-1 complex in blue, shelterin complex in orange except for POT1 in black. Values are the average of the 3 biological replicates. D. Heat map representing changes in levels of selected proteins involved in DNA damage (MRN complex in red, 9-1-1 complex in blue) and HDR proteins (purple) after POT1 knockout induction. Proteins marked with * pass t-test, but not t-test with Benjamini-Hochberg correction (p < 0.05, q < 0.09). Data information: 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells.
Figure 2Schematic illustration of a single human rDNA gene repeat and positions of the primers used for ChIP.   In Jurkat cells, a ChIP assay was performed with control IgG, NF45 and NF90 antibodies, and then the precipitated DNA was analyzed using qPCR with the aforementioned primers. The relative rDNA fold-enrichment was normalized to control IgG treatment. (n=3) The human rDNA promoter region contains one ARRE2-like sequence. The red letters in gray shaded area indicate the probe sequences used in panel D. Gray shaded area indicates the upstream control element. Blue color indicates the core promoter region. EMSA assay. Lane 1) Biotin-labled rDNA probe. Lane 2) Biotin-labled rDNA probe + nuclear protein. Lane 3) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled specific competitor. Lane 4) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled mutant competitor. Lane 5) Biotin-labled rDNA probe + nuclear protein + NF90 antibody. Lane 6) Biotin-labled rDNA probe + nuclear protein + NF45 antibody. The details were described in Materials and Methods section. The relative luciferase activity of HEK293T cells transfected with empty vector (EV) or FLAG-NF90 (NF90) in combination with rDNA
Figure 4(A) A real-time OR-ID assay showing the association-dissociation curves of surface-immobilized Palbociclib with recombinant STING protein. The data shown are representative of n=3 biological replicates. (B) Structure of synthesized biotin-conjugated Palbociclib. Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) and the lysates of HEK293T cells transfected with Flag-STING (C) The data shown are representative of n=3 biological replicates. Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) mouse peritoneal macrophages (D) The data shown are representative of n=3 biological replicates. Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) THP1 cells (E). The data shown are representative of n=3 biological replicates. (F) Immunoblotting of the streptavidin precipitates of the mixture of biotin-c-diAMP (2 μM) and recombinant SUNO-tagged STING protein (10 μg/mL) in the presence of DMSO and increasing concentrations of Palbociclib (0, 1, 10 μM). The data shown are representative of n=3 biological replicates. (G) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STING and HA-STING in the presence of DMSO and indicated concentrations of Palbociclib. The data shown are representative of n=3 biological replicates. Representative immunofluorescent assay of STING and ERGIC in mouse peritoneal macrophages stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) (H). Scale bar, 25 μm. STING and ERGIC in mouse peritoneal macrophages stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) The quantification data is shown in (I). The data shown are mean ± SD from indicated number of cells from 1 representative of n=3 biological replicates. Statistical significance was determined by two-tailed Student's t-test. ****, P<0.0001. (J) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STEEP and HA-STING in the presence of DMSO or Palbociclib (10 μM). The data shown are representative of n=3 biological replicates. (K) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STING and HA-TBK1 in the presence of DMSO or indicated concentrations of Palbociclib. The data shown are representative of n=3 biological replicates.
Fig.Figure 4(A) Survival of Drosophila S2 cells treated with preheating, 100 µM NAT, and 100 µM NAT with 10 mM 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) after heat stress at 42oC for 60 min (data are means ± SEM; n=10). After pretreatment with chemicals for 12 h, those were removed by changing the medium. *P<0.05, **P<0.01 vs. PBS. (B) Survival of Drosophila S2 cells pretreated with 100 µM NAT, 100 µM NAT with 30 µg/ml antimycin A, and preheating with or without 30 µg/ml antimycin A 48 h after heat stress at 42oC for 60 min (data are means ± SEM; n=10). After pretreatment with chemicals for 12 h, they were removed by changing the medium. *P<0.05 vs. PBS. (C) Effects of 100 µM NAT on MitoRed localization in mitochondria of S2 cells. Upper: representative images of time-dependent changes in MitoRed localization in mitochondria after adding NAT. Scale bar: 20 µm. Lower: relative intensities of MitoRed fluorescent signals in mitochondria were quantified by microplate reader. Different letters above bars represent significant differences [P <0.05 (data are means ± SEM, n=12)]. (D) ROS concentrations in S2 cells after adding 100 µM NAT (data are means ± SEM; n=12). *P<0.05 vs. zero time. (E) Graphic depicting the participation of NAT in triggering mitohormesis in animal cells. (F) Effects of dsRNA targeting of FoxO on NAT-dependent expression of Keap1 (data are means ± SEM; n=12). *P<0.05, **P<0.01 vs. without NAT. Note that NAT feeding did not increase Keap1 expression in FoxO knockdown flies. (G) Effect of NAT on growth of HCT116 colon cancer cells in mice (BALB/cSlc-nu/nu, 6 wks old, female) (data are means ± SEM; n=5). *P<0.05, **P<0.01 vs. without NAT. Test mice began to freely drink 5 mg/ml NAT solution for 7 d before transplantation of the tumor cells on day 0: the average amount of NAT consumed by each mouse was about 8.8 mg/day (0.4 - 0.5 g/kg weight/day). HCT116 cells (5.0 x 106 cells) were transplanted into each test mouse using a 1 ml syringe with a 27G needle.
Figure 8A, Co-IP assay of HDA15-TAP with TOC1-GFP and 5X-GFP in N. benthamiana. HDA15-TAP was immunoprecipitated with IgG resins and digested by HRV-3C protease. HDA15 and TOC1 were detected by α-MYC and α-GFP, respectively. B, Quantification of the interaction strength in A. Results from 5 independent trials were averaged. Error bar indicates SEM, asterisk indicates significant difference (*p<0.05, student's t test). C and D, Hypocotyl lengths and free-running periods of indicated plants lines. Data of ~75 plants from 3 independent experiments were averaged, different letters indicate statistically significant differences (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test). 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. E, Expression of PIL1, AT5G02580 and CDF5 in 3-d-old SD-grown seedlings at ZT18. Data from 3 independent experiments were averaged. Error bars indicate SEM. Different letters denote statistically significant differences based on Wilcoxon test (p<0.05).
Protein map of the yeast endocytic coat based on detected FRET proximities of coat-associated proteins.Figure 3A Protein proximity map of the endocytic coat-associated proteins. B Enlarged view of the map focused on selected proximities of clathrin subunits, End3, and Sla2 and Pan1 shortened and full-length proteins. C Enlarged view of the map focused on Gts1 proximity network.
The N-terminal region of FRH is unstructured in the absence of FRQ.Figure 3(A) Comparison of the FRH N-terminus (large cell structure, magenta) to that of Mtr4 (green) aligned on their RecA domains. Only the additional elements of FRH are shown (cyan). The FRH N-terminal extension has little ordered secondary structure, although regions 143-147 and 129-135 form small turns that interact with the helicase core. (B) The very N-terminus of FRH extends away from the RecA-2 domain to interact with a symmetry related molecule in the crystal (below), whereas the Mtr4 N-terminus turns back to form an outer β−strand. Center panel shows the crystal contacts, with close-ups for each region highlighted as insets on the left and right.
Figure 5Ribbon diagram of IAPV-IRES/80S complex viewed from the 40S colored by pairwise root-mean-square deviation displacements observed between the classes indicated on the left. Class 1 is unrotated while classes 2 and 3 exhibit a small rotational movement of the 40S. Top, general overview of the non-rotated IAPV-IRES/80S class 1 structure. IAPV-IRES is depicted as solid Van Der Walls surface colored according to the secondary structure motifs. The PKI (green) is solidly anchored to the A site. Bottom, close up view of the IAPV-IRES PKI inserted in unsharpened map. Zoomed view of A3760, a nucleotide belonging to the helix 69 (H69) of the 28S rRNA interacting with PKI. Final refined model inserted in the post-processed map is shown. This interaction is not disrupted along the small fluctuations of the 40S. An apical view along the axis of the PKI of a superposition of class 1 versus class 2 shows the IRES displacements are minimal and are followed by the H69 which constantly interacts with the IRES.