EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Volcano plot of AP-MS data using HA-BET1 or Ile51Ser. (B) Co-IPs using HA-tagged constructs expressed in HEK293 cells. As baits HA-tagged proteins were immunoprecipitated with anti-HA beads. IP samples and cell lysates were subjected to immunoblotting with α-HA, α-ERGIC-53, α-BET1, α-SEC22b, α-GOSR2 and α-GAPDH antibodies, respectively. (C) Quantification of three independent replicates of Co-IP experiments. Protein levels of the Co-IP samples were normalized to BET1-HA-levels. Data represented are mean ± SD of three biological replicates. Significant differences were checked by one-way ANOVA followed with Tukey`s post-hoc test. *=p≤0.05. (D) Endogenous Co-IP of BET1 and ERGIC-53. Immunoblots of HEK 293T cell lysates (+ lysate) or lysis buffer (-lysate) incubated with (+) or without (-) BET-1 antibody followed by precipitation with protein A/G beads. ERGIC-53 signal was only present in samples with lysate and antibody. whereas * denotes unspecific signals from beads used for precipitation. As a positive control GOSR2 antibody was used. (E) Subcellular localization of ERGIC-53 in fibroblasts by indirect immunofluorescent confocal microscopy. Representative immunofluorescence images of ERGIC-53 (green) and GM130 (red) in fibroblasts from P1, P2 and a control cell line. Scale bar: 20 µm. PCC (right panel) for GM130 versus ERGIC-53 (C1: n=74, C2: n=95, C3: n=59, P1: n=72 and P2: n=76). (*** = p≤ 0.001, one-way ANOVA was followed by Tukey's post-hoc test). Shown are means ± SEM. Immunofluorescence images shown are one example of a total of two biological replicates. (F) Colocalization of ERGIC-53 and BET1 in control and P2-derived fibroblasts. Representative immunofluorescence images of ERGIC-53 (green) and BET1 (red) in fibroblasts from P2 and a control cell line. Scale bar: 20 µm. PCC (right panel) for BET1 versus ERGIC-53 (C1: n=72, C2: n=46, C3: n=56 and P2: n=37). (*** = p≤ 0.001, one-way ANOVA was followed by Tukey's post-hoc test). Data represented are mean ± SEM of two independent experiments. (G) Immunofluorescence images of control fibroblasts (C1) and patient fibroblasts (P1 and P2) treated with Brefeldin A for 40 min and Golgi recovery for indicated time points. Golgi reconstitution was analyzed by immunofluorescence microscopy of the cis-Golgi marker GM130. Cells displaying an intact Golgi are marked with a plus (+). Scale bar: 20 µm. (H) The number of cells with intact Golgi was divided by the number of total cells per image and plotted against time (three biological replicates). Dots are means ±SD. For quantification, a sigmoidal fit was performed with the software GraphPad Prism 7. Dashed lines indicate extrapolated plateaus. LogIC50 ±95% confidence interval: C1 = 59.38±2.31 min, P1 = 72.89±1.65 min, P2 = 68.57±1.929 min. Significant differences were assumed when 95% confidence interval do not overlap.
promotes the differentiation of neural progenitors in the regenerating spinal cord (A) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa.Figure 5(A) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa. After sectioning, the samples were labelled with antibodies against Sox3 (green), EdU (magenta) and DAPI (blue). White arrowheads show Sox3 positive extensions. (B) Sections of spinal cords at 5 dpa in the stump (non-regen.) or in the regenerate (regen) labelled with anti-Sox3 (green), anti-Acetylated Tubulin (AcTub, magenta) and DAPI (blue). White arrowheads show Sox3 positive extensions. (C) Quantification of the images shown in (A). The ratio of Sox3+ per total number of cells (DAPI) in the spinal cord was determined in the stump (non-regen.) and the regenerating spinal cord (regen.) in wt and foxm1-/- tadpoles (n=3 to 8, with an average of 15 sections per data point). (D) Quantification of the proportion of cycling progenitors (Sox3+EdU+/DAPI, S3+/E+/DAPI), the proportion of progenitors having divided (Sox3+EdU+/Sox3+, S3+E+/S3+) and the proportion of progenitors self-renewal (Sox3+EdU+/EdU+, S3+E+/E+) in the regenerate. The graph represents the mean +/- SD from 9 tadpoles (wt) and 8 tadpoles (foxm1-/-) with an average of 18 sections per data point. (E) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa. After sectioning, the samples were labelled with antibodies against Myt1 (green), EdU (magenta) and DAPI (blue). (F) Quantification of images shown in (E). The graph represents the mean +/- SD from 3 tadpoles with an average of 12 sections each.
Figure 6Volcano plots displaying shRNA-Cct3- and shRNA-luciferase-infected LSK cells cultured either in normal glucose (upper graph) or low glucose conditions (lower graph). Dots represent all the NanoString analyzed transcripts. Red dots are UPR and ER stress related genes. The UPR and ER stress genes significantly down-regulated only under low glucose conditions in shCct3 cells compared to controls are indicated near the lower graph. For better visualization two dots with q ≤ 0.001 have been removed from the volcano plot (see them in Dataset EV4). Y-axis indicates the false discovery rate (FDR) (q- value), and the X-axis indicates the log2 fold changes. The FDR-based method of p-value adjustment was conducted to calculate the q-values by nSolver software using Benjamini-Hochberg methods. Significance is established by q-value < 0.05 and indicated by the dotted horizontal line. n= 3 mice. ;UPR and ER stress" genes are significantly enriched (only down-regulated) in shCct3 cells compared to controls under low glucose conditions (**P < 0.01 using two-sided Chi-square test). The graph shows that the ratio between Xpb1(spliced) / Xpb1(unspliced) is lower (i.e., lower levels of UPR) in LSK which have Cct-3 down-regulated compared to controls only when cultured under low glucose conditions. Error bars are s.d. from the mean. P < 0.001 (three asterisks) and n.s. indicates not significant using two-tailed Student's test with equal sample variance. n = 3 mice.
Figure 5A minimal ecological scenario of alternating periods of feast and famine is used to study overall fitness of different strategies. Periods of famine and feast can vary between scenarios. Based on the observed relation between growth and death (Figs. 1 and 2), we assume that bacteria cannot independently choose growth and death rate, but are forced to trade-off growth against death according to the exponential relation γ(μ) = 0.21 d − 1exp (1.0 h μ). The optimal growth rate, i.e. the one that maximizes fitness over a cycle of famine and feast, depends on the ratio of feast and famine periods. For long periods of feast interrupted by short periods of famine, E. coli the optimal strategy is to grow fast. For long periods of starvation intermitted by short periods of growth, the optimal strategy is long survival and slow growth. For the scenario of panel A and B, $\frac{T_{-}}{T_{+}} = 48$, the optimum is μ = 0.86 h − 1 and γ = 0.5 d − 1, see dashed line.
Periods of the locomotor activity rhythm of flies after silencing the expressions of studied genes.Fig 3  Tor, Tsc1, Rheb, Atg5 and Atg7 genes expression was silenced in per-positive cells. Period of locomotor activity rhythm was measured in DD conditions (mean +/- SE). Representative actograms were presented. As a control, the progeny of per-Gal4 and UAS-Val10-GFP crossing was used. After silencing the Tor gene, the period of locomotor activity rhythm was significantly shorter than in the control [U Mann-Whitney Test, *** for p≤0,001]. Silencing the Tsc1 gene caused a slight lengthening in the period of the rhythm.
PBP4 forms a PG multienzyme complex with NlpI and PBP1A/LpoA.Figure 5A-B. PBP4 has different interaction sites for PBP1A/LpoA and NlpI as shown by a single concentration MST assay. Plots show the FNorm or fluorescence values of fluorescently labelled PBP1A or LpoA with or without PBP4, NlpI or PBP4-NlpI. Values are mean ± standard deviation of three independent experiments.
Neutrophils induce paracrine senescence via telomere dysfunction a) Histograms representing distribution of individual telomere FISH intensities at day 4 and 16 following co-culture with neutrophils.Figure 2a) Histograms representing distribution of individual telomere FISH intensities at day 4 and 16 following co-culture with neutrophils. Dotted blue lines represent median telomere FISH fluorescence. Data are from n=3 independent experiments b) Representative micrographs of telomere FISH in metaphases c) Mean telomere FISH intensity at 4 and 16 days following co-culture with neutrophils represented as mean±s.e.m of 3 independent experiments; d) Representative Immuno-FISH micrographs (using telomere‐specific (CCCTAA) peptide nucleic acid probe and anti-γH2A.X antibody). Arrows indicate co-localization between telomeres and γH2A.X; e) Mean number and % of Telomere-associated DDR foci (TAF). Data are mean±S.E.M. of 3 independent experiments; f) Population doublings of hTERT expressing MRC5 fibroblasts or controls following co-culture with primed neutrophils (data are representative of 6 independent experiments); g) histograms representing distribution of individual telomere FISH intensities in MRC5 fibroblasts expressing hTERT 16 days following neutrophil co-culture. Dotted blue lines represent median telomere FISH fluorescence.; h) Representative Immuno-FISH micrographs (using telomere‐specific (CCCTAA) peptide nucleic acid probe and anti-53BP1 antibody) 20 days following co-culture; i) quantification of mean TAF and % TAF in MRC5 fibroblasts 8 and 20 days following neutrophil co-culture. Data are mean±S.E.M. of 3 independent experiments; J-L) mRNA expression of p21 (j), IL-6 (k) and IL-8 (l) 20 days after neutrophil co-culture; Data are mean±S.E.M. of 2-3 independent experiments. m) western blot and quantification of p21 and LaminB1 20 days after neutrophil co-culture. Data are mean±S.E.M. of 2 independent experiments.
Taxanes induce apoptosis and mitotic defects within human hair follicle K15+ epithelial stem/progenitor cell nichesAi) Paclitaxel treatment promotes mitotic arrest (orange arrows/pH3+ cells) and apoptosis (white arrows/caspase 3+ cells), within the K15+ bulge and K15+ proximal bulb outer root sheath (pbORS) stem/progenitor cell compartments of the human hair follicle (HF).Figure 4Ai) Paclitaxel treatment promotes mitotic arrest (orange arrows/pH3+ cells) and apoptosis (white arrows/caspase 3+ cells), within the K15+ bulge and K15+ proximal bulb outer root sheath (pbORS) stem/progenitor cell compartments of the human hair follicle (HF). 20 µm scale. Aii) High magnification montage demonstrating mitotic arrest (orange arrow) and apoptosis/caspase 3 positivity (white arrow) within the K15+ bulge. 10 µm scale. pbORS - proximal bulb outer root sheath. B) Representative immunofluorescence images of heightened cleaved caspase 3 immunoreactivity within the K15+ bulge following extended paclitaxel organ culture experiments 50 µm scale. EC - extended cultures. C) Graph showing significantly (p = 0.029 [**]) increased K15/caspase 3 double+ cells within the bulge following extended paclitaxel HF organ cultures. Welch's t-test performed using N of 8-9 HFs from 3 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error. D) K15+ cells of the human HF bulge express Ki-67 during extended organ culture experiments. Paclitaxel treatment did not significantly affect the number of bulge K15/Ki-67 double+ cells. Unpaired t-test performed using N of 8-9 HFs from 3 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error. E) Representative double immunofluorescence images of elevated γH2A.X immunoreactivity within the K15+ bulge following extended paclitaxel organ culture experiments (see methods). 50 µm scale. EC - extended cultures. F) γH2A.X analysis showing a significant (p = 0.065 [**]) increase in the number of cells with DNA double-strand breaks in the K15+ bulge following extended organ culture experiments. Mann Whitney U test performed using N of 5-6 HFs from 2 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.
Consequences of fusion-fission events.Figure 4(A) Schematic illustrations of mitochondrial tracking in four different experiments (i-iv) and corresponding Δψm traces (iv). Colored arrows indicate generation of hyperpolarizing (red) and depolarizing (green) daughters during fission. Note that after a fission event, fusion (black arrows) preferably occurred in the hyperpolarized daughter mitochondrion. (B) Depolarized mitochondria are selectively targeted for autophagy after a multi‐hour time lag. APs are labeled with LC3:GFP, which translocates from the cytosol to the AP's isolation membrane. MTR, a membrane potential dye that stains mitochondria irreversibly and is retained during depolarization, is used to pulse label mitochondria in INS1 cells (14 min, 50 nM) at different time points before detection of APs content. At the time set for detection, cells were treated with pepstatin A (10 μM) and E64d (10 μM) for 30 min to arrest digestion inside the APs, and then subjected to confocal microscopy (see schematic illustration of the Materials and methods). MTR pulse is used here to report on Δψm during the staining period. While mitochondria outside APs show bright MTR fluorescence, those localized in APs varied in fluorescence based on the time at which they were pulsed with MTR. Note that mitochondria inside APs have dim MTR FI if they were pulsed with MTR 1 h before autophagy was detected (circle, top panel), but bright MTR FI if pulsed 24 h before detection of autophagy (circle, bottom panel). Scale bar, 10 μm. (C) Distribution of MTR FI (given in Δψm) of AP‐localized mitochondria at different times before autophagy. Values are relative to cell's average MTR FI. In the x‐axis, a zero value represents the average Δψm and positive values represent depolarized mitochondria.
Super-active receptors are insensitive to NTD-mediated allosteric modulation.Figure 3Schematic representation of the super-active GluN1-E698C/GluN2B-L795C receptor locked in the "rolled" conformation, showing the NTD binding sites for spermine, zinc and ifenprodil, positive (+) and negative (-) allosteric modulators, respectively. Open circles indicate that the binding site is on the other side of the GluN2B NTD. The allosteric inhibitor H+ is also indicated, although its binding site remains i Zinc, ifenprodil and pH dose-response curves of wild-type (WT) GluN1/GluN2B receptors and mutant GluN1-E698C/GluN2B-L795C receptors. For comparison, zinc and ifenprodil dose-response curves of GluN1/GluN2B receptors lacking the whole GluN2B NTD (GluN1 WT/GluN2B-delNTD) are also shown (dashed lines; data from Rachline et al, 2005) Spermine (200 µM, pH 6.3) potentiation of WT GluN1/GluN2B and mutant GluN1-E698C/GluN-L795C receptors. Spermine (200 µM, pH 6.5) sensitivity is also shown for WT GluN1/GluN2B receptors and receptors lacking either the GluN1 (GluN1-delNTD/GluN2B WT) or GluN2B (GluN1 WT/GluN2B-delNTD) NTD (data from Mony et al, 2011) Glutamate and glycine dose-response curves of WT GluN1/GluN2B receptors and mutant GluN1-E698C/GluN2B-L795C receptors
Figure 5Quantification of sulfide levels in cerebrum and kidneys of Coq9+/+, Coq9R239X and Coq9Q95X mice (A). Qualitative measurement of hydrogen sulfide in cerebrum and kidneys of Coq9+/+ and Coq9R239X mice (B).
Figure 6A. HeLa cells were starved for 8 h with HBSS and subsequently exposed to SA (30 min, 300 µM), and immunostained for Sec24C or TRAPPC2 and eIF3. Scale bar 10 µm. B. Analysis of the proliferation status of the cells after 8 h starvation in HBSS. Top, representative images of starved and non-starved (CTRL) cells using EdU incorporation (see Materials and Methods). Scale bar 100 µm. Bottom, quantification of EdU incorporation in starved cells as a percentage of incorporation in control fed cells, N=3, mean ± s.d. C. HeLa cells were seeded at different confluency, treated with SA and stained for Sec24C and G3BP as an SG marker. Scale bar, 10 µm. Flow cytometry (FACS) analysis (right panels) was performed to evaluate the distribution of cell cycle phases in HeLa cell populations seeded at different confluency. The graph shows quantification of Sec24C mean intensity in SG puncta (normalized for the cytosolic Sec24C) at the indicated cell confluency. Mean ± s.e.m. of a representative experiment out of 5 biological replicates. n= 50-80. ns: not significant; ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test. D. Differentiation of podocytes. Western blot of growing and differentiated podocytes. Synaptopodin (SYNPO) was used as a differentiation marker, GAPDH as a loading control. E. Growing and differentiated podocytes were treated with SA (300 µM, 30 min) and stained for TRAPPC2 or Sec24C and eIF3. Scale bar, 10 µm. The graphs show quantification of TRAPPC2 and Sec24C (mean intensity) in SGs. Mean ± s.e.m. of three independents experiments. ****p<0.0001, Student's unpaired two-tailed t-test. F. Western blot analysis of total (RB) and phosphorylated (p-RB) retinoblastoma in growing (non-differentiated) versus differentiated podocytes. ß-actin was used as loading control. G. CDK kinase activity in growing and differentiated podocytes. Western blot using a specific antibody recognizing phosphoSer-CDK substrates was used on total cell lysates.
Figure 2A-B. LC-ESI-MS/MS chromatograms of midguts of flies carrying Act5Cts-GAL4-driven UAS-lacZ (control, A) or Las RNAi (B). C. Quantification of the content of ALA in midguts of flies with indicated genotypes using LC-ESI-MS/MS. Error bars indicate the SD of three independent experiments. Data information: Error bars represent SDs. Student's t-tests, ****p < 0.0001, and non-significant (NS) represents p > 0.05. D-F. Immunofluorescence images of the midgut section from the R4 region in Drosophila carrying esgts-GAL4-driven UAS-lacZ (D, control), Las RNAi (E), or Las RNAi in response to ALA administration (F). esg-GFP (green) indicates ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm G. Quantification of the number of esg-GFP+ cells, Dl+ cells, and pH3+ cells in experiments (D-F). n is indicated. The numbers of quantified guts from left to right are 17, 19, 18, 17, 19, 18, 17, 19, and 18. Data information: Error bars represent SDs. Student's t-tests, ****p < 0.0001, and non-significant (NS) represents p > 0.05. H. Quantification of the luciferase activity of midguts with indicated genotypes and manipulation. Error bars show the SD of six independent experiments. Data information: Error bars represent SDs. Student's t-tests, ****p < 0.0001, and non-significant (NS) represents p > 0.05. I-J. Immunofluorescence images of control (FRT40A, I) and Las RNAi (J) MARCM clones (green, outlined by white dotted lines) 10 days after clone induction (ACI). Pdm1 staining (red) was used to visualize ECs. K. Quantification of the ratio of Pdm1+ cells per clone of MARCM clones with indicated genotypes. n is indicated. Each dot corresponds to one clone. L. Quantification of MARCM clone size of experiments in (I-J). n is indicated. Each dot corresponds to one clone. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm (D Error bars represent SDs. Student's t-tests, ****p < 0.0001, and non-significant (NS) represents p > 0.05.
Identification of human S/T kinases modulating the ERα-14‑3‑3 phosphorylation‑dependent interactionA.Figure 6A. Schematic of phospho-dependent interaction of the estrogen receptor ERα with 14‑3‑3β. Phosphorylation of ERα promotes its interaction with 14‑3‑3 proteins. This interaction removes ERα from the E2-stimulated ERα dimerization and consequently inhibits ERα-DNA interactions and gene expression. A protein kinase that can modulate the binding of ERα to 14‑3‑3 proteins is unknown. B. Results of the phospho-Y2H screen to identify kinases modulating the interaction of the ERα with 14‑3‑3β. The interaction of ERα (prey) with 14‑3‑3β (bait) is promoted by the co-expression of wildtype kinases of PAK5, CDK17, BUB1 and NEK6, visible by the yeast growth on selective media in comparison to the control (three biological replicas are shown). The coexpression of the kinase‑dead mutants of PAK5 and CDK17 also promoted the interaction of ERα and 14‑3‑3β. In contrast the kinase‑dead mutant versions of BUB1(K821M) and NEK6(K74M&K75M) did not promote the Y2H interaction. C. Phosphorylation of ERα in mammalian cells. ERα was co-expressed with the indicated wildtype kinases or kinase‑dead versions in HEK293 cells and immunoprecipitated. Phosphorylation status of ERα was analyzed with an in-gel phosphostain (Pro-Q® Diamond phosphoprotein gel stain, Invitrogen), relative to the whole protein stain (SYPRO®Ruby protein gel stain, Invitrogen). D. Interaction of ERα and 14‑3‑3β in a GST pull-down assay. GST‑tagged 14‑3‑3β was expressed in E. coli and immobilized on beads. PA‑tagged ERα was co-expressed with either wildtype PAK5 or NEK6, the kinase‑dead (KD) version or without a kinase in HEK293 cells. Immobilized 14‑3‑3β was used to pull PA‑tagged ERα from the HEK lysates. Co-precipitation of ERα was successful in the presence of wild kinases, but not in their absence or with kinase‑dead mutant versions. E. Effect of NEK6 co-expression on ERα gene activation. Relative luciferase activity of ERα in the presence and absence of 1 nM E2 (17β-estradiol) was measured. A dose‑dependent reduction (ng plasmid transfected) in E2-Erα‑dependent luciferase expression was observed in HEK293T, MCF-7 and US-OS cancer cell lines. This effect was not observed with the NEK6(K74M&K75M) kinase‑dead version. Graphs represent ≥ 5 biological replicates, each derived from averaging 6 technical replicates. Error bars indicate SD across the biological replicates.
IBMPFD mutant expression or autophagic inhibition redistributes TDP-43 to the cytosol in mouse skeletal muscle.Figure 9.(A) TDP-43 immunostaining of quadriceps skeletal muscle from 15-mo-old control (Cont) or VCP-WT, -RH9, or -RH12 transgenic mice. A single myofiber is outlined in white. (B) TDP-43 immunostaining of quadriceps skeletal muscle from 3-mo-old mice treated with saline or 50 mg/kg/d intraperitoneal chloroquine (Chlq) for 4 wk. (A and B) Insets show one myonuclei from each field. Bars, 30 µm.