EMBO/sd-character-level-ner huggingface dataset:
Rap2b inhibits the autophagy induced by S.Figure 10(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b wt (upper panel) or GFP-Rap2b ΔAAX (lower panel). Twenty-four hours after transfection, they were infected for 4 h with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−)+pHla). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods and immediately visualized by confocal microscopy. These data are representative of three independent experiments. (B) Quantification of the percentage of infected cells presenting LC3 puncta upon the infection. * p<0.05, *** p<0.001 (paired Student's t-test, n = 50 cells/condition). These data are representative of three independent experiments. (C) Quantification of the percentage of bacteria decorated with Rap2b wt upon the infection. ** p<0.01 (paired Student's t-test, n = 30 cells/condition). These data are representative of three independent experim
Figure 5(A) Stopped-flow assay measuring relative rates of lactic acid transport into reconstituted vesicles containing wild-type or mutant transporter (green, control with empty vesicles; red, wild-type; blue, K35A; cyan, F90A; magenta, F94A; yellow, K177A; olive green, H230A; and gray, H230N). (B) Relative rates of lactic acid transport based on reswelling rates of proteoliposomes. Each data point is an average of five different light scattering data sets. Error bars depict standard deviation for these five light scattering data. All of the transport rates from the mutant transporters are significantly different compare from that of wild-type PfFNT (K35A, P = 2.8 x 10-3; F90A, P = 4.4 x 10-7; F94A, P = 4.9 x 10-6; K177A, P = 1.2 x 10-3; H230A, P = 4.5 x 10-7; H230N, P = 2.0 x 10-6; student's t-test) (green, control with empty vesicles; red, wild-type; blue, K35A; cyan, F90A; magenta, F94A; yellow, K177A; olive green, H230A; and gray, H230N).
Blockage of microtubule growth by actin filaments on isolated centrosomes.Figure 4Schematic illustration of the first dynamic assay: sequential addition of tubulin followed by tubulin and actin on isolated centrosomes. Representative images showing microtubules (top line) and the merged images of actin and microtubules (bottom line) for the two steps of the assay; in the presence of tubulin only (left column) and in the presence of tubulin and actin (right column). Scale bar: 10 µm. Quantification of the differences in the number of microtubules per centrosome between the two stages of the experiment described above on centrosomes capable (first condition), or not (second condition), to grow actin filaments. Data were collected from a single experiment; asters without actin filaments: n= 29; asters with actin filaments: n= 13. Data were analyzed using Mann-Whitney test. Schematic illustration of the second dynamic assay: tubulin is added to measure centrosome nucleation capacity and washed out. Then actin is added followed by actin and tubulin. Representative images showing microtubules (top line) and the merged images of actin and microtubule (bottom line) during the three steps of the assay; in the presence of tubulin only (left column), in the absence of tubulin and presence of actin (middle column) and in the presence of tubulin and actin (right column). Scale bar:10 µm. Arrowheads indicate microtubules unable to re-grow after assembly of actin filaments. Quantification of the differences in the number of microtubules per centrosome between the first and last steps of the experiment described above (panels D and E) on centrosomes capable (first condition), or not (second condition), to grow actin filaments. Data were pooled from 2 independent experiments; asters without actin filaments: n= 24; asters with actin filaments: n= 13. ****:P<0.001 Student t-test.
R-III reduces BDL-induced liver fibrosis.Figure 9Liver sections from control, BDL, and BDL/R-III-treated mice were stained with Sirius red. Scale bar, 200 μm.. Hydroxyproline content in the livers (μg/g liver). *P-value, two-sample t-test (n = 10) (compared to BDL-treated mice); BDL + R-III (10 μg): 0.031, BDL+R-III (5 μg): 0.026.
NAF‐1 does not affect BIK‐initiated apoptosis, but influences BIK‐induced autophagy in the absence of caspase activation.Figure 3(A) H1299 neo and HA‐BCL‐2b5 cells treated with control (CTRL) or NAF‐1 shRNA were either mock infected or infected with Ad‐BIK in the absence or presence of 50 μM zVAD‐fmk. Cell lysates were analysed by immunoblot. (B) Caspase activity was measured using the fluorescent substrate DEVD‐AMC. The results represent the average±s.d. of three independent experiments. (C) Prolonged BIK expression and caspase inhibition induces autophagy, which is enhanced by knockdown of NAF‐1. H1299 neo and HA‐BCL‐2b5 cells treated with CTRL or NAF‐1 shRNA were infected with Ad‐BIK in the presence of zVAD‐fmk for the indicated periods of time. Cell lysates were analysed by immunoblot. Levels of LC3 II were normalized to actin levels by densitometry analysis. Graph depicts normalized LC3 II levels of each lane.
Figure 7ATP measurement on 48hR trunk extracts shows that cct3A RNAi under starved conditions contains less ATP than controls. Error bars are s.d. from the mean and asterisks indicate P < 0.05 (one asterisk) and n.s. indicates not significant using two-tailed Student's test with equal variance. n = 3 replicates (5 planarians per replicate). The Kaplan-Meier curve demonstrates increased survival of starved cct3A(RNAi) animals when treated with palmitic acid/palmitoylcarnitine (PA/PC). Percentages indicate the number of survivals at the indicated time points. Three asterisks indicate P < 0.001 with log-rank (Mantel-Cox) test. Images are representative surviving animals for the different conditions in (B). At the bottom are the number of planarians with the phenotype shown. The rest of planarians are dead at this point. Scale bars indicate 500 µm. In our proposed model the levels of ER stress (UPR) are basal during regeneration in feeding conditions at 72hR and not essential for stem cell proliferation and regeneration. Planarians obtain energy and lipids from administered food, including exogenous fatty acids (exo FA). During starved conditions, ccts increase the levels of the UPR and this allows proper stem cell proliferation and regeneration when exogenous fatty acids are not available.
Figure 6(A) Digitonized mitochondria from doxycycline-treated 143B cells and COX2∆ cybrids were analyzed by 2D-BN/SDS-PAGE and western blot with the indicated antibodies. (B) Mean incorporation rates of COX subunits into SC I+III2plus from COX2∆ cybrids. Signals from three independent experiments were quantified and normalized by CII. Time-point values are expressed as percentages of the untreated cells (SS) and indicated as mean ± SD. Control (143B) values for subunits COX4, COX5A and COX6C were obtained from (Moreno-Lastres et al., 2012). C-D SDS-PAGE (C) and BN-PAGE (D) analyses from 143B cells and COX2∆ cybrids transiently transfected either with a COX5B-Myc-DDK construct or with the empty vector, followed by immunoblotting with antibodies against COX5B and CII subunit SDHA (loading control). The identities of MRC complexes and SCs are as in Figure 1. The red arrow points to the accumulation of over expressed COX5B bound to SC I+III2 in control cells.
Figure 1A Schematic representation of the integrated mini virus (A1 J-Lat Tat-GFP) utilized in the primary screen of small molecules. Expression of the GFP reporter is under control of the HIV-1 LTR promoter in this cell line. B Arrayscanimages of untreated cells (left panel), or cells treated with PMA for 24 hours (right panel: Green-GFP; Blue-Hoechst; Red-PI). C Distribution of GFP expression produced by treatment with compounds from the CCBN, LCGC and DIVERSet libraries (black), or untreated cells (grey). Results are presented as percent GFP expression relative to a positive control reference sample treated with PMA (green) and determined from three biological replicates (n=3).
a) GFP-LC3 HeLa cells were cultured in fed conditions or starved using EBSS plus 10 μM hydroxychloroquine and treated with Vps34 inhibitor.figf4(a) GFP-LC3 HeLa cells were cultured in fed conditions or starved using EBSS plus 10 μM hydroxychloroquine and treated with Vps34 inhibitor. Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). One representative image, acquired with an iCyte imaging cytometer, for three independent experiments is depicted (X40). White arrows indicate GFP-LC3 spots, which correspond to the autophagosomes. Scale bars, 10 μm. (b) Histograms show the percentage of positive cells and GFP intensity, measured using an imaging cytometer for different compound concentrations on GFP-LC3 HeLa cells. Black and gray histograms correspond to fed and starved conditions, respectively. For GFP intensity, the values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments. (c) Wild-type HeLa and H1299 cell lines were cultured either in fed or starved conditions with EBSS and 10 μM hydroxychloroquine (HCQ), as indicated. Cells were treated at the same time with SAR405 at different concentrations. LC3-II was measured by western blotting, and GAPDH was used as a loading control. One representative experiment for three independent experiments is shown. Full-length blots are shown in Supplementary Figure 18. (d) Histograms show the LC3-II/GAPDH ratio for each condition in the two cell lines. Black and gray histograms correspond to fed condition and EBSS conditions, respectively. The addition of hydroxychloroquine at 10 μM is indicated. Values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.
Figure 7Besides of the well-known NAFT-IL-2 T cell activation pathway, NFAT translocates to nucleolus, cooperatively with NF45/NF90 promote rDNA transcription upon T cell activation. Inhibition of rDNA transcription by NF45/NF90 knockdown or a specific polymerase I inhibitor CX5461 suppressed T cell activation and skin and heart allograft rejection.
TGFβ/ActivinB signaling promotes cell proliferation in Group 3 MB cell lines.Figure 41603MED cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2 and β-actin upon inhibition of TGFβ/Activin signaling using LY364947 and SB431542 inhibitors for 24 hours. Bar graphs on the right panel represent the quantification of the relative level of P-Smad2 (P-S2) to β-actin. Bars represent the mean ± SD. Number of replicates is n≥3. 1603MED cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Growth curve experiments showing cell proliferation upon TGFβ/Activin signaling inhibition. The p-values were determined by unpaired t-test and two-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001. 1603MED cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labelling at 48 hours upon inhibition. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S and G2/M phases). Bars represent the mean ± SD. Number of replicates is n≥3. 1603MED cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Percentage of apoptotic cells measured by FACS analysis of cleaved caspase3 48 hours after TGFβ/Activin signaling inhibition. Bars represent the mean ± SD. Number of replicates is n≥3. D283 cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2 and β-actin upon inhibition of TGFβ/Activin signaling using LY364947 and SB431542 inhibitors for 24 hours. Bar graphs on the right panel represent the quantification of the relative level of P-Smad2 (P-S2) to β-actin. Bars represent the mean ± SD. Number of replicates is n≥3. D283 cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Growth curve experiments showing cell proliferation upon TGFβ/Activin signaling inhibition. The p-values were determined by unpaired t-test and two-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001. D283 cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labelling at 48 hours upon inhibition. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S and G2/M phases). Bars represent the mean ± SD. Number of replicates is n≥3. D283 cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Percentage of apoptotic cells measured by FACS analysis of cleaved caspase3 48 hours after TGFβ/Activin signaling inhibition. Bars represent the mean ± SD. Number of replicates is n≥3.
a) Wild-type (WT),.figf7(a) Wild-type (WT), mxl-3(ok1947), hlh-30(tm1978) or mxl-3(ok1947);hlh-30(tm1978) double mutant were used in longevity epistasis analyses, revealing that hlh-30 suppresses the mxl-3 extended lifespan phenotype. Animals were incubated at 20°C and transferred every other day until cessation of reproduction. The cumulative survival curve is depicted. Kaplan-Meier statistics, calculated using SSPS 17, are also shown. (b) Live Nile red quantification of wild-type and lipl-1lipl-3 double mutant worms shows that lipl-1 and lipl-3 contribute to the clearance of lipids from the endocytic pathway. Mean+s.e.m. signal intensity is shown as percentage relative to wild type; significant differences are indicated; n = 4 independent experiments. (c,d) Animals overexpressing LIPL-1 or LIPL-3 were incubated at 25 °C and transferred every other day until cessation of reproduction. Data presented as in Fig. 7a show that activated lysosomal lipolysis extends C. elegans lifespan. a Survival presented as mean lifespan (LS) ± s.e.m. b Number of uncensored animals (animals that crawled off the plate, bagged, exploded or became contaminated were censored).
Figure 1(A) Genome-wide ubiquitin binding profiles identify numerous regions of H2B-ubiquitylation in WT cells and distinct sites of non-H2B ubiquitylation ('ubiquitin-hotspot', ub-hotspot, ub-HS) which persist in h2b-K123R and rad6∆ strains, and increase in cdc48 mutants (cdc48-6). A 90 kb stretch of chromosome XIII (ChrXIII) is depicted. Chromatin immunoprecipitation was performed using the FK2 ubiquitin antibody (see also Appendix Fig. S1A) and enriched DNA was analyzed on NimbleGen arrays (Chip-chip). DNA from non-specific IgG-ChIP experiments served as background control. Significantly enriched regions are marked by bars above the respective ChIP-chip tracks and are summarized in Dataset EV1. Data represent means from two independent replicates, except for ubiquitin (FK2) in rad6∆ and IgG-ChIP in WT (n = 1). All experiments, including those using cdc48-6 and other temperature-sensitive (ts) alleles, were performed at 30°C (semipermissive temperature for ts-alleles) unless stated otherwise. (B) Seven ub-HSs show strong correlation between ubiquitin binding and Slx8 enrichment (Slx8-9myc ChIP). 16-kb-windows of the indicated regions centered around the ub-HSs are depicted for ubiquitin and Slx8 binding profiles. Ubiquitin (FK2) data for WT and cdc48-6 are from the same experiment as depicted in (A). Data represent means from two independent replicates. (C) Schematic representation of the 16 yeast chromosomes (I-XVI) with positions of the ub-HSs marked by red triangles. Vertical bars indicate positions of centromeres. (D) Slx8 and Slx5 are recruited to ub-HSs. DNA from Slx8-9myc (top) or 3HA-Slx5 (bottom) ChIP experiments of the indicated strains was analyzed by quantitative real-time PCR (ChIP-qPCR) at selected ub-HSs. Data represent mean ± standard deviation (SD, n = 2). Consistent with a previous study, we did not observe any Slx8 enrichment at centromeres (van de Pasch et al, 2013), however, we note that we could not confirm the reported centromere binding of Slx5 (see also Appendix Fig. S2E). (E) K48-linked ubiquitin chains accumulate in a cdc48 mutant (cdc48-3). Ub-K48 ChIP followed by qPCR for same loci as in (D) are depicted. See also Fig. EV1C-D. Data represent means ± SD (n = 2). (F) Slx5 and Slx8 are required for ub-HS formation. Ub-K48 ChIP-qPCR in strains lacking one subunit of the Slx5/Slx8 complex (slx5∆, slx8∆). Data represent means ± SD (n = 3). (G) Ufd1 and Npl4 act in concert with Cdc48 to remove ubiquitylated proteins from ub-HS sites. Ub-K48 ChIP for the indicated strains grown at the semi-permissive temperature of 30°C. Data represent means ± SD (n = 2).
Figure 1(A,B) Level of expression of borgs and septins in young and aged LT-HSCs. mRNA levels of borgs and septins were normalized to the level of expression of GAPDH and borgs are shown relative to the level of Borg 2 expression in young LT-HSCs. Septins are shown relative to the level of septin 7 expression in young LT-HSCs. At least three biological replicates (n=3) are shown. Error bars indicate mean±SEM, unpaired t-test, **P<0.01, ***P<0.001, ****P<0.0001. (C) Percentage of cells polarized for septin 7 among young, aged and aged LT-HSCs treated with CASIN (5µM). At least 3 biological repeats, at least 50 cells were scored per sample. Error bars indicate mean±SEM, unpaired t-test, **P<0.01, ***P<0.001. (D,E) Representative immunofluorescence microscopy images of the distribution of septin 7 (red) or borg3 or borg4 (green) in young, aged and aged LT-HSCs treated with CASIN (5µM). Nuclei were stained with DAPI (blue), scale bar= 5µm.
Structural basis of Sir4 H‑BRCT interaction with phospho-peptides A) The electrostatic surface potential covering a range from -4 kT/e (red/acidic) to +4 kT/e (blue/basic) is mapped onto the Sir4 H‑BRCT structure to highlight the basic patch on the H‑BRCT surface.Figure 3The electrostatic surface potential covering a range from -4 kT/e (red/acidic) to +4 kT/e (blue/basic) is mapped onto the Sir4 H‑BRCT structure to highlight the basic patch on the H‑BRCT surface. Sir4 H‑BRCT is shown in two orientations rotated by 180° and selected residues are labeled. Superpositions of Sir4 H‑BRCT-Esc1pS1450 (grey cartoon, peptide as sticks in atom colors), Sir4 H‑BRCT-Ty5pS1095 (blue cartoon, peptide as sticks in atom colors), and Sir4 H‑BRCT-Ubp10pT123 (green cartoon, peptide as sticks in atom colors) peptide complex structures reveal the highly similar conformation of peptide binders. Esc1pS1450, Ty5pS1095, and Ubp10pT123 peptides used for co-crystallization are indicated with residues modeled in the structures in bold and residues that contribute most binding to Sir4 H‑BRCT boxed. Detailed view of the Sir4 H‑BRCT-Esc1pS1450 peptide interaction. Sir4 H‑BRCT is displayed as orange model with residues involved in peptide binding shown as sticks (orange, atom colors). The Esc1pS1450 peptide is presented as sticks in grey (atom colors). Electrostatic interactions are highlighted with green dotted lines while main hydrophobic contacts are shown as dotted lines in black. Detailed view of the Sir4 H‑BRCT-Ty5pS1095 peptide interaction. Sir4 H‑BRCT is displayed as in (C) while the Ty5pS1095 peptide is presented as sticks in blue (atom colors). Protein-peptide interactions are labeled as in (C). Detailed view of the Sir4 H‑BRCT-Ubp10pT123 peptide interaction. Sir4 H‑BRCT is displayed as in (C) while the Ubp10pT123 peptide is presented as sticks in green (atom colors). Protein-peptide interactions are labeled as in (C). MST analysis of the binding interactions between Sir4 H‑BRCT and Cy5-labeled phospho (filled circles) and non-phospho peptides (empty circles) from Esc1, Ty5 and Ubp10. KD = equilibrium dissociation constant. ΔFnorm values were divided by the amplitude of the saturation level, resulting in the fraction bound (from 0 to 1) for each data point. Data are represented as the mean ± SEM from ≥3 independent measurements.
Figure 7A CUEDC2 ablation reduced I/R injury, which was induced by 30 minutes of ischemia followed by 24 hours reperfusion. AAR, area at risk; LV, left ventricular area, IRI, area of I/R injury. The ratios of AAR to LV, IRI to AAR and IRI to LV are shown. * p < 0.0001 compared with WT, n = 15 mice per group. (top panel) Representative cross-sectional slices derived from the hearts stained by TTC. Area of I/R injury is indicated in light pink. B Representative images of ventricular myocardium sections from WT and Cuedc2-/- mice exposed to sham operation or I/R injury (2 hours after the onset of reperfusion following 30-min ischemia). Green, TUNEL-positive myocytenuclei; red, Troponin I-stained cardiomyocytes; blue, DAPI-stained nuclei. Scale bar, 50 μm. C Quantitative analysis of apoptosis. * p < 0.0001, n = 8 mice per group. D SerumLDH levels in sham-operated mice or those subjected to ischemia and a 4-hour reperfusion. Data from 3 separate experiments was analyzed by one-way analysis of variance. * p < 0.0001, n = 13 mice per group. E Echocardiography analysis of left ventricular dimensions and cardiac function at 1 week post-I/R in mice. ** p < 0.0001, n = 15 mice in the sham-operated group and n = 10 mice in the I/R group.
Figure 7(A) Animal protocol. (B) The levels of CRAMP in serum (n = 8). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (C) The levels of CRAMP in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (D) Representative images of intestinal damage by H&E staining. Scale bar: 200 μm. The graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (E) Western blot and densitometry analyses of tight junction proteins (claudin-1 and occludin) and zonulin in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (F) Intestinal permeability was assessed by measuring FITC-dextran (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (G) The frequency of NKG2D+ IELs among CD3+CD4+ population (n = 8). Data information: Data were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (H) The frequency of CX3CR1+ macrophages among CD45+CD11b+ population (n = 8). Data information: Data were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. (I) The percentages of three subsets of CD45+CD11c+ DCs (n = 8). (J-L) The percentages of (J) Foxp3+ Treg, (K) T-bet+ Th1 and (L) RORγt+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8). Data information: Data were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.