EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Representative confocal micrograph of HeLa cells transfected to produce either mCherry (red), GFP-ACAP1 (green) and Arf6-HA (blue; left hand panels) or mCherry-BspF (red), GFP-ACAP1 (green) and HA-Arf6 (blue; right hand panels) and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 µm and 2 µm (insets). (B) Representative Western blot analysis of co-immunoprecipitations of myc-ACAP1 and Arf6-HA in the presence or absence of HA-BspF. HeLa cells were transfected to produce Arf6-HA and combinations of myc-ACAP1 and HA-BspF, or not, and myc-ACAP1 was immunoprecipitated using anti-myc-conjugated magnetic beads. Input lysates (6% of post-nuclear supernatants) and co-immunoprecipitates were separated by SDS-PAGE and probed for Arf6-HA, HA-BspF and myc-ACAP1 by Western blotting. Quantification of the Arf6/ACAP1 ratio was performed by densitometric analysis. Data are means ± SD of 3 independent experiments. The asterisk indicates a statistically significant difference (P = 0.0017, unpaired Student's t test) between BspF-producing and control conditions. (C) Quantification of Arf6 activity (GTP-Arf6) in HeLa cells transfected to produce either mCherry and Arf6-HA or mCherry-BspF and Arf6-HA by G-LISA. Data are means ± SD of n=3 independent experiments, normalized to mCherry-producing controls. The asterisk indicates a statistically significant difference (P = 0.0026, unpaired Student's t test) between BspF-producing and control conditions. (D) Bacterial replication in BMMs transduced to either produce GFP, Arf6Q67L-GFP or Arf6T27N-GFP and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria for 24 h. Data are means ± SD of n=4 independent experiments, in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions. (E) Bacterial replication in BMMs transduced to either produce GFP, GFP-ACAP1 or GFP-ACAP1R448Q and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria for 24 h. Data are means ± SD of n=3 independent experiments, in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.
Figure 4(A) FoxM1, Plk1, and Cdc25B mRNA expression in stem and progenitor cells with and without AhR, n=4 mice per group. (B) FoxM1, Plk1, and Cdc25B mRNA expression in WT stem and progenitor cells with and without TCDD, n=3-4 mice per group. (C) Schematic representation of the FoxM1 promoter region containing a putative dioxin response element. Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT colonic organoids treated with DMSO or TCDD, n=3 or 4 per group. A DNA fragment amplified in a gene desert on mouse chromosome 6 was used as a negative control. Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT and KO crypts (E) treated with DMSO or TCDD, n=3 or 4 per group. (F) FoxM1 luciferase activity determination for young adult mouse colonocytes (YAMCs) treated with DMSO or 10 nM TCDD for 1 d, n=3 per group. (G Expression of FoxM1 and PLK1 at mRNA level, , n>=3 independent organoids per treatment. H) Expression of FoxM1 and PLK1 at protein level, n>=3 independent organoids per treatment. (I) Representative brightfield images of mouse colonic organoids generated from WT Lgr5+-GFPhi stem cells treated with DMSO or 5µM FDI-6. Scale bar 200 µm. (J, K) Quantification of organoid forming efficiency (J) and organoid area (K) from WT colonic stem cells treated with DMSO or 5µM FDI-6. Data are represented as mean ± SEM in (K). (L) Organoid expression of Lgr5 in response to FDI-6. Lgr5 expression level was dramatically suppressed by FDI-6 in a dose-dependent manner, n>=3 per treatment. (M) Representative brightfield images for secondary organoid formation following treatment with DMSO or 5 µM FDI-6 for 2 d. Scale 200 µm.
Figure 1(A-B) Spinal cord sections from 5-month-old WT and Tmem106b-/- mice were stained with anti- neurofascin, anti- LAMP1 and anti-Cath D antibodies. Accumulation of LAMP1 and CathD positive vacuoles at the distal end of AIS was observed in Tmem106b-/- motor neurons. Representative images from three independent mice were shown. Scale bar = 10 µm. n=3. Data presented as mean ± SEM. Unpaired Student's t test. ****, p<0.0001. (C-F) Western blot analysis of p62, ubiquitin (Ub), TDP-43 and p-TDP-43 in RIPA- and urea-soluble fractions from brain (C and E) and spinal cord (D and F) of 16-month-old WT and Tmem106b-/- mice. n=5. Data presented as mean ± SEM. Unpaired Student's t test. *, p<0.05, ***, p<0.001, ****, p<0.0001.
Figure 1A. Pull-down assays of yTBP (61-240) with Kap95p, Kap114p and Kap121p. GST-fused yTBP (61-240) was incubated with recombinant Kap95p, Kap114p and Kap121p. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. B. Band intensities of Kap -βs from the SDS-PAGE gels in (A) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP. C-E. ITC titration curves (upper) and binding isotherms (lower) of (C) Kap114p, (D) Kap95p, and (E) Kap121p with yTBP (61-240). Salt concentration and Kd values are indicated. F. Cartoon representation of the Kap114p crystal structure. The A and B helices composed of each HEAT repeat are shown as gray cylinders. The HEAT8 hairpin, HEAT19 loop and additional HEAT18/19 loop are highlighted in blue, red and green, respectively. A 45o rotated view is shown on the right. Overall dimensions of the Kap114p structure are indicated. G. Superimposition of Kap114p (gray) with cargo-bound Cse1p (light orange; PDB code: 1WA5). The HEAT8 hairpin and HEAT19 loop of Kap114p are colored blue and light green, respectively. H. Schematic representation of the Kap114p domain configuration using the same color code as shown in F.
Figure 6(A) Confocal microscopy images of untreated (UT) or treated with Ciliobrevin D (CilioD) or AZ82 or both treatments simultaneously (CilioD + AZ82) ECs and stained for endogenous EEA-1 (in green) and LAMP-1 (in red) to visualize EEs and LEs, respectively, and Draq5 (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm. (B) Distribution of distance to nucleus overtime, normalized on cell size, quantified by image (data correspond to movies EV3 and EV5) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during AZ82 treatment in ECs, compared to untreated cells (UT). Results are from two independent experiments for a total of 853 early endosomes in 6 untreated cells (142 ± 22 endosomes per cell) and 355 early endosomes in 7 AZ82 cells (51±12 endosomes per cell). Data are analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***. (C) Distribution of retrograde velocity overtime, quantified by image (data correspond to movies EV3, EV4 and EV5) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during Ciliobrevin D (CilioD D) or AZ82 treatment in ECs, compared to untreated cells (UT). Results are from three independent experiments for a total of 580 EEs in 6 UT cells (97 ± 18 endosomes per cell), 432 EEs in 9 CilioD cells (48 ± 15 endosomes per cell) and from two independent experiments for a total of 223 EEs in 7 AZ82 cells (32 ± 6 endosomes per cell). Data are analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for UT and CilioD treated ECs p≤ 0,01 ** and for UT and AZ82 treated ECs p≤ 0,001 ***. (D) Distribution of distance to nucleus overtime, normalized on cell size, quantified by image (data correspond to movies EV3, EV5 and EV6) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during AZ82 treatment (as in b), alone or in combination with CilioD treatment (CilioD + AZ82), compared to untreated EC cells (UT). Results are from two independent experiments for a total of 853 early endosomes in 6 UT cells (142 ±22 endosomes per cell), 355 EEs in 7 AZ82 cells (51 ± 12 endosomes per cell) and 1106 EEs in 8 CilioD+AZ82 cells (136 ± 42 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for UT and AZ82 treated ECs p≤ 0,001 *** (orange) and for CilioD and CilioD + AZ82 treated ECs p≤ 0,001 *** (green).
Figure 6A Simulation of SDD model, where after reaching steady-state (black line, insets), either production was stopped (left) or degradation increased (right). The gray arrow shows the direction of time, the color of the lines reflects the mCherry/sfGFP ratio (y axis). where the SDD model was run until reaching steady state (red circle) and then production and/or degradation parameters were perturbed The y-axis shows the mean mCherry/sfGFP ratio (proxy for average protein age) in the anterior region of the embryo (0 to 25% of embryo length), and the x-axis represents the mean green fluorescence intensity in the same region (age/levels diagram). C During the cleavage divisions the embryos define reproducible trajectories in the age/levels diagram. The trajectories of 4 embryos from n.c. 10 to gastrulation are shown (each embryo depicted with a different symbol and in a different panel). The time resolution of the time-course was 6 min. The data points are colored according to the division cycle. A time window of approximately 30 min where the trajectories stay (compatible with a steady-state), is marked with dotted-grey circles. All imaged embryos that managed to start gastrulation were included in the analysis. D The normalized trajectories from the embryos overlap. The intensity values were normalized for each embryo to the intensity values at the mid n.c. 14 The gray line depicts the average trajectory.
In the presence of nutrients, MXL-3 represses the expression of the lysosomal lipase genes, which contribute to breaking down fats through lipophagy.figf8In the presence of nutrients, MXL-3 represses the expression of the lysosomal lipase genes, which contribute to breaking down fats through lipophagy. Upon fasting, MXL-3 is transcriptionally repressed and delocalizes from the nuclei, releasing the repression of the lysosomal lipases. Concomitantly, HLH-30 translocates from the cytoplasm to the nucleus and hlh-30 expression is induced, in part by an autoregulatory mechanism. In the nucleus, HLH-30 enables the activation of lipophagy through the induction of lysosomal lipase and autophagy genes. The activation of this transcriptional program enables utilization of internal reserves of energy, and promotes survival of starvation.
Pol λ fails to discriminate against dATP misincorporation opposite 8-oxo-dG(syn).Figure 3(A) Substrates used for crystallization of the 8-oxo-dG:dATP insertion complex. The primer terminal base (ddC) is shown in cyan. The incoming dNTP (dATP) and corresponding templating 8-oxo-dG are shown in magenta and green, respectively. (B) Overview of the 8-oxo-dG:dATP insertion complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). The primer terminal ddC and the incoming dATP are shown in cyan and magenta, respectively. Key residues that form the nascent base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The catalytic metal A and the nucleotide binding metal B are shown in red and neon, respectively. (C) 8-oxo-dG (green) establishes a Hoogsteen base pair with dATP (magenta). Asn513 and Arg517 (purple) interact with the minor groove of the nascent base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ). (D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, the primer terminus in cyan and the incoming dATP in magenta. A water molecule (red) and Mg2+ (neon) are occupying the metal A and B sites, respectively.
Figure 6 (a) Cluster formation assays were conducted in presence or absence of 20µM mCherry-LC3B or mCherry, either pre-mixed with p62 or added 1" after the addition of GST-4xUb. Quantification of particle number (left) and size (right) are shown (b) Cluster formation assays were conduced with mCherry-p62 wt or LIR mutant in presence of GST-4xUb. Left: representative images of the samples two minutes after the addition of GST-4xUb. Middle: quantification of particle count. The dashed line indicates the time point of images displayed on the left. Right: input gel of the samples after reaction. The relative position of marker bands is indicated. For all graphs, averages and SDs from at least three independent replicates are shown (c) Top left: Western blots of lysates of CRISPR/Cas9 genome-edited STG-p62 wild type and LIR mutant cells. Clone Lir1B was used for the experiments shown in Figure 6c. Bottom left: Representative pictures of STG-p62 WT or LIR mutant Hap1 cells treated with Puromycin for 90 minutes and stained with anti-Ub conjugates antibody (FK2). Right: quantification of cytoplasmic p62 puncta per cell (top), and of their co-localization upon puromycin treatment (bottom). Averages and SDs from three independent experiments are shown. In total, at least 1440 number of cells were counted per condition
Figure 2(A)Knockdown of SIRT5 inhibits NADPH production in HEK293T cells. The ratio of [NADPH/NADP+] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test). (B) Knockdown of SIRT5 suppresses GSH production in HEK293T cells. The ratio of [GSH/GSSG] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test). (C) Knockdown of SIRT5 leads to higher ROS levels in HEK293T cells. ROS levels were determined in the indicated stable cells as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments ***p<0.001 (two-tailed unpaired t-test). (D) Knockdown of SIRT5 leads to higher sensitivity to Paraquat. The expression levels of PARP and Caspase-3 were determined by western blot analysis. (E) SIRT5 protects HEK293T cells from ROS-induced cell death. The indicated stable HEK293T cells were treated as mentioned above in (D), and then cell viability was determined by counting the remaining adherent cells. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test). (F) Cartoon representation of major NADPH-producing enzymes in the cell. As shown, G6PD, 6PGD, ME1, IDH1 and MTHFD1 are located in cytosol, while IDH2 and MTHFD2 are located in mitochondria. (G) The activities of G6PD/6PGD and IDH1/2 are reduced in stable HEK293T cells with SIRT5 knockdown. Activities of the indicated endogenous proteins were determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05, **p<0.01, ***p<0.001, n.s.: not significant (two-tailed unpaired t-test). (H) G6PD and IDH2 are activated by SIRT5 in vitro. Upon pre-incubation with SIRT5 in vitro, the activities of the indicated ectopically expressed proteins were determined as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01, n.s.: not significant (two-tailed unpaired t-test).
Figure 1Percentage of the total signal intensity attributed to phosphopeptides (blue bars; 11 unique peptide ions) and non-phosphorylated peptides (grey bars; 25 unique peptide ions) following SAX fractionation at (A) pH 6.0, (B) pH 6.8 and (C) pH 8.0, with later fractions consisting of up to 100% phosphopeptide ion signal. Representative pHis-containing myoglobin peptide (LFTGHPETLEK; solid line), and its non-phosphorylated counterpart (LFTGHPETLEK; dashed line), quantified across all 16 SAX fractions; SAX was performed at pH 6.0 (red), pH 6.8 (blue) or pH 8.0 (green). % of the total peak area of each individual peptide across the gradient is plotted for each fraction. In order to assess pHis stability and effects of SAX separation in a complex mixture (black), SAX was also repeated at pH 6.8 with phosphorylated myoglobin spiked into a human cell lysate prior to digestion. Data for the five pHis-containing myoglobin peptides is shown in Fig. EV3.
Figure 5A Validation of biomarker genes related to autophagy in FOXO3-knockdown Bel-7402 cells by RT-PCR. 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 and are representative of 3 independent experiments. B Protein level of FOXO3 in naïve HepG-2 and sorafenib-resistant HepG-2 cells under hypoxia for 48h. C Protein levels of LC3 I/II in FOXO3-knockdown SMMC-7721 cells. D Overexpression of FOXO3 in sorafenib-resistant HepG-2 cells by Western blot. E Protein levels of LC3 I/II in METTL3-knockdown & FOXO3-overexpression SMMC-7721 cells under hypoxia for 48 h. F Electron micrographs of METTL3-knockdown SMMC7721 cells and METTL3-knockdown & FOXO3-overexpression SMMC7721 cells under hypoxia for 48 h. G Representative Immunostaining images of LC3 in METTL3-knockdown SMMC7721 cells and METTL3-knockdown & FOXO3-overexpression SMMC7721 cells under hypoxia for 48 h. Scale bar, 200μm. The quantification of the numbers of GFP-LC3 puncta/cell by Imaris in G-1. H Representative Immunostaining images of LC3 in naïve HepG-2 cells and sorafenib-resistant HepG-2 cells under hypoxia for 48 h. Scale bar, 200μm. The quantification of the numbers of GFP-LC3 puncta/cell by Imaris in H-1. I The IC50 of METTL3-knockdown SMMC7721 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition. 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 and are representative of 3 independent experiments. J The IC50 of naïve HepG-2 cells and sorafenib-resistant HepG-2 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition. 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 and are representative of 3 independent experiments. K Cell survival assay of METTL3-knockdown SMMC7721 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition. L Cell survival assay of naïve HepG-2 cells and sorafenib-resistant HepG-2 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition.
Influence of the transcription elongation rate on the requirement of Sen1 Nter for non-coding transcription termination.Figure 7A) Decreasing the elongation rate alleviates the termination defects associated with deletion of Sen1 Nter. Northern blot assays performed in a Sen1-AID, ∆rrp6 strain carrying a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in former experiments. Where indicated, cells were treated with 50 mg/L of 6-azauracile (6AU) for 2h. Representative gel of one out of two independent biological replicates.The U4 RNA is used as a loading control. B) Model for the role of the N-terminal domain and the NIM in the control of Sen1 action at ncRNAs. Nrd1 and Sen1 are likely independently recruited to the elongation complex. The recruitment of Nrd1 and Nab3 is mediated by the recognition of specific RNA sequences and enhanced by the interaction of Nrd1 CID with the S5P-CTD via the CID. Sen1 would be recruited either by unspecific binding to the nascent RNA or by possibly transient protein-protein interactions with RNAPII. The interaction of Sen1 NIM with Nrd1 CID also enhances Sen1 recruitment. The interaction of Sen1 Nter with the CTD could facilitate the loading of Sen1 to the nascent RNA in the vicinity of paused RNAPII. Sen1 would translocate along the nascent RNA to induce dissociation of the elongation complex. The release of Sen1 from Nrd1 would be required for the CID to be available to recruit TRAMP for subsequent processing/ degradation of the released RNA.
a–c) Reprogramming is more efficient after MBd enrichment.figf8(a-c) Reprogramming is more efficient after MBd enrichment. Differentiated cells (dH1f) and embryonic fibroblasts (IMR90) are reprogrammed after stable expression of either NBR1 shRNA or shNT. Emerging iPSC colonies are scored on the basis of TRA-1-60 expression. Cells depleted of NBR1 to increase MBd levels show an increase in iPSC colony formation (a,b, dH1f, 3.1±0.5-fold, n=15, P=0.00035; IMR90, 3.4±0.8-fold, n=3, P=0.02; data are mean±s.e.m.) but insignificant changes in autophagic activity (c) over shNT control. (b) Representative plates with TRA-1-60-immunostained iPSC colonies. The immunoblot (c, top) and densitometry (c, bottom; percentage of autophagic flux) show representative results (n=3); α-tubulin, loading control. (d) MCF-7 side-population (SP) cells have a significantly higher percentage of MBd+ cells over the non-side-population (MP; P=0.0015, n=3; data are mean±s.d.). (e,f) MBd enrichment in cancer cells leads to increased anchorage-independent growth. MKLP1-GFP-expressing HeLa cells are separated into 'MBd-high' and 'MBd-low' subpopulations. An increase in the 'MBd high' over 'MBd low' ratio is associated with an increase in soft-agar colony formation (e). No significant difference was observed when the enrichment of the MBd-high subpopulation was less than threefold. More soft-agar colonies are formed when MBds are enriched by NBR1 depletion (NBR1 shRNA) in HeLa (f, left; P=0.0012, n=3) and mouse 134-4 cells (f, right; P=0.0086, n=3); control, shNT. Data are mean±s.d., and the colony number (e,f) is the sum of INT-violet-stained colonies from ten random fields. (g) Model for MBd fate in cells. The newly formed MBd is preferentially inherited by the daughter cell with the older centrosome (top panel). The inherited MBd (black ring) is recognized by binding of the NBR1 autophagic receptor (grey circle) with the midbody protein CEP55 (magenta). The MBd is then encapsulated by the autophagosome (yellow circle), and degraded after fusion of autophagosome and lysosome (red circle) in differentiated cells. This pathway prevents MBd accumulation. In contrast, stem cells efficiently accumulate MBds through successive divisions and evasion of NBR1-mediated autophagy. Furthermore, differentiated and stem cells possess overall high and low autophagic activity, respectively. Uncropped images of blots are shown in Supplementary Fig. S6.