EMBO/sd-character-level-ner huggingface dataset:
Figure 3Confocal microscopy images of co-localization analysis of the BNIP3-proficient (shCntl, n=27 cells) or deficient (shBNIP3, n=31 cells) B16-F10 cells transiently transfected with GFP-LC3 and immunostained for TOMM20 (mitochondrial marker). Colocalization events of GFP-LC3 puncta (green) with TOMM20 (red) are highlighted with white arrows. The percentage of GFP-LC3 puncta colocalizing with TOMM20 was calculated in n cells from 3 biologically independent experiments and analyzed with Mann-Whitney's non-parametric test. Scale bars represent 10µm. Schematic representation of the metabolic approaches used to characterize B16-F10 cells 1H-NMR stands for proton-based nuclear magnetic resonance, GC-MS for gas chromatography coupled to mass spectrometry, OCR for oxygen consumption rate and ECAR for extracellular acidification rate. Basal OCR per µg of protein of B16-F10 cells in the presence of galactose (25mM galactose, 2mM glutamine) medium using the Seahorse technology (n=3) analyzed using an RM one-way ANOVA (Geisser-Greenhouse correction) with Holm-Sidak's multiple comparisons test. Basal ECAR per µg of protein of B16-F10 cells assayed in glucose (25mM glucose, 2mM glutamine) medium using the Seahorse technology. Extracellular lactate (E), extracellular (F) and intracellular (G) glucose levels from B16-F10 cells detected using 1H-NMR (n=3) analyzed using a RM one-way ANOVA (Geisser-Greenhouse correction) with Holm-Sidak's multiple comparisons test. 13C incorporation in intracellular lactate from B16-F10 cells fed with 13C-labelled glucose for 24h and detected using GC-MS (n=7). The graph shows the percentage of lactate detected without any carbon isotopically labeled (m0), with one (m0+1), two (m0+2) or three (m0+3) carbons labeled. m0 and m0+3 conditions were analyzed using Friedman's test with Dunn's multiple comparisons test. Glut1, Hk2, Pkm1, Pkm2, Pdk1, Mct1, Ldha, Ldhb and Mct4 transcript levels measured by qPCR from the corresponding B16-F10 cell lysates. Transcript expression is represented as the fold-change relative to their corresponding shCntl value. Heatmap shows the average values in each condition (n=4) and they were analyzed with a one-sample T-test only against shCntl, except for Glut1 shATG5 that was analyzed with a Wilcoxon rank test.
Induction of autophagy genes by heat stress.Figure 2Five weeks-old Arabidopsis wild-type Col-0 plants were placed in the 22°C and 45°C growth chambers and total RNA was isolated from leaf samples collected at indicated times. Transcript levels were determined using real-time qRT-PCR. Error bars indicate SE (n = 3).
Phosphorylation of spaghetti‐squash activator (Sqa) at Thr‐279 is required for Atg1‐mediated myosin II activation.Figure 3(A) Characterization of Atg1‐dependent phosphorylation sites on Sqa. 293T cells transfected with Flag‐tagged Atg1 or Atg1‐KR were subjected to immunoprecipitation with anti‐Flag antibodies, followed by in vitro kinase assays with bacterially expressed Sqa‐K2, Sqa‐K2‐T194A, Sqa‐K2‐T239A, and Sqa‐K2‐T279A as substrates. Atg1 but not Atg1‐KR was autophosphorylated (top panel). Relative phosphorylation levels of substrates were quantified. Data are represented as mean±s.e. of triplicates. (B-D) Phosphorylation of Sqa at Thr‐279 is critical for the kinase activity of Sqa. (B) HA‐tagged Sqa or Sqa‐T279A immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant spaghetti‐squash (Sqh) WT and A20A21, in an in vitro kinase assay. (C) Clonal expression of Sqa but not Sqa‐T279A (GFP‐positive cells) in the larval wing imaginal discs resulted in a marked increase in phospho‐MRLC staining (blue) and actin reorganization (red). Bar, 20 μm. (D) The larval fat body of denoted genotypes were dissected, lysed, and subjected to western blot analysis using anti‐phospho‐MRLC and anti‐Sqh antibodies. For quantification, the levels of MRLC phosphorylation in each genotype were measured with ImageJ and normalized to the Sqh levels
scRNA-seq captures the expression profiles of effectors genes in the late stages of Drosophila embryogenesis.Figure 1(A) Single cells were isolated from Drosophila embryos 14-14.5h after egg deposition (AED). Isolated cells were processed through a 10x Genomics instrument. After sequencing the resulting libraries, the reads were aligned and processed using the standard pipeline from 10x Genomics. The single-cell gene expression profiles were used to map the cells to known cell types by comparing against the available in situ hybridization patterns from the Berkeley Drosophila Genome Project. (B) A tSNE projection of the scRNA-seq data is shown in the middle, and the known tissues to which the clusters were assigned to are graphically illustrated outside. Marker genes for each tissue type are shown in parentheses.
Figure 4a) Schematic representation of the two constructs used. LEFT. GST-tagged HHARIRBR with its native RING1 domain that does not induce closed conformations of UbcH5~Ub. RIGHT. GST-tagged HHARI construct in which the RING1 domain was replaced with the Ubox domain of E4BU to generate the UBR-hybrid that promotes closed conformations of UbcH5~Ub. b) LEFT. Auto-ubiquitination assays were performed with wild-type HHARIRBR or an active site-dead mutant (C357A- HHARIRBR). Products were visualized by western blotting against GST. The zero time point was taken immediately prior to ATP addition, all other times are post-ATP addition. RIGHT. Identical assays as shown on left were performed with the UBR-hybrid. The active site-dead mutant (C357A) retains substantial auto-ubiquitination activity signifying that UbcH5~Ub is able to transfer Ub directly onto the GST-UBR-hybrid construct, bypassing the RING2 active site Cys.
Characterization of cell lines used: metabolic profile, mitochondrial membrane potential and morphology.Figure 1A) Metabolic profiles of HeLa cells in dependence on IF1 levels under glycolytic and OXPHOS conditions generated by an automatic flux analyzer measuring oxygen consumption rates and extracellular acidification rates. n=3 independent experiments (error bars = SD). B) Mitochondrial membrane potential (∆ψm) under the influence of overexpressed IF1-H49K and the absence of IF1 in IF1-KO cells. To estimate ∆ψm, HeLa cells were stained with TMRE (7 nM). In addition, HeLa cells were stained with the membrane potential-independent dye MitoTracker™Green (MTG, 100 nM) for normalization to the mitochondrial area. Calculated TMRE/MTG ratios from n=4 independent experiments are displayed for each cell. Statistics: one-way ANOVA with post hoc Scheffé test, *, p≤0.05, **, p≤0.01, ***, p≤0.001. C) MiNA analysis of HeLa cells stained with the membrane potential-independent dye MitoTracker™Green (MTG, 100 nM) to visualize and determine the mitochondrial area (= mitochondrial footprint). Left: HeLa WT cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Co-localization shown in yellow. Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). D) Left: HeLa IF1-KO cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). E) Left: HeLa IF1-H49K cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). Co-localization shown in yellow. Scale bars = 10 µm. F) F) Mitochondrial footprint in µm2 determined via MiNa analysis of HeLa cells. Cells were stained with the membrane potential-independent dye MitoTracker™Green (MTG, 100 nM). G) Mean branch length in µm determined via MiNA analysis. H) Number of networks determined via MiNA analysis. I) Number of individuals determined via MiNA analysis.
a) Mouse fibroblasts expressing the KFERQ-mCherry1 photoactivatable reporter without (None) or with 20 μM of the indicated compounds imaged 16 h after photoactivation.figf5(a) Mouse fibroblasts expressing the KFERQ-mCherry1 photoactivatable reporter without (None) or with 20 μM of the indicated compounds imaged 16 h after photoactivation. Insets show higher-magnification images. Nuclei are labeled with DAPI. Scale bars, 10 μm. (b) Quantification of the effect of increasing concentrations of GR1 on the same cells. Untreated cells and cells treated with 40 μM ATRA are also shown. Representative images are shown in Supplementary Figure 7. Graph shows the average number of fluorescent puncta per cell, quantified in >50 cells. All values are mean ± s.e.m. (c) Mouse fibroblasts were cotransfected with the human RARα (hRARα) receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection. Values show relative luciferase units (RLU) detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12h . (d) Mouse fibroblasts were cotransfected with the human RARα (hRARα) receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection.Cells transfected as in c were treated with 100 nM of ATRA alone or in the presence of the indicated concentrations of the three retinoid derivatives or the antagonist BMS614 for 12 h. Values are shown as RLU. (e) Mouse fibroblasts were cotransfected with the human RXR receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection. Values show relative luciferase units detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12 h. (f) Mouse fibroblasts were cotransfected with the human RXR receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection. Values show relative luciferase units detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12 h. Cells transfected as in e were treated with 10 μM of ATRA alone or in the presence of the indicated concentrations of the three retinoid derivatives or the antagonist BMS614 for 12 h. Values are shown as RLU. Values in c-f are mean ± s.e.m.; n = 4-6. (g) Left, immunoblot for LC3-II in cells treated with 20 μM of the retinoid derivatives and protease inhibitors (PIs), as labeled. Actin is shown as loading control, and full-length blots are shown in Supplementary Figure 21. The amount of LC3-II in untreated cells, expressed as fold change relative to control (middle), and the increase in LC3-II after PI treatment (LC3-II flux, right), expressed as fold change, were calculated from the densitometric quantification of immunoblots. Values are mean ± s.e.m.; n = 3.
Figure 4(A-B). qRT-PCR analysis of the relative expression of ICAM-1 and EPCR mRNA in non-treated PLB985 cells (control), ICAM-1 knock-down (ICAM-1kd) and EPCR knock-down (EPCRkd) PLB985 cells. (C). Flow cytometric analysis (FACS) of surface ICAM-1 (α human mAb, see methods) expression in control non-treated PLB985, ICAM-1kd and EPCRkd PLB985 cells. (D). Flow cytometry quantification of the percentage of neutrophils that became GFP+ following incubation with GFP+ iRBC of control (untreated PLB985 cells), ICAM-1kd and EPCRkd PLB985 cells. (E). Short-term (6h) killing assay of NF54-luciferase iRBCs incubated with PLB985 neutrophils in the presence of anti-CD11b and anti-ICAM-1 blocking (mAb 15.2) and non-blocking (α human mAb, see methods) antibodies. (F). Inhibition of PLB985 neutrophil (%) phagocytosis of GFP+ iRBCs in the presence of anti-CD11b and anti-ICAM-1 (mAb 15.2) blocking antibodies. The inhibition was quantified by flow cytometry (FACS). (G). Short-term (6h) PLB985 neutrophil killing of NF54-luciferase iRBCs in the presence or absence of soluble ICAM-1-Fc fusion protein (sICAM-1-Fc). Soluble RAGE (sRAGE) protein was used as a negative control.
during DNA replication through damaged DNA.Figure 7Mrc1 is an integral component of the replisome that activates Rad53 when replication forks slow down or pause at damaged templates. In contrast, Rad9 is a mediator of DNA damage that signals post-replicative DNA damage. High doses of MMS (0.1%) induce a rapid stalling of replication forks and a robust induction of the Mrc1 pathway, leading to the efficient repression of late origins, as it is the case in HU-arrested cells. In cells exposed to a lower dose of MMS, forks progress further and replicons merge, leading to the extinction of the Mrc1 pathway. Maintenance of Rad53 activation depends on the induction of the Rad9 pathway by post-replicative DNA lesions. Induction of a limited number of DSBs (as in the case of Zeocin) activates the Rad9 pathway and represses both initiation and elongation at otherwise unchallenged forks.
Figure 5Scatterplot of mRNA expression and ribosome profiling data from control and YTHDF1 knockdown cells. The upregulated (red) and downregulated (blue) genes in translational efficiency (TE) are highlighted. The relative m6A peak coverage in control cells for transcripts grouped according to TE changes after YTHDF1 knockdown. POI: peak over input. Down, genes significantly downregulated in YTHDF1 knockdown cells (<0.5 fold). Unchanged, genes not significantly changed. Up, genes significantly upregulated (>2 fold). The upper and lower quartiles and the median are shown for each group. Mann-Whitney test. **p<0. 01. ***p<0. 001. Violin plots showing TE change between control and YTHDF1 knockdown cells for not-methylated (non-m6A) and methylated (m6A) transcripts. The upper and lower quartiles and the median are indicated for each group. Mann-Whitney test. Cumulative distributions of TE change for non-m6A and m6A transcripts as in (C). Kolmogorov-Smirnov test. Heatmap showing the TE of Wnt signaling components in control and YTHDF1 knockdown cells. The values show the fold change of the indicated color of the heatmap.
Figure 7(A) Principal Component Analysis of IBP parameters in HC (black circles) and APBD (red circles) fibroblasts. (B) Morphological characterization of lysosomes based on lysotracker staining of n=4 HC and n=4 (biological replicates) APBD patient skin fibroblasts starved for 48 h and treated or not with 50 uM 144DG11 for 24 h. Values are deviations from untreated HC. 144DG11 has reduced lysotracker mean intensity, integrated intensity (DXA), and area in both HC and APBD fibroblasts (***p<0.0001, One-Way ANOVA with Sidak's multi-comparison post-hoc test). (C) Functional (mitochondrial membrane potential (TMRE parameters)) and biomass (mitotracker) characterization of mitochondria in HC and APBD cells treated and analyzed as in (B). Mean (***, p<0.0001) and integrated (***, p<0.0006) TMRE intensities, and mitochondrial biomass (***, p<0.0001), were reduced in APBD v HC. 144DG11 increased TMRE mean (**, p<0.009, **, p<0.006) and integrated (*, p<0.01, **, p<0.001) intensities in APBD and HC cells, respectively and mitotracker intensity in APBD cells only (***, p<0.0001). Data represent n=3 biological replicates. Statistical differences analyzed by One-way ANOVA with multiple comparisons. (D) HC and APBD patient cells, starved for 48 h and then treated with 50 uM 144DG11 for 24 h in full medium, were subjected to bioenergetic characterization. 144DG11 treatment significantly increased basal and maximal (following FCCP) oxygen consumption rate (OCR), and coupling efficiency ((OCR drop following Oligomycin)/(Basal OCR)), in both HC and APBD fibroblasts, and ATP production (basal OCR - oligomycin OCR) only in APBD fibroblasts (Figure EV5). A representative experiment (n=3 biological replicates) based on n=6 technical replicates. (E) Volcano plots of the proteins affected by APBD and 144DG11 under starvation or glycogen burden conditions. Analysis is based on cells derived from n=3 APBD and n=3 HC subjects. (F) Venn diagrams of proteins down-modulated by APBD and up-modulated by 144DG11 and vice versa under starvation (48) and glycogen burden (48+24) conditions. (G) Gene ontology of proteins up-modulated (left) and down-modulated (right) by 144DG11.
Figure 2Western blot analysis of whole cell extract for the indicated conditions. Quantitative analysis of Rod levels using LiCor technology is indicated below. The asterisk indicates an unspecific band. Analysis of ZW10 and Mad1 kinetochore levels in the indicated cells and conditions. Kinetochore signal is normalized to CREST signal. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown. Representative still images of unperturbed mitosis for parental cells, Rod C cells and Rod CR cells with time in minutes indicated. CFP-Histone 3 was used here as the chromosome marker. Scale bar, 5 μm. Time from NEBD to exit of indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped. Time from NEBD to exit of indicated conditions with cells treated with taxol. Red circles represent cells still arrested in mitosis when the filming stopped.
GEMC1 interacts with E2F4/5 and Multicilin through distinct domains.Figure 6A. GEMC1 interacts with E2F4 and DP1. Western blot of HA or FLAG tagged protein expression in the input lysates are shown in the top panel. FLAGimmunoprecipiation (IP) of GEMC1co-IPs DP1 (α-HA, lane 5) and E2F4 (α- HA, lane 6). The interaction is enhanced upon co-expression of HA tagged E2F4 and DP1 (α-HA, lane 7). B. GEMC1 interacts with E2F5 but not E2F1. Co-IP of E2F5 (α-HA, lane 3) and E2F5-DP1 (α-HA, lane 6) but not E2F1 or E2F1-DP1 (α-HA, lanes 2 and 5). In the FLAG-IP panels, a short (top) and long (second from top) exposure of the Western are shown. C. Identification of the domain required for E2F4 interaction. GFP tagged GEMC1 and mutant forms were expressed and IPs were performed against GFP. Wild type GEMC1 (lane 5) or GEMC1 lacking the CC domain (lane 6) pulled down E2F4-DP1. A GEMC1 protein lacking the TIRT domain or GFP alone did not IP E2F4-DP1 (lanes 7, 8). D. Mutation of the single amino acid analogous that identified in MCIDAS in human RGMC patients (G313D) impairs the interaction with E2F4-DP1 (compare land 5 and 6). E. GEMC1 interacts with Multicilin through its CC domain. Wild type GEMC1 or a mutant lacking the TIRT domain pull down Myc-tagged Multicilin (anti- Myc blot, lanes 4 and 6) while a CC domain mutant does not (anti-Myc blot, lane 5). F. Schematic summary of the GEMC1 mutants used in A-E and their effect on EDG complex or Multicilin interactions.
High resolution maps of dynactin's shoulder and pointed end A.Figure 1Schematic showing a dynein-dynactin-adaptor complex on microtubules Schematic showing the domain architecture of dynactin. Density improvements during processing. For each step, density shown is taken from the same p50 arm helix in the shoulder. Map of the shoulder region. Density of the shoulder is colored in blue. Map of the pointed end including Arp 11 (yellow); p62 (orange); p25 (brown) and p27 (light brown).
Parabulin inhibits parasite invasion and replication.Figure 4(A) Evaluation of vertebrate cell toxicity using the MTT assay which detects metabolic activity proportionate to the number of viable cells in a sample. The absorbance measurements for all treatment conditions were normalized to the null control value which is set to 100% growth. Sub-confluent human fibroblasts were used to visualize potential compound effects during proliferation. The results represent the average of 9 readings (3 biological replicates with 3 technical replicates of each treatment) ± standard error of the mean. (B) Replication of GFP-expressing T. gondii tachyzoites in individual intracellular vacuoles can be followed by live cell imaging. Since vacuoles containing more than eight tachyzoites are not always easy to score, we tallied vacuoles above eight as being 8+ in size. At 36 hours, both null and vehicle control samples show most vacuoles harbor eight or more tachyzoites (three or more doublings). Vacuoles in parabulin-treated cultures contain significantly fewer parasites, indicating that replication is slowed. The graph shows three biological replicates with values normalized to 100% for each condition and each replicate, error bars represent standard error of the mean between experiments. (C) The parabulin EC50 value for T. gondii growth was determined using a plaque assay to measure a reduction in plaque area relative to control conditions. Error bars represent standard error of the mean. The results represent the average of 18 readings (6 biological replicates with 3 technical replicates of each treatment) ± standard error of the mean. (D) Phase contrast of extracellular parasite cultures. Serial passage of T. gondii in 40-80 μM parabulin induces accumulation of aberrant tachyzoite forms. These arise with MT defects that disconnect coordinated nuclear division and daughter cell budding.
Figure 8Representative images showing colony formation in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown or LATS2 knockout. hOSEs at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before imaging. Quantitative data showing colony numbers in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown or LATS2 deletion. hOSE cells at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before counting colonies under a microscope. Representative images showing tumorigenesis (red circle area) of hOSE-YAPS127A cells with or without pRB knockdown or LATS2 deletion. Tumors formed by hOSE-YAPS127A cells with RB1 knockdown or LATS2 deletion are shown in red circle area. Right panel showing representative tumors formed by hOSE-YAPS127A-LATS2KO and hOSE-YAPS127A-shRB1 cells. Please note that no tumor xenografts formed in hOSE-YAPS127A alone group. Representative images showing expression of YAP, Ki67 and LATS2 in hOSE-LATS2KO-YAPS127A cell-derived tumors examined by immunohistochemistry.
Overview of MhsT structure and function.Figure 1A) MhsT is shown in a ribbon representation (scaffold helices in blue, bundle helices in red, and TM5 in dark grey, sodium ions shown as purple spheres, L-4-Fluoro-phenylalanine, bound between unwound region of TM1 and TM6, shown as green spheres. B) Schematic representation of the MhsT structure (LeuT fold). L-4-Fluoro-phenylalanine shown as gray sticks. Remaining color coding same as in panel A. C) Results of a competitive uptake assay showing inhibition of 0.2 µM L-[3H]Trp or L-[3H]Leu uptake in MhsT-WT-expressing MQ614 cells, measured for 30 seconds in the absence (-) or presence of 12 µM of the natural amino acids (the one-letter code is used) or 4-fluoro-phenylalanine (4FP). Data are the mean ± S.E.M. of three independent experiments each performed as technical triplicates.
Figure 5Representative areas from human PDAC tissue showing expression of HNF1β in G1, G2 and G3 areas. The red dotted line indicates the borders of a G3 area. The percentage of HNF1β-positive cells in areas of different grade from a PDAC tissue microarray is shown. Colocalization of HNF1β and FOXA2 in human PDAC areas. PDAC sections from three different patients were stained with the anti-HNF1β antibody, stripped and re-stained with the anti-FOXA2 antibody. The red dotted areas on the left represent poorly differentiated (G3) tumor regions.