EMBO/sd-character-level-ner huggingface dataset:
– Tau protein mediates the deleterious effect of stress on the dendritic morphology and PSDs of newborn granule neuron.Figure 5A Schematic diagram of the experimental design. B Representative images of 8-week-old newborn granule neurons of WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice infected with a PSD95-GFP-expressing retrovirus. In the WT CNP image a schematic representation of Sholl´s analysis is shown.C-D Quantification of total dendritic length (C) and Sholl´s analysis (D) of 8-week-old newborn granule neurons (n= 3 mice per experimental condition). E-F Representative images of an 8-week-old newborn granule neuron, in which proximal dendrites are highlighted (E), and of PSDs (green) in proximal dendrites belonging to WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice (F).G-H Quantification of the number of PSDs/µm (G) and PSD area (H) in each dendritic branching order of 8-week-old newborn granule neurons (n= 3 mice per experimental condition). I-J Representative Western Blot (I) and quantification (J) of the levels of GluR1 subunit of AMPA receptors in hippocampi of WT and Tau-/- mice subjected or not to the Porsolt test (n= 6 mice per experimental condition). K Representative image showing 8-week-old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field (indicated by a white square). L-M Representative images (L) and quantification of the area (M) of MFTs of 8-week-old newborn neurons in the CA3 region of WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice (n= 3 mice per experimental condition). Arrows indicate MFTs.
Figure 4(A-E) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of condensin (35oC) in smc2-8 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of condensin (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05; **p < 0.01; ***p < 0.001. (F) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of condensin.
Figure 6A Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 cells stimulated with poly (I:C) or poly (dA:dT) at the indicated time points. B Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 and L929 cells infected with VSV-eGFP at the indicated time points. C, D qRT-PCR analyses of the mRNA abundance of IFNAR1 and IRF3 in A549 cells transfected with poly (I:C)-LMW (C) or infected with VSV-eGFP (D) for 16 hrs, followed by treatment with Act D (5 μg/mL) for the indicated times. E qRT-PCR analyses of the proportion of IRF3 and IFNAR1 mRNAs in polysome fractions from A549 cells transfected with poly (I:C)-LMW for 16 hrs. F Relative luciferase activities of IFNAR1-3' UTR or IRF3-5' UTR with wild-type (WT) m6A sites in 293T cells that were transfected with poly (I:C)-LMW or infected with VSV-eGFP for 24 hrs. Renilla luciferase activity was measured and normalized to firefly luciferase activity. G Immunoblot analyses of IRF3 and IFNAR1 in shCtrl and shWTAP A549 cells, followed by infection with VSV-eGFP at the indicated time points. H, Immunoblot analysis of IRF3 and IFNAR1 in A549 cells transfected with poly (I:C)-LMW or infected with VSV-GFP for 16 hrs and then treated with PR-171 or DMSO for additional 4 and 8 hrs. Representative images from 3 independent biological experiments are shown (H), protein abundance of IRF3 and IFNAR1 in A549 cells transfected with poly (I:C)-LMW or infected with VSV-GFP for 16 hrs and then treated with PR-171 or DMSO for additional 4 and 8 hrs. the relative levels of IFNAR1 (left) and IRF3 (right) are presented as the mean ± SD (I).
Figure 1(A) Picrosirius red (PSR) staining of collagens (red) in PDAC tissue following surgical resection. Scale bar represents 200 µm. (B) Quantification of PSR in low-grade (1-2) and high-grade (3) tumors indicated in panel A as percentage of area. n=15 primary PDAC samples. Data information: data are represented as mean±SD, Student's t-test. (C) Quantification of tumor cell percentage as scored by pathologists in low- and high-grade PDAC indicated in panel A. n=15 primary PDAC samples. Data information: data are represented as mean±SD, Student's t-test. (D) PSR and α-SMA staining of indicated PDAC cell lines which were injected in NSG mice. Scale bar represents 200 µm for PSR and 100 µm for α-SMA images. (E) Quantification of stainings indicated in panel D. Three tumor ROIs of a tumor derived from epithelial cell lines (Capan-2 and AsPC-1) and mesenchymal cell lines (MIA PaCa-2 and PANC-1) are displayed and averages were pooled for analysis. Data information: data are represented as mean±SD, Student's t-test. (F) IHC staining of α-SMA in 3D organotypic co-cultures of PS-1 cells and indicated PDAC cell lines. Scale bar represents 100 µm.
Figure 3(a - c) Kymograph of salt-resistant Scc1GFP-TEV-cohesin diffusing past (a) TetO-DNA-bound TetRHalo-TMR. (a - c) Kymograph of salt-resistant Scc1GFP-TEV-cohesin diffusing past (b) DNA-bound Halo-TMREcoRIE111Q. (a - c) Kymograph of salt-resistant Scc1GFP-TEV-cohesin diffusing past (c) a DNA-bound TMR-labelled nucleosome. (d) Kymograph of salt-resistant Scc1GFP-TEV-cohesin failing to bypass DNA-bound QDotEcoRIE111Q. DNA was stained with Sytox Orange.
Figure 6(A) Maximum intensity projection shows trajectories of moving P. berghei, P. yoelii and P. falciparum sporozoites (green), engaging with CD31-labeled vascular endothelia (magenta). Scale bars, 25 μm. See movies EV4 - EV6. (B) Lymphatic and blood vessel invasion of P. berghei, P. yoelii and P. falciparum sporozoites moving in mouse dermis. Combined invasion events observed in 4 min videos recorded at 5, 10 and 20 min after intradermal injection of sporozoites (left panel) and 60 and 120 min after intradermal injection (right panel). n = number of videos scored for each Plasmodium species. To view a blood vessel entry event by P. yoelii or P. falciparum, see movies EV7 and EV8. There were no statistically significant differences in lymphatic or blood vessel entry among the three Plasmodium species at 5 to 20 min after sporozoite inoculation whereas there was a statistically significant increase in lymphatic entry by P. falciparum sporozoites compared to P. berghei and P. yoelii sporozoites at 60 to 120 min after inoculation (Kruskal-Wallis test, p<0.05).
Figure 7(A) Schematic to illustrate the goal of this experiment. (B) Schematic illustration of the experiments (C) Representative immunofluorescence images showing exogenous CENP-AmRFP-DHFR reloading in the presence (control), but not in the absence (sample) of endogenous CENP-A and CENP-C. Cells with centromeric CENP-A are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP-A. (D) Quantification of relative number of cells showing centromeric eCENP-AmRFP-DHFR in the indicated conditions. Error bars represent SEM from 3 independent experiments. (E) Bar plot of CUT&RUN qPCR quantification using CENP-A antibody and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats. (F) Representative immunofluorescence chromosome spreads showing reloading of exogenous eCENP-CmRFP-DHFR in the presence (control) and in the absence (sample) of endogenous CENP-A and CENP-C. Cells were arrested with Colcemid for 3 h prior to spread. Cells with centromeric CENP-C are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP-C. Chromosomes in inset are highlighted using a white dashed line. (G) Quantification of relative number of cells with eCENP-CmRFP-DHFR in the indicated conditions. Each dot represents one independent experiment with at least 30 cells per condition. (H) Bar plot of CUT&RUN qPCR results using CENP-C antibody and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats. Each dot represents one independent experiment.
Figure 4A. Experimental scheme for B-G. Whole-cell patch-clamp recordings were made from neurons in CA1 of organotypic hippocampal slices treated with inactive control, GSK3α or GSK3β inhibitor, or AR-18 (all at 10 µM). EPSCs were elicited with CA3 Schaffer collateral stimulation, and low-frequency stimulation (LFS) was used to induce LTD. For inactive control, GSK3α and GSK3β inhibitor compounds, the experiment was performed blind with respect to treatment group. B. Representative traces for C, D, where baseline responses (dark trace) are taken between 10 and 0 minutes before LTD induction, and responses after LTD induction (light trace) are taken between 40 and 45 minutes after the end of LTD induction. C. Time course of the peak AMPAR-mediated synaptic response to Schaffer collateral stimulation before, during, and after the induction of LTD by LFS (300 pulses at 1 Hz), in control (inactive control), GSK3α (blue) and GSK3β (orange) inhibitor, and AR-18-treated neurons (yellow). Drug treatment was for 1 hour before recording at 10 µM for all compounds. Error bars represent SEM. D. Quantification of average synaptic depression from the last 5 minutes of the time course shown in C. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test. E. As B, for F and G. F. Time course of the peak AMPAR-mediated synaptic response to Schaffer collateral stimulation before, during, and after the induction of mGluR-dependent LTD by PP-LFS (900 paired pulses, separated by 50 ms, at 1 Hz), in control (inactive control) and GSK3α inhibitor-treated neurons (blue). Drug treatment was for 1 hour before recording at 10 µM for all compounds. Representative traces for synaptic responses before (dark trace) and after (light trace) LTD induction are shown above the time course. Error bars represent SEM. G. Quantification of average synaptic depression from the last 5 minutes of the time course shown in F. Error bars represent SEM. No statistically significant difference was found, according to the Mann-Whitney U test.
Figure 2SAGA is phosphorylated in response to nutrient starvation. (A,B) 4-20% gradient SDS-polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium (R) or starved for 45 minutes (S). SAGA was purified using endogenously TAP-tagged Ada1. A fraction of each eluate was loaded and either stained with silver, to detect all proteins (A),. A strain without any TAP tag is shown as a negative control for the purification. The graph on the left of the gel in B shows the fluorescence intensity of the phospho-specific stain, which was quantified along the left lane in blue (rich) and the middle lane in red (starved), using ImageJ. The area of one peak, which corresponds to the bands marked with arrowheads and was coined p55, is 1.8-fold larger in SAGA purified from starved cells, as compared to rich conditions. Below is an anti-HA immuno-blot (IB) of each eluate, to reveal the amount of Ada1-HA bait recovered. Shown are gels that are representative of 3 independent experiments. A.U., arbitrary units. SAGA is phosphorylated in response to nutrient starvation. (A,B) 4-20% gradient SDS-polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium (R) or starved for 45 minutes (S). SAGA was purified using endogenously TAP-tagged Ada1. A fraction of each eluate was loaded and either stained with Pro-Q® Diamond, which stains phosphorylated proteins (B). A strain without any TAP tag is shown as a negative control for the purification. The graph on the left of the gel in B shows the fluorescence intensity of the phospho-specific stain, which was quantified along the left lane in blue (rich) and the middle lane in red (starved), using ImageJ. The area of one peak, which corresponds to the bands marked with arrowheads and was coined p55, is 1.8-fold larger in SAGA purified from starved cells, as compared to rich conditions. Below is an anti-HA immuno-blot (IB) of each eluate, to reveal the amount of Ada1-HA bait recovered. Shown are gels that are representative of 3 independent experiments. A.U., arbitrary units.
Accumulated shorter LSP transcripts in TEFM-depleted mitochondria do not contribute to mtDNA replication.Figure 4In organello transcription analyses using heart mitochondria isolated from 8-week-old control and Tefm knockout mice (n= 5 mice for each group). The steady-state levels of 12S rRNA and tRNAV were used as endogenous controls, due to their unchanged levels. VDAC was used as loading control for the equal input of mitochondrial extracts. This experiment has performed five times with either two or three replicates. Northern blot analyses of 7S RNA level in 4- and 8-week-old control and Tefm knockout mouse hearts (n= 12 mice at each time-point for each group). 18S rRNA was used as loading control. RNA from tissue-specific Mterf4 knockout mouse hearts and tissue-specific Polrmt knockout mouse hearts were loaded as positive and negative control, respectively. Southern blot analyses of mtDNA to obtain 7S DNA in 8-week-old control and Tefm knockout mice. Heart mitochondria from 3 control or knockout mice were pooled for preparation of mtDNA. Equal amounts of mtDNA shown on the top of the gel, were loaded as the endogenous control. This experiment has performed three times. In organello replication assays were performed on heart mitochondria isolated from 8-week-old control and Tefm knockout mice (n= 5 mice for each group). VDAC was used as loading control. Northern blot analyses of the 7S RNA levels in 4- and 8-week-old control and Tefm knockout mouse hearts (n= 12 mice for each group). 5S rRNA was used as loading control. RNA from tissue-specific Mterf4 knockout mouse hearts and tissue-specific Polrmt knockout mouse hearts were loaded as positive and negative control, respectively. Mapping 3' termini of mitochondrial 7S RNA/early LSP transcripts in control (L/L) and Tefm knockout (L/L, cre) mouse hearts at the age of 8 weeks. A 3' RACE was performed using linker-ligated RNA and a forward primer corresponding to the first 45 bp from the transcription initiation site at LSP. CSB, conserved sequence block.
Figure 2(a-c) Dimension reduced single cell transcriptomic data is visualized through multiple Uniform Manifold Approximation and Projections (UMAPs). The colors illustrate (a) patient cohorts, (b) disease status, (c) and cell type identity (d) Differential gene expression between end stage lung disease patients and controls across cohorts was compared for the indicated cell type identities. The color code demonstrates similarities of gene expression changes calculated by Pearson correlation of the t-value coefficient, which represents differences in the likelihood of detection for any gene between health and disease. (e) The heatmap shows the top 79 genes differentially expressed in the indicated cell type identities. (f-h) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in (f) alveolar epithelial cells, (g) fibroblasts, and (h) macrophages when compared to control tissue. The boxes represent the interquartile range, the horizontal line in the box is the median, and the whiskers represent 1.5 times the interquartile range (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11).
Figure 1(A) Proteolytic processing of Opa1. L1 is cleaved by both Oma1 and Yme1L, while L2 is only cleaved by Oma1. (B) Western blotting of WT and Drp1-KO MEFs with or without FCCP treatment (30 min) using the indicated antibodies. (C) Quantification of band intensity. Values are average ± SD (n = 3). Significance was calculated using ANOVA with post-hoc Tukey: **p<0.01, ***p<0.001.
Figure 3(A) Assay of FoxA2 mRNA by RT-PCR in Tcf7l1 null cells on day 1 to 4 of differentiation without CH. Average and SD of three independent experiments. (B) Gene expression analysis of endodermal associated genes by RT-PCR in control wild-type and Tcf7l1 null cells on day 4 of differentiation in the presence and absence of 3 µM CH. Average and SD of three independent experiments. (C) FoxA2 (green) and Oct4 (red) immunostaining of Tcf7l1 null ES cells on day 4 of differentiation in the presence or absence of 3 µM CH. DAPI staining in blue. (D) Assay of CXCR4, Hex and Sox17 mRNA by RT-PCR in control wild-type and Tcf7l1 null cells on day 6 of differentiation in the presence and absence of 3 µM CH. Average and SD of three independent experiments.
Fig.Figure 3Control of epidermal autophagy by TOR signalling. (A) Epidermis of third instar larvae expressing the autophagosome marker GFP-Atg8a together with constructs for up- or down-regulating the autophagy pathway in the epidermis. Healthy epidermis contains few autophagosomes, but artificially activating autophagy through overexpression of Atg1 or blocking TOR signalling leads to accumulation of autophagosomes. (B) Higher magnification of the areas marked by magenta boxes in (A). Control of epidermal autophagy by TOR signalling. (C) Quantification of Atg8a puncta in an area of 10000 µm2, n=6-8 larvae each genotype. We assumed unequal sample size and unequal variances and calculations were performed. Values are presented as box plots. Box plot elements are: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. (D, E) Transmission electron micrographs of sections through the epidermis of larvae with elevated autophagy at two different magnifications. AP: autophagosome; Ax: cross-section of an axon; BL: basal lamina; C: cuticle; HD: hemidesmosome; M: mitochondrion; N: nucleus. F Disruption of epidermal morphology after upregulation of autophagy. The membrane marker Src-GFP is lost from large areas of the epidermis and nuclei have lost their regular spacing. Image in control and rictori panels are from the time laps in Appendix Fig S5B and Movie EV12. G Schematic for temporally controlled transgene expression and imaging in (H). Gene expression is induced at the end of the second larval instar, live imaging started 6 h later and continued for an additional 6 hours. H Example of membrane dynamics after time-controlled Atg16B expression. Src-GFP containing material appears to be taken out of and eventually detached from lateral cell membranes (arrows). Over the period of observation, abnormal accumulation of GFP is seen in the nuclei (cyan asterisks). t = 0 is 6h after A58-Gal4 activation. Image from Movie EV9.
Figure 6A. Approximately 1x109 CFU of Fn 12230, US1 (∆fadA), and lam suspended in carboxymethylcellulose (CMC) were orally administered to C57BL/6 mice three times a week for 10 weeks. CMC alone was administered as a control. Maxillae from mice inoculated with CMC alone (n=5), Fn 12230 (n=5), US1 (∆fadA; n=5), and lam (n=4) were harvested and fixed in 4% paraformaldehyde and stored in 70% ethanol, followed by microCT (μCT) scanning using a Scanco vivaCT 80 system at 55 kVp, 145 µA, and 250 ms integration time. Shown in the figure are reconstructed grayscale images. ImageJ was used to measure difference in bone height from the cementoenamel junction (CEJ) to the alveolar crest between palatal roots of first and second molars (see arrows). B. The average bone loss of each group shown in (A), with the lines above each bar representing standard deviations. *p< 0.05. **p< 0.01 (t-test).
a) Percentage of HeLa cells transfected with vectors encoding GFP or GFP–IRGMd that were rounded 48 h post-transfection.figf7(a) Percentage of HeLa cells transfected with vectors encoding GFP or GFP-IRGMd that were rounded 48 h post-transfection. (b) HeLa cells transfected with plasmids encoding GFP or GFP-IRGMd were stained with 7-AAD after 48 h and percentages of GFP+ cells that were also 7-AAD+ were quantified. (c) HeLa cells transfected with vectors encoding GFP-IRGMd versus GFP-IRGMdS47N and YFP-IRGMb (vector encoding GFP only was transfected as a control) were stained with 7-AAD after 48 h, and percentages of GFP+ (for IRGMd and IRGMS47N) or YFP+ (for IRGMb) cells that were also 7-AAD+ were quantified. (d) HeLa cells were transfected with plamsids encoding GFP-IRGMd, GFP-IRGMDS47N or YFP-IRGMb (vector encoding GFP only was transfected as a control), and after 48 h were stained with propidium iodide (PI). Percentages of GFP+ or YFP+ cells that were also PI+ were quantified. (e) Images of HeLa cells transfected with vectors encoding GFP-IRGMd (bottom) were stained for active caspases 3 and 7 using FLICA dye. Staurosporine (STS; middle images) was used as a positive control. Cells were transfected with vectors encoding GFP only as a control (top). (f) HeLa cells were transfected with plasmids encoding GFP-IRGMd and were stained with antibodies against HMGB1 (absence of nuclear HMGB1 stain is a marker of HMGB1 release) or DAPI. Top: images indicate GFP-IRGMd (left), HMGB1 (middle) and DAPI (right) localization. Bottom: phase-contrast microscopy image of cells (left) and image merged from other panels (right). Scale bars, 5 μm. (g-i) Cells were transfected with plasmids encoding GFP or GFP-IRGMd, and control or DRP1 siRNA, as indicated. After 48 h, (g) cells were stained with 7-AAD and percentages of GFP+ cells that were also 7-AAD+ were quantified, (h) rounded cells were quantified, or (i) cells were stained with HMGB1 and HMGB1+ nuclei were quantified. Data are means ± s.e.m. (n = 3). Asterisk indicates P 0.05 and double asterisk indicate P 0.01 (t-test).
Figure 6(a, b) Scatter plots stratify genes based on the protein lung function associations (y-axis) and cell type specific ILD associations (x-axis). The size of the dots represents the detection level of each gene in the corresponding cell type. Colors highlight genes with marginal associations at the protein and RNA levels. (c, d) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in (c) basal cells, and (d) alveolar epithelial cells when compared to controls. The boxes represent the interquartile range, the horizontal line in the box is the median, and the whiskers represent 1.5 times the interquartile range (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11). (e, f) The scatter plots show the positive correlation of the indicated proteins in BALF (MS-intensity, x-axis) with meta lung function (y-axis). (g-j) Immunofluorescence analysis of the indicated proteins and cell type markers in IPF (n=3) and control samples (n=3).