EMBO/sd-character-level-ner huggingface dataset:
Figure 4U2OS cells transfected with siCTL or siPRMT5 were subjected to DRIP-qPCR analysis. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; and **: p < 0.01. Western blotting of whole cell extracts from siCTL, siPRMT5, siDDX5 or siDDX5/ siPRMT5 transfected cells. U2OS cells transfected with siCTL, siPRMT5, siDDX5 or siDDX5/ siPRMT5 were subjected to DRIP-qPCR analysis. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; and **: p < 0.01.
Figure 4Neutralization of SARS-CoV-2 by the indicated VHH antibodies at nanomolar (A and B) or picomolar (C and D) concentrations. The neutralization experiment was performed ;n" indicates the number of independent biological replicates
Inhibition of fission attenuates mitochondria‐specific autophagy (mitophagy).Figure 6(A) Confocal images of FIS1 RNAi, DRP1‐DN and control cells coexpressing the AP marker LC3:GFP (green), and stained with MTR (red, control and FIS1 RNAi group) or mtDsRed (red, DRP1‐DN) and treated with pepstatin A and E64d to arrest digestion in late APs. A magnified area within a FIS1 RNAi cell shows APs with and without mitochondria (white and blue circles respectively). Scale bar, 5 μm. (B) Quantitative analysis of AP‐containing mitochondria in INS1 cells expressing FIS1 RNAi or DRP1‐DN. In each group the number of AP‐containing mitochondria was normalized to its control and found to be significantly different (*P0.001; **P0.001). (C) Altering fission does not impair ER autophagy and AP or lysosomal mass. INS1 cells expressing LC3:GFP were stained with ER Tracker to assess AP‐containing ER. The number of AP‐containing ER was not significantly different between control and FIS1 RNAi cells (P=0.51; n=11). Lysosensor stain (75 nM, 30 min) reveals no difference between control and FIS1 RNAi cells (P=0.21). The total AP number in DRP1‐DN cells is similar to that of the control (n=11, P=0.53).
Figure 3a) Schematic drawing of the lentiviral vectors used. Abbreviations as in Figure 1; EF1α prom: the human elongation factor-1α gene promoter; CD19CAR: chimeric antigen receptor (CAR) targeting the pan-B cell marker CD19 (for detailed information see Materials and Methods). b) A schematic timeline of the experimental setting. c) Percentage of CD19CAR expression on CD3+ T cells from each independent donor. d) Histograms of CD19 CAR expression on T cells form one representative donor before and after expansion. e) CD107a expression on transduced T cells (CD3+) after exposure to CD19+ target cells (Daudi) for 24 h (n=6). f) IFN-γ production in the supernatant was analyzed by ELISA after 24 h of exposure of transduced T cells to target cells (n=6). g) Relative viability of target cells (CD19+ Daudi), as an indication of T cell killing efficacy, were measured at different effector T cell to target cell ratio (E:T ratio) and normalized to the signal from target cells alone (n=6). h) Transduced T cells were labeled with a fluorescent cell membrane dye and co-cultured with CD19+ Daudi cells for 4 days. Dye dilution upon T cell division was monitored by flow cytometry and representative histograms are shown. i-m) Mice were treated by intravenous injection of NILV-S/MAR(Mock), LV(CD19CAR) and NILV-S/MAR(CD19CAR)-transduced T cells (8x106 cells/injection) at days 18, 21, 24 after tumor implantation. Tumor growth was monitored by caliper measurements. i-k) Tumor volume of individual mice in each treatment groups are presented. l) The average tumor volume of each treatment group is presented and the values on day 37 were statistically compared (n=12 in LV group, n=9 in NILV-S/MAR group and n=11 in Mock control group). m) The survival of mice is shown as Kaplan-Meier curves. n-p) Representative images showing infiltration of adoptively transferred human T cells into tumors. T cells were visualized by staining for human CD3 (red) and nucleus (blue). q) Quantification of CD3+ T cells from each treatment group is presented (n=5).
Sir4 RKR residues are important for heterochromatin silencing and proper localization of Sir4 at the nuclear periphery A) Example images of Nup49-RFP Sir4-GFP in wild-type (wt) cells (GA-10372; row 1); sir4 RKR cells (GA-10373; row 2); wt cells transformed with either empty plasmid (row 3), plasmid overexpressing wt Sir4 H‑BRCT (row 4), or plasmid overexpressing RKR mutant Sir4 H‑BRCT (row 5).Figure 4Example images of Nup49-RFP Sir4-GFP in wild-type (wt) cells (GA-10372; row 1); sir4 RKR cells (GA-10373; row 2); wt cells transformed with either empty plasmid (row 3), plasmid overexpressing wt Sir4 H‑BRCT (row 4), or plasmid overexpressing RKR mutant Sir4 H‑BRCT (row 5). The bar indicates 2 μm. Quantification of mean fluorescence intensity of Sir4-GFP using >100 cells. Error bars show the SD from 3 independent experiments. Quantification of cells containing Sir4 clusters at the nuclear envelope (NE). Data are represented as mean ± SD from 3 independent experiments where >100 cells were counted. Top: Schematic representation of the TEL7L::URA3 reporter used to assay silencing at telomeres. Bottom: wt cells (GA-503) transformed with either an empty plasmid (row 1), plasmid overexpressing wt Sir4 H‑BRCT (row 2), or plasmid overexpressing RKR mutant Sir4 H‑BRCT (row 3). Cells lacking endogenous sir4 (GA-5822) (row 4). Cells were grown in the absence of uracil and plated as 1:5 serial dilutions onto control YPAD or YPAD lacking uracil plates and imaged after 2 days. Top: Schematic representation of the HMR-EΔB::TRP1 reporter gene locus used for assaying silencing at the HMR locus. Bottom: wt cells (GA-485) transformed with plasmids as in panel D. Cells lacking endogenous sir4 (GA-6888) (row 4). Cells were grown in the absence of tryptophan and plated as 1:5 serial dilutions onto YPAD or YPAD lacking tryptophan plates and imaged after 2 days.
Bcl-2 blocks apoptosis during catagen of the hair follicle cycle.Figure 5A Histology of P19 catagen backskin HF of control (Ctr) and Bcl-2EOE mice. RS: retracting strand, Scale bar, 50 µm. B Analysis of length and width of retracting strands of control (Ctr) and Bcl-2EOE mice (n=4; multiple measurements/biological replicate; whiskers: min and max; boxes: median ± first and third quartile; **** p<0.0001, two-tailed unpaired Student's t-test). C Itgα6 and aCas3 Immunofluorescence staining of catagen tailskin HF of control (Ctr) and Bcl-2EOE mice (n=4 biological replicates). Dashed lines: retracting strands. Scale bar, 100 µm. D Quantification of aCas3+ cells in the RS of P19 Ctr and Bcl-2EOE mice (n=4 biological replicates; mean ± standard deviation (SD); **** p<0.0001, two-tailed unpaired Student's t-test). HF: hair follicle. E Hair-coat recovery in Ctr and Bcl-2EOE mice following depilation (n=3 biological replicates). F,G qRT-PCR for mRNA expression of the quiescence-related markers Bmp6 (F) and Fgf18 (G) in epidermis of control (Ctr) and Bcl-2EOE mice (P27), (n=4 biological replicates; mean ± standard error of mean (SEM); * p<0.05, *** p<0.001, two-tailed unpaired Student's t-test).
Figure 9A. Inhibition of Cdk1 at anaphase I. CDC20-mAR and CDC20-mAR cdc28-as1 cells were released from the metaphase I-arrest with CuSO4 at 7 h in SPM (t = 0) and treated with 1NM-PP1 at t = 40 min to inhibit Cdc28-as1 (arrows). Top, time-lapse series from the imaging of Mpc70-GFP, SPBs (Cnm67-RFP), and nuclei (TetR-RFP). Bottom, quantification of meiotic events. B. cdc20ts-mAR PDMC1-AMA1 PHSL1-CDH1 cells were released from the metaphase I-arrest with CuSO4 (t = 0) at 25°C. Subsequent incubation at 25°C allows progression through meiosis II (left), while a shift to 37°C at t = 50 min causes arrest at metaphase II (right). Mpc70-GFP and the PSM marker RFP-Spo2051-91 were imaged in cells fixed at the indicated times. RFP-Spo2051-91 is expressed from an episomal plasmid (Diamond et al., 2009). Images represent ≥ 90% of cells harboring the plasmid. Scale bar, 2 μm. Data information: data are representative of two independent experiments. C. cdc20ts-mAR PDMC1-AMA1 PHSL1-CDH1 cells containing CDC28 or cdc28-as2 were released from metaphase I at t = 0 and shifted to 37°C at t = 50 min to cause arrest at metaphase II. 1Na-PP1 was added at t = 100 min to inhibit Cdc28-as2. Microtubules (GFP-Tub1), Mpc70-GFP, and RFP-Spo2051-91 were imaged in cells fixed at the indicated times. Images represent ≥90% of cells carrying the RFP-Spo2051-91-plasmid. Scale bar, 2 μm. Data information: data are representative of two independent experiments. D. The regulatory network controlling MP assembly at different stages of meiosis. Green arrows: activation. Dashed, green arrows: latent activation overwhelmed by inhibition. Red, bar-headed lines: inhibition. Items in light grey are inactive or absent. Block arc: SPB outer layer. X: Cdc20-substrate that blocks PSM formation at metaphase II. See text for details.
a) Diagrams of the deletion–mutation constructs of Keap1 (left) and the corresponding immunoprecipitation assays (right).figf1(a) Diagrams of the deletion-mutation constructs of Keap1 (left) and the corresponding immunoprecipitation assays (right). Each Flag-tagged mouse Keap1 and mutant was expressed in HEK293T cells. At 22 h after transfection, lysates were prepared and immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS-PAGE and analysed by immunoblotting with anti-Flag, anti-p62, anti-Nrf2 and anti-actin antibodies. Data are representative of three individual experiments. (b) Diagrams of the deletion-mutation constructs of p62 (left) and the corresponding input (upper right) and pull-down assay (lower right). The MBP-tagged mouse p62 deletion mutants conjugated to amylose (AM) resins were incubated with purified GST-tagged mouse Keap1-DC. The pulled-down complexes with the MBP-p62 mutants were subjected to SDS-PAGE and revealed by staining with Coomassie brilliant blue. The bands corresponding to MBP-p62 and its mutants are indicated by black dots. Red arrowheads indicate the band corresponding to GST-Keap1-DC. For details of construct 14 see Supplementary Information, Fig. S3. (c) Alignment of the Keap1-interacting regions (KIR; red line) and the LC3-recognition sequences (LRS; green line) of p62 homologues in various species. Black and grey boxes indicate identical amino acid residues with complete and partial conservation, respectively. (d) Immunoprecipitation assays. Flag-tagged p62, KIR-deleted p62 (p62 ΔKIR) and a p62 mutant defective in oligomerization (p62 K7AD69A) were expressed in primary mouse hepatocytes by the adenovirus system (left) or in HEK293T cells by transfection (right). Cell lysates were immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS-PAGE and analysed by immunoblotting with anti-p62 and anti-Keap1 antibodies. The bands corresponding to Flag-p62, endogenous p62, Keap1 and actin are indicated. The data shown are representative of three separate experiments. (e) Interaction of endogenous p62 with Keap1. Lysates prepared from the human hepatocellular carcinoma cell line Huh-1 were immunoprecipitated with anti-p62 antibody or anti-Flag antibody (negative control) followed by immunoblotting with antibodies against p62 and Keap1. The bands corresponding to endogenous p62, Keap1 and IgG heavy chain (IgG H.C.) are indicated. The data shown are representative of three separate experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.
Figure 7A. Western blot analysis of lysates from Pmp22+/- and WT sciatic nerves at P10 shows that Akt phosphorylation (S473) is increased in Pmp22+/- nerves, results are mean ± SEM, p=0.0317, two-tailed Mann Whitney U test, representative of two independent experiments. B. Western blot analysis of Tace using lysates from Pmp22+/- and WT sciatic nerves at different stages of development. The arrowhead indicates the pro-protein and the arrow shows the cleaved active form of Tace. Representative of two independent experiments. B. Tace activity as measured in Pmp22+/- and WT sciatic nerves at P30 (n=6 mice per genotype, results are mean ± SEM, p=0.0070, two-tailed Mann Whitney U test). C. Representative images of semithin section analysis of sciatic nerves at P45 from Pmp22+/- saline and Pmp22+/- Niaspan-treated mice. Niaspan was administered daily by i.p. injection at 160mg/Kg starting at P15 for 30 days.C'. The percentage of tomacula in sciatic nerves was assessed by ultrastructural analysis, results are mean ± SEM, n= 6 animals per condition, p=0.0022, two-tailed Mann Whitney U test, representative of two independent experiments.D. G-ratio analysis on semithin sections of sciatic nerves shows that increased myelin thickness in Pmp22+/- sciatic nerves at P45 is restored to normal values following Niaspan treatment. Mean g-ratio values, WT saline-treated: 0.69 ± 0.002, 2052 fibers, n=5 animals; Pmp22+/- saline-treated: 0.66 ± 0.003, 2643 fibers, n=6 animals; Pmp22+/- Niaspan-treated, 0.68 ± 0.002, 2311 fibers, n=6 animals. WT saline as compared to Pmp22+/- saline-treated, p<0.0001; Pmp22+/- saline-treated as compared to Pmp22+/- Niaspan-treated, p=0.0002; Pmp22+/- Niaspan-treated as compared to WT saline, p=0.0899; repeated measures ANOVA.E. The number of myelinated fibers is similar between the 3 groups analyzed, p=0.4362, nonparametric one-way ANOVA, followed by Dunn's post hoc test.
Figure 4A. Schematic flow overview of the process for ER junction quantification. B. Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD-GFP (green) and SiR-lysosome-stained endosomes/lysosomes (orange). COS7 cells in the middle panels overexpress mCherry-RILP (magenta). The COS7 cells in the lower panels overexpress mCherry-TMEM55B (magenta). Zoom insets show region of interest containing the ER (TMD-GFP) (green) and junction analysis of the skeletonized ER where the number of ER junctions representing the quantified average. Scale bars: 15µm. zoom insets: 3µm. C. Quantification of the number of ER junctions per µm2 from panel (B) as resulted from ER junction analysis. Graph represents mean ±s.d. of analyses at multiple locations within one cell (n=15 from 3 independent experiments). Significance two-tailed student t-test. ***P<0.001.
Figure 6A. Distribution of enriched GO terms (Biological Processes) in J34c DE genes grouped into nine functional categories. Bar chart represents the number of expected and observed hits for GO terms associated with 'Immune response'. B. Gene interaction network for J34c DE genes (filled nodes) and additional highly connected genes (transparent nodes). Node size reflects the number of network connections.
Figure 7 MTT assay was performed to assess cell viability 24 hours post treatment. Results are normalized relative to untreated controls and presented as survival rate percentage. The indicated mean and SE for the results are determined from three biological replicates (n=3) and technical duplicates. A JurkatTat cells were treated with the indicated concentrations of the PH compounds. MTT assay was performed to assess cell viability 24 hours post treatment. Results are normalized relative to untreated controls and presented as survival rate percentage. The indicated mean and SE for the results are determined from three biological replicates (n=3) and technical duplicates.B PBMCs from healthy donors were treated with the indicated concentrations of the PH compounds. MTT assay was performed to assess cell viability 24 hours post treatment. Results are normalized relative to untreated controls and presented as survival rate percentage. The indicated mean and SE for the results are determined from three biological replicates (n=3) and technical duplicates.C PBMCs from healthy donors were treated with the indicated concentrations of the PH compounds in combination with 10 nM PEP005.
Figure 1A. Dose-response relationship between icv LPS injection and mice weight loss (n=6 to 12). Data are expressed as means ± SEM. *p<0.05, **p<0.01, ***p<0.001, color-coded asterisks indicate a significant difference from the experimental condition assigned to the respective color-coded curve. B, C. Analysis by real-time PCR Arrays of interleukins, cytokines (B) gene expression in the hypothalamus 6h after an acute icv injection of LPS vs saline in WT mice (n=6 per group). LPS-induced fold up-regulation vs saline condition was calculated using the ΔΔCT method according to the manufacturer's protocol. B, C. Analysis by real-time PCR Arrays of CC chemokine ligands and receptors (C) gene expression in the hypothalamus 6h after an acute icv injection of LPS vs saline in WT mice (n=6 per group). LPS-induced fold up-regulation vs saline condition was calculated using the ΔΔCT method according to the manufacturer's protocol.
ROS accumulation, STAT3 activation and late-onset spontaneous development of hepatocellular carcinoma in PR-SET7ΔHepA mice.Figure 5A Analysis of reactive oxygen species (ROS) accumulation in frozen liver sections from 120-day-old (P120) and 240-day-old (P240) PR-SET7ΔHepA mice and control littermates (WT). B Immunohistological staining of liver sections from P240 animals with phospho-STAT3 antibody. C Western blot analysis of liver extracts from P45, P120 and P240 mice with STAT3 and phospho-STAT3 antibodies. D Macroscopic appearance of livers in P240 PR-SET7ΔHepA mice and control littermates (WT). E Serum alanine aminotransferase (ALT) levels in P45, P120 and P240 mice. Bars represent mean values of ALT levels and SEM from liver extracts of five individual mice. *P-value < 0.01. F Representative hematoxylin and eosin staining of liver sections from P240 PR-SET7ΔHepA mice and control littermates (WT). Two different magnifications are shown. G Relative mRNA levels of selected oncofetal genes. Bars represent mean Afp, H19, GPC-3 and CTGF mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 240-day-old mice. The data are presented as fold over values obtained with wild-type samples. *P-value < 0.01.
a) A representative ITC profile of the titration of Keap1-DC with p62M7 (residues 168–391).figf3(a) A representative ITC profile of the titration of Keap1-DC with p62M7 (residues 168-391). The upper panel shows the raw ITC thermograms and the lower panel shows the fitted binding isotherms. (b) Immunoprecipitation assays. Flag-tagged Keap1 was co-expressed with increasing concentrations of green fluorescent protein (GFP)-p62 (lanes 3-6) in HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS-PAGE and analysed by immunoblotting with anti-Flag, anti-p62 and anti-Nrf2 antibodies. The bands corresponding to Flag-Keap1, endogenous p62, Nrf2 and actin are indicated. Data are representative of three independent experiments. (c) The competitive p62 activity against Keap1 was measured by luciferase assay. The expression plasmids for Nrf2, Keap1 and p62 wild-type (w.t.) or its mutants were transfected into Hepa1 cells along with pNqo1-ARE reporter plasmid and pRL-TK as an internal control. At 36 h after transfection, the luciferase activity was measured in accordance with the instructions provided by the manufacturer. Assays were performed twice in triplicate. Data are means and s.d. for six determinations. (d) Immunoblot analysis. Flag-tagged p62 and its mutants defective in interacting with Keap1 were overproduced in wild-type and p62−/− primary mouse hepatocytes by the adenovirus system. At 48 h after infection, total cell lysates and nuclear fractions were prepared and subjected to immunoblot analysis with the antibodies specified. The bands corresponding to Flag-p62, endogenous p62, Keap1, Nrf2, Nqo1, actin and Lamin B are shown. Data are representative of three independent experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11. (e) Quantification of mRNA levels of the detoxification enzymes Nqo1, Gstm1 and Cyp2a5 in hepatocytes overexpressing Flag-p62 and its mutants. Total RNAs were prepared from non-infected or infected hepatocytes and reverse transcribed into their respective cDNAs, which were used as templates in real-time PCR analysis. Values were normalized to the amount of each mRNA in the non-infected hepatocytes. The experiments were performed three times. Data are means ± s.d. for three experiments.
Figure 1Survival curve of N. furzeri in the FLI facility. Recording of deaths starts at age of 5 wph, which corresponds to sexual maturity, and the colored dashed lines indicate the three age groups analysed in this study (5 animals/group), namely 5 weeks post hatching (wph, young, sexual maturity), 12 wph (adult) and 39 wph (old, past median lifespan) of a wild-derived strain that exhibits a median lifespan of 7-8 months. Principal component analysis (PCA) of brain samples based on the abundance of all proteins identified by label-free mass spectrometry. The smaller dots represent individual samples and the larger dots the centroids of each age-matched group. Ellipses represent 95% confidence intervals. The percentage of variance explained by the first two PC axes is reported in the axis titles. Global protein-transcript correlation for each sample, grouped by age. RPKM and iBAQ values were used to estimate transcript and protein levels from matched RNA-seq and TMT-based proteomics data obtained from the same animal. An ANOVA test was performed to evaluate significance among the age groups (mean correlation at 5 wph: 0.48; at 12 wph: 0.43; and at 39 wph: 0.33; p = 3.05e-07, n=5 per age group). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range. Scatter plot of log2 fold changes for genes differentially expressed both at transcript and protein levels (adj. p < 0.05). The color gradients indicate gene density in the regions where individual points overlap. Numbers of genes in each quadrant and the value of Pearson's coefficient of correlation, r, are reported for each graph. Solid lines represent a spline fit (r = 0.505 for genes significantly affected at both transcript and protein levels, p < 2.2 x 10-16, D) (r = 0.126, p = 0.007, E). Mechanisms affecting protein abundance during aging. Bar plots are based on all the proteins affected in either one of the age comparisons (adj. p < 0.05). Proteins were divided in the following five groups: (i) proteins and transcripts with significant and consistent changes (dark brown), (ii) proteins with significant changes, and with consistent changes of the transcripts (light brown), (iii) proteins with no transcripts detected (dark gray), (iv) proteins with transcripts whose translation is potentially regulated by miRNAs (light gray), as assessed by the workflow displayed in (Figure EV2D), (v) all the remaining proteins that we classified as regulated by other post-transcriptional mechanisms (violet). pgs = protein groups. Bar plots representing enriched KEGG pathways among genes that showed significant changes at both transcript and protein levels in aging. Genes were grouped according to the four possible patterns of transcript and protein regulation, as visualized by their positions in the four quadrants shown in (D) and (E), respectively. Only pathways significantly enriched (FDR < 0.05) are shown. The complete list of enriched pathways is reported in Dataset EV4.