EMBO/sd-character-level-ner huggingface dataset:
Figure 3A MS/MS analysis of UbcH7 in the absence (top) or presence (bottom) of PknG to reveal ubiquitination sites. Tryptic peptides spanning residues ranging from 74 to 100 were identified as the high-confidence peptides possessing di-Gly modifications. The ubiquitinated residue (Lys82) in the peptide sequence is highlighted in red. The peak heights are the relative abundances of the corresponding fragmentation ions. Matched amino terminus-containing ions (b ions) are indicated in red, and matched carboxyl terminus-containing ions (y ions) are indicated in blue. Only the major peaks that were identified are labeled. B Ubiquitin conjugation assay of UbcH7 or its mutants catalyzed by GST-tagged PknG at 37°C for 30 min. Reaction products were immunoblotted using antibodies against UbcH7 (top panel), Ub (middle panel), and the GST tag on PknG (bottom panel). C In vitro ubiquitin discharge assay of UbcH7~Ub with or without PknG. The reaction products were immunoblotted as in B. D In vitro isopeptidase assays of PknG with or without ubiquitin-aldehyde (Ub-Ad). The reactions were performed at 37°C for 30 min and analyzed as in B. E In vitro ubiquitin conjugation assay of UbcH7 by PknG, PknG P57A, or PknG I87A. The reaction products were immunoblotted as in B. F In vitro ubiquitin discharge assay of UbcH7~Ub by PknG or its isopeptidase activity-dead mutant (PknG I87A). The reaction products were immunoblotted as in B.
Active translation is important for HXT2 mRNA release to the bud.Figure 2A, Inhibiting translation by either applying Cycloheximide (CHX) or Verrucarin A (VerruA) leads to the retention of HXT2 mRNA in the mother cell after MAT. Chloro: chloroform; solvent control for VerruA; NT: not treated; control for CHX. FISH of CHX or VerruA-treated cells. Chloro: chloroform; solvent control for VerruA; NT: not treated; control for CHX. C CHX treatment leads to increased transcript stability. Quantitative PCR of HXT2 mRNA in CHX-treated cells in comparison to prt1-1 mutant cells. The data represents 3 independent experiments and the standard deviation is given. D Translation of HXT2 mRNA is more active late in the cell cycle. The HXT2 mRNA level bound to either di-/trisomes and polysomes (≥ 4 ribosomes) over monosomes was determined by extraction of mRNA from cells elutriated and harvested at G1/S or M-phase followed by qPCR. At G1/S similar levels of the HXT2 mRNA were found in the different ribosome fractions, whereas in M-phase more mRNA was bound to polysomes, suggesting a more active translation in M-phase. E The localization of HXT2 mRNA is independent of the Signal Recognition Particle (SRP). Deletion of SRP54 does not affect the retention in the mother cell or the equal distribution of HXT2 mRNA.
Figure 4Model for Integrator cleavage of nascent piRNA precursors associated with promoter-proximal Pol II. Cleavage results in the production of a short capped RNA, and a 3′ degradation fragment. Signal of 5′ P small RNA 5′ ends mapping to piRNA loci as a function of the distance to piRNA TSSs, after removal of reads corresponding to mature 21U-RNAs (see Methods). The signal is normalized to counts per million of non-structural mapped reads. A peak of 5′ P ends centered at +38 from piRNA TSSs is observed. Distributions of counts per million of 5′ P ends mapping from +25 to +50 of piRNA TSSs across the different genotypes and conditions. Two independent biological replicates are shown. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range. Length distribution of putative 3′ degradation fragments with 5′ P ends mapping at +28-+58 of the 5′ U of annotated 21U-RNAs. Number of loci with detected cleavage fragments in N2 empty vector nematodes across percentile bins of increasing termination signal strength (top panel). Distributions of fragment read counts per locus for detected loci across percentile bins of increasing termination signal strength (bottom panel). Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range. The data shown here corresponds to one of two N2 EV replicates, see Appendix Figure S6 for this analysis across replicates and genotypes.
Figure 4A. Volcano plots of RNA sequencing data of genes that pass a threshold false discovery rate (FDR) < 5%, with log10 fold-change (FC) in expression in 4HT treated ROCK1:ER expressing cells relative to GFP:ER expressing cells (left) or 4HT treated ROCK2:ER expressing cells relative to GFP:ER expressing cells (right) versus log10 adjusted p value (n=3). ROCK1:ER+4HT vs GFP:ER+4HT; 3828 genes. ROCK2:ER+4HT vs GFP:ER+4HT; 4481 genes. B. Venn diagram of genes with FDR < 5% and > 2-fold expression changes for ROCK1:ER+4HT vs GFP:ER+4HT and ROCK2:ER+4HT vs GFP:ER+4HT. C. Heatmap of 33 differentially expressed genes in the MetaCoreTM process network Cell adhesion: Cell-matrix interaction. High relative expression in red and low relative expression in blue. D. MMP average RNA sequence reads. Mean ± SEM (n=3) * adjusted p-value <0.05. E. Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-) or 1 µM 4HT for 24 h (left). Means ± SEM (n=4), p value by unpaired t test. Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-), 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h (right). Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.F. Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-) or 1 µM 4HT for 24 h (left). Means ± SEM (n=4), p value by unpaired t test. Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-), 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h (right). Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test. G. MMP10 immunohistochemistry stained sections of human pancreas adenocarcinoma. Scale bar = 100 µm (left). Histoscores of MMP10 staining (right) in normal pancreas (n=5), and pancreas adenocarcinoma grade 1 (n=23), grade 2 (n=27) and grade 3 (n=13). One-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.
Figure 7(A) A549 cells were transfected, for 48 hours, with a mCherry-DFCP1 plasmid and then infected, for 4 hours, with P. aeruginosa (WT, ΔSTY, ΔS, ΔTY, SG-A-, SG-A+ or SG+A-) and the mCherry puncta number per cell was analyzed by fluorescence microscopy. Representative immunofluorescence images were showed, and scale bars represent 10 µm. (B) Quantitative analysis of mCherry-DFCP1 puncta by visually count more than 100 cells for each sample. Each bar represents mean value of from three independent experiments and error bars represent standard deviation. The significance of differences between assays was determined using ANOVA with Dunnett multiple-comparison posttest, NS: not significant; *p ≤ 0.05; **p ≤ 0.01.
Public bladder single cell dataset.Figure 3.two-dimensional visualization of bladder cell atlas by UMAP. Expression pattern of canonical markers used for identifying bladder cell types. ACE2 expression across 12 cell subgroups in the bladder dataset.
Figure 4A.-D. 15N-labeled diubiquitin probes were titrated with the UBA domain up to molar ratio of 8:1 (UBA : Ub2). NMR spectra were recorded for (A) the distal moiety in K63-Ub2, (B) the proximal moiety in K63-Ub2, (C) the distal moiety in K48-Ub2 or (D) the proximal moiety in K48-Ub2. e. CSPs of I44 were plotted over UBA domain concentration. f. KD values for binding of the UBA domain were determined from results in (E) assuming two independent but different binding sites.
DCVs are present in axonal, dendritic and synaptic compartments.Figure 3Representative confocal images and zooms of a single isolated hippocampal neuron (DIV14) immunostained for the endogenous DCV marker ChgB (green), dendritic marker MAP2 (red) and ß3-tubulin (blue) as neurite marker. Areas of zooms are indicated in (A). Closed arrows: dendritic ChgB staining, open arrows: axonal ChgB staining. Scale bars: 20 µm (A), 5 µm (B, C). Zoom of immunostaining for the excitatory synaptic vesicle protein VGLUT1 and DCV marker ChgB. Closed arrows: synaptic ChgB staining, open arrows: extra-synaptic ChgB staining. Scale bar: 5 µm. Zoom of immunostaining for two synaptic proteins VGLUT1 and Synapsin I/II. Scale bar: 5 µm. Manders' coefficients of colocalization analysis between ChgB, ß3-tubulin and MAP2. Manders' coefficients of colocalization analysis between ChgB and VGLUT1, with VGLUT1-Synapsin I/II colocalization as positive control and VGLUT1-Synapsin I/II rotated 90° as negative control. Number of ChgB puncta per neuron in dendrites, axons and the total pool (dendrites + axons). Wilcoxon matched-pairs signed rank test: n.s. = not significant, p = 0.75. Total dendritic and axonal length (mm) per neuron. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001. ChgB puncta per μm dendrite and axon per neuron. Lines next to individual data points show mean ± SEM. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001. Mean ChgB fluorescence intensity per puncta in the total neuron (dendrites + axons) and in dendrites only. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001. Histogram of fluorescence intensity of ChgB puncta in the total neuron (dendrites + axons) and in dendrites only. ChgB puncta per μm dendrite and axon per neuron. Lines next to individual data points show mean ± SEM. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001. Mean ChgB fluorescence intensity per puncta in dendrites and axons. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001. Distribution of DCVs in dendrites and axons of sparse-labeled network cultures. (P) ChgB puncta per μm dendrite or axon. Lines next to individual data points show mean ± SEM. Mann-Whitney U test: **** p = < 0.0001. (Q) Mean ChgB fluorescence intensity per puncta in dendrites and axons. Unpaired t-test: *** p = < 0.0001. Analysis of DCVs in axons and dendrites of mCherry labeled mouse Dentate Gyrus (DG) granule cell tissue after stereotaxic viral injections and post-hoc immunostained for ChgA as DCV marker. (R) Representative mouse brain slice with labeled DG granule cells (mCherry-filler, red) and immunostained for ChgA (green). ChgA puncta were measured in dendrites and axons at indicated areas. Scale bar: 250 µm. (S) ChgA puncta per μm dendrite or axon in DG granule cells. N = 1 mouse (6 dendrite regions, 7 axonal regions).
Figure 2(A) Histological analysis of plaque-associated BACE1 immunoreactivity in male (n=10) and female (n=8) APP23 mice. BACE1 area covered was normalised to 4G8-positive area covered of the same image (left). Right: representative images, scale bar = 50 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.3724. (B) Stereological quantification of the number of cortical GFAP-positive astrocytes in male (n=10) and female (n=8) APP23 mice (left). Right: representative images of GFAP staining, scale bar = 200 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, *p=0.0134. (C) Quantification of activated microglia within 30 µm from plaque borders. Top: Numbers of Iba1-positive microglia were normalised to the size of the nearest 4G8-positive plaque and quantified in male (n=10) and female (n=8) APP23 mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.0576. Bottom: Histogram representing Clec7a staining intensity within plaque-associated Iba1-positive microglia in male (n=10) and female (n=8) APP23 mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for each single bin, p=N.S.. Right: representative images, scale bar = 40 µm. Data points were taken from graphs analysing APP23p40+/+ against APP23p40-/- mice. (D) Radial intensity profiles of Iba1 and 4G8 around the center of the nucleus of plaque-associated Iba1-positive microglia in male (n=10) and female (n=8) APP23 mice. Iba1 intensity declines until a radius of ~6 µm, marking the cell periphery. 4G8 intensity peaks inside the cell (~4 µm), but stays high outside the cell. This is very likely due to the close proximity to 4G8 positive plaques. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for the number of binned radii shows no significant difference between both 4G8 traces.
Figure 5(A) Time-lapse 3D-SIM imaging of T6SS assembly, contraction and disassembly by visualizing TssB-sfGFP in cells of A. baumannii DSM30011. Data information: Yellow arrows indicate examples of contact-dependent sheath foci or paired sheath assemblies. Scale bars and time intervals are indicated. (B) Time-lapse 3D-SIM imaging of sheath dynamics (TssB-sfGFP) in the absence of TslA in A. baumannii DSM30011. Data information: Scale bars and time intervals are indicated. (C) Time-lapse 3D-SIM imaging of sheath dynamics (TssB1-sfGFP) of B. thailandensis E264. (D) Time-lapse 3D-SIM imaging of sheath dynamics (TssB1-sfGFP) of B. thailandensis E264 in the absence of tagM1. Data information: Yellow arrows indicate examples of contact-dependent sheath foci or paired sheath assemblies. Scale bars and time intervals are indicated.
Figure 2Schematics of layer-specific polysome profiling comparing, here, the WT versus hyl1 columella. Profiling was conducted with RPL18 expressed under each cell-type-specific promoter, or the ubiquitous 35S promoter, providing a control for the total roots' signal. Representative confocal images of cell-type-specific promoter::GFP transcriptional fusions in hyl1 vs WT roots. Hierarchical clustering of validated miRNA targets between different cell layers in either WT or hyl1 roots. Total number of up-(pink) or down-(blue)-regulated transcripts in hyl1 versus WT background, compared to the total number of validated miRNA targets (grey) isolated via total, or cell-type-specific, polysome profiling. qRT-PCR validation of three distinct classes of direct miRNA target transcripts from total root-, total root polysome- or epidermis-specific polysome-associated RNA. The AT2G37050 kinase is positively regulated by LAC17. Scale bars: 50μM. All qPCR values were normalized to the internal control ACTIN2. miRoot snapshots of whole-root (left) and layer-specific (right) LAC17 mRNA accumulation in WT versus hyl1, as assessed by polysome profiling.
Ame1 and Mif2 are Mtw1c receptors that promote outer kinetochore assembly.Figure 1Left panel: Comparison of kinetochore assembly plans. Generally, inner kinetochores are represented by the constituted centromere associated network (CCAN) of which Ame1 and Mif2 are the only essential subunits in S. cerevisiae. Right panel: Among other functions Ame1 and Mif2 both act as receptors for the outer kinetochore component Mtw1c using short binding motifs at their N-termini. An Ame1∆N is lethal, while a Mif2∆N mutant is viable. Two different scenarios for Mtw1c-receptor configurations are indicated.
Figure 4Swim speed during acquisition and reversal learning is comparable in all groups (each point represents one day with 6 trials). During acquisition learning and after platform reversal to the opposite quadrant, AAV-Venus injected cDKOs (red circles) show considerably longer escape latency compared to AAV-Venus injected LM controls (white circles). AAV-APPsα injected cDKO mice (green circles) show an overall intermediate performance (genotype F(2, 46)=6.361, p=0.0036) and that differs significantly at day 5 of reversal learning compared to AAV-Venus injected cDKO mice (genotype x day F(8,184)=5.266, p<0.0001; **p<0.01). Measurements of swim path length confirms the training performance deficit of cDKO mice compared to LM controls. During the reversal phase, the performance of AAV-APPsα injected cDKOs is significantly improved at day 5 (genotype F(2,46)=16.70, p<0.0001, genotype x day F(8,184)=5.788, p<0.0001; *p<0.05, p<0.01). In the probe trial, AAV-Venus injected cDKOs (red) spend significantly less time in the target zone compared to AAV-Venus (###p<0.001) injected LM control animals (white) and failed to prefer it significantly over control zones in adjacent quadrants. Note that expression of APPsα (green) largely rescues the deficits of cDKOs. N=22 (LM controls: AAV-Venus), N=18 (cDKOs: AAV-Venus), N=18 (cDKOs: AAV-APPsα). Data represent mean ± SEM. Dashed lines: chance level. Data were analyzed using a mixed ANOVA model with conditions (LM control: AAV-Venus, cDKO: AAV-Venus and cDKO: AAV-APPsα) as between subject factor. Within subject factors were added to explore the dependence of genotype effects on day and place. Significant interactions and main effects were further explored by pairwise FDR-corrected two-tailed Student's t-tests. # indicates significant differences between LM control and cDKO injected with AVV-Venus, * between cDKO injected with AAV-Venus or APPsα, ● between LM control injected with Venus and cDKO injected with AAV-APPsα nsp>0.05, */#/●p<0.05, **/##/●●p<0.01, ***/###/●●●p<0.001.
Figure 1A. Gene locus of LincGET and Dyei. LincGET is between Dyrk1b and Gm18672, and Dyei is between Dyrk1b and Eid2. There are one GLN sequence and some ERV fragments of MERVK or MERVL between Dyrk1b and Gm18672. AATAAA is the polyadenylated signal site. B. Single strand RT-PCR (SSRT-PCR) results for LincGET. LincGET is transcribed from the top strand. Primers used in strand-specific reverse transcription are shown in green, while primers used in PCR are shown in red. About 50 early 4-cell embryos were used for each experiment and three experimental replicates were performed. C. 3′ RACE and 5′ RACE results for LincGET and Dyei. Gene-specific primers (F4, F5, R4, and R5) are shown in Figure EV1B. * indicates the bands corresponding to the correct band of 3′ RACE for LincGET. About 200 early 4-cell embryos were used for each RACE experiment and three experimental replicates were performed. D. Subcellular localization analysis of LincGET and Dyei by RNA fractionation and TM-qPCR analysis. The results show that LincGET and Dyei locate in the nucleus and are mainly associated to chromatin. The error bars represent s.e.m. Chro, chromosome. Nuc, nucleoplasm. Cyto, cytoplasm. Gapdh and Xist respectively act as cyto and chro control. About 500 early 4-cell embryos were used for each experiment and three experimental replicates were performed. E. miRNA reverse northern blot for LincGET and Dyei. miRNAs were isolated and amplified by RT-PCR after adding double adaptors, then southern blot (reverse northern) was performed. It shows no evidence of small RNA products, indicating that LincGET and Dyei do not function as pre-miRNA. The miR1982 was used as a miRNA positive control. About 400 early 4-cell embryos were used for each experiment and three experimental replicates were performed. F. Expression pattern of LincGET and Dyei at different stages of preimplantation mouseembryos by TM-qPCR. The error bars represent s.e.m. About 50 embryos of each stage were used and three experimental replicates were performed. G. RNA-FISH in early 2- to 16-cell embryos for LincGET and Dyei. The results show that LincGET is present in the nucleus of late 2- to 4-cell embryos and weakly expressed in early 8-cell embryos, while Dyei is present in the nucleus of 2- to 4-cell embryos. E2C, early 2-cell stage (n = 6 for each probe). L2C, late 2-cell stage (n = 7 for each probe). E4C, early 4-cell stage (n = 6 for each probe). L4C, late 4-cell stage (n = 5 for each probe). E8C, early 8-cell stage (n = 4 for each probe). L8C, late 8-cell stage (n = 5 for each probe). 16C, 16-cell stage (n = 4 for each probe). BL, blastocyst (n = 5 for each probe). Scale bar, 50 μm. Three experimental replicates were performed.
Figure 6Kinetic analyses of EC migration in an ex vivo aortic ring angiogenesis model. Upper panels are snapshots of time-lapse imaging in which EC nuclei are labeled by SYTO dye (green). In analyzed regions framed by dotted lines, the centroids of the first five EC nuclei from the tip at 0 min are pseudocolored in magenta and those of the remaining EC nuclei are shown in cyan. Initial discrete labeling becomes intermixed over time (see also Movie EV1). Lower panels show trajectory analyses representing normalized positional changes of individual ECs during vessel elongation. Scale bar, 100 µ Quantification of the kinetics of EC migration. Values of forward and reverse migration for RhojWT/WT are normalized to 1. n = 33 vessel branches per group. Quantification of vessel elongation (left graph). n = 33 vessel branches per group. Scatter plot shows relationship between "vessel elongation" and "reverse migration" in RhojGFP/GFP aortic rings as evaluated by a Pearson correlation coefficient. Schematic representation for construction of Rhoj mutant mice. Cre-loxP-mediated genetic recombination of the Rhoj-flox allele generates the Rhoj-KO allele by excising the RhoJ cDNA-polyA cassette inserted into exon 1 of the Rhoj gene. Labeling for YFP/GFP (green), CD31 (red), and ETS transcription factor ERG (blue) in retinal vascular fronts of P5 CAG-MerCreMer:R26R-EYFPflox/WT and CAG-MerCreMer:Rhojfloxflox mice after intraperitoneal (i.p.) injections of 10 µg of 4-hydroxytamoxifen (4OHT) at P1. Scale bar, 50 µm.   Relative contribution of YFP/GFP-positive ECs to the tip positions. n = 16 per group. Values for CAG-MerCreMer:R26R-EYPflox/WT are normalized to 1.
GW274150 treatment reduces muscle wasting in the C26 model.Figure 3Effect of GW on tumor weight. (left) Representative image of Western blot analysis of 3NT staining and Stain Free imaged total protein levels from quadriceps muscle. (right) Quantification of the 3NT to total protein ratio (saline n=5, C26 n = 4, GW 5mg/kg n=5, and C26 GW 5mg/kg n=5). Percent body weight change from day 0 to day 16. (D-G) Weight of tibialis anterior (D), gastrocnemius (E), quadricep (F) and soleus (G) muscle normalized to initial body weight. (left) Representative photomicrographs of gastrocnemius muscles sections from control and C26 mice treated with or without GW taken after H&E staining. Scale bars = 100 μm. (right) frequency histogram showing the distribution of muscle fiber minimum feret diameter in the gastrocnemius muscles from control and C26 mice treated with or without GW (saline n=4, C26 n = 4, GW 5mg/kg n=3, and C26 GW 5mg/kg n=4). A total of 600-700 fibers per muscle were used for the analysis. Statistical comparisons, mean, and standard deviation of the mean are shown in the histogram legend. Change in grip strength from before tumor cell injection (day 0) and before endpoint collection (day 16).
Figure 2Venn diagrams representing the genes up regulated (yellow) and down regulated (purple) by PIFs or BES1/BZR1 (BES), and the direct target loci (green) of these transcription factors. Gene sets derive from our bes1-D and bes1-D;pif4pif5 microarray analyses and from published RNA-seq studies of pifq, bzr1-1D and bri1-116 mutants (Appendix Tables S1 and S2) . The direct target PIF and BES datasets were obtained from published ChIP-chip or ChIP-seq experiments with these factors (Appendix Tables S3 and S4). Histogram bars represent the percentage of PIF-, BES- and PIF+BES- regulated genes that are directly bound by these transcription factors, as compared to all the Arabidopsis genes (total genes, in gray) (Chi-squared test, ** indicates p-value <0.01, ns indicates not significant differences). Heat map and fold-change graph representation of the genes identified in A. Expression data were obtained from Genevestigator and heatmap and clustering analyses were performed using the MeV software. PIF+BES-UP indicates genes up-regulated by PIF and BES and identified as targets of both the PIF and BES factors. BES-DOWN are BES repressed targets, while BES-UP correspond to BES induced targets. The PIF-UP cluster includes PIF induced genes directly bound by the PIF factors. Percentages of occurrence of the indicated motifs in the PIF, BES or PIF+BES target loci, as compared with all Arabidopsis genes (genome, in gray)