EMBO/sd-character-level-ner huggingface dataset:
Figure 2A Representative images of GFP-gephyrin and GFP-G375D, expressed in hippocampal neurons at 9 + 4 days in vitro. Inset shows higher magnification. Scale bar = 20 µm.B Relative distribution of cluster density of gephyrin variants on dendrites from transfected neurons. Number of gephyrin clusters in each dendrite is normalized to 20 µm segments. C Representative segments of dendrites expressing GFP-gephyrin or GFP-G375D and immunostained with the presynaptic marker VGAT. Arrowheads show co-localization of gephyrin and VGAT clusters. Scale bar = 10 µm.D Quantification of co-localization between gephyrin and VGAT clusters (gephyrin-VGAT 77.4±2.9 %, G375D-VGAT 30±4.4 %). 31 GFP-gephyrin and 30 GFP-G375D neurons from three independent cultures were used for quantifications. Results are expressed as mean ± SEM
| Kugel number is decreased by Notch inhibition but increased by Wnt inhibition or activation.Figure 8Kugel number was significantly decreased by inhibition of Notch signalling by 12h treatment with 50µM DAPT (****p<0.0001; control n=24 embryos 9.38 ± 1.77 (mean ± s.e.m.), DAPT n=24 embryos 0.92 ± 0.28 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test). Mean kugel diameter was not statistically significantly altered by DAPT treatment (p = 0.0832; control n=225 kugeln from 24 embryos 8.64 ± 0.54 (mean ± s.e.m.); DAPT n=22 kugeln from 24 embryos 6.88 ± 0.86 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Student's t-test). A transgenic dll4 reporter line showed no indication of different dll4 expression at the sites of kugeln (white arrowheads pointing to kugeln; n=122 kugeln from 23 3dpf embryos; 2 experimental repeats). Studying Notch signalling in the Notch reporter line Tg(TP1glob:venusPest)s940, showed high Notch levels in the mid-brain, but, again, no difference in expression at the sites of kugeln (n=45 kugeln from 19 4dpf embryos; 2 experimental repeats). Kugel number was statistically significantly increased by inhibition of Wnt signalling by 4h treatment with 10µm XAV-939 (***p = 0.0003; control n=22 embryos 1.32 ± 0.24 (mean ± s.e.m.); XAV-939 n=21 embryos 4.29 ± 0.71 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test). Kugel diameter was not statistically significantly altered by XAV-939 (p = 0.4098; control n=29 kugeln from 22 embryos 8.78 ± 0.76 (mean ± s.e.m.); XAV-939 n=90 kugeln from 21 embryos 7.99 ± 0.56 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Student's t-test) Kugel number was statistically significantly increased by activation of Wnt signalling by 4h treatment with 10µm GSK3 inhibition XV (p 0.0359; control n=22 embryos 1.04 ± 0.34 (mean ± s.e.m.); GSK3 inhibitor n=21 embryos 4.24 ± 1.28 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test). The diameter of kugel was not statistically significantly altered by GSK3 inhibition (p 0.5555; control n=23 kugeln from 22 embryos 7.26 ± 0.89 (mean ± s.e.m.); GSK3 inhibitor n=89 kugeln from 21 embryos 8.09 ± 0.97
Figure 3Splenocytes from C57BL/6 mice were freshly harvested. Resting and activated T cells, monocytes, macrophages, DCs and B cells were obtained as in Figure 2. The resting and activated immune cells were stained with biotinylated TAPBPL-Ig or control Ig，followed by streptavidin PE, as well as anti-CD4, CD8, CD11b, F4/80, CD11c, B220 or CD19 antibody to identify immune cells. Representative flow cytometric profiles showing the binding TAPBPL-Ig or control Ig to freshly harvest and activated immune cells (n=3). Significance was calculated by two‐way ANOVA with Tukey test. * P<0.05 compared with control Ig. ** P<0.05 compared with resting cells. Splenocytes from C57BL/6 mice were freshly harvested. Resting and activated T cells, monocytes, macrophages, DCs and B cells were obtained as in Figure 2. The resting and activated immune cells were stained with biotinylated TAPBPL-Ig or control Ig，followed by streptavidin PE, as well as anti-CD4, CD8, CD11b, F4/80, CD11c, B220 or CD19 antibody to identify immune cells. statistical analysis showing the binding TAPBPL-Ig or control Ig to freshly harvest and activated immune cells (n=3). Significance was calculated by two‐way ANOVA with Tukey test. * P<0.05 compared with control Ig. ** P<0.05 compared with resting cells. HEK-293 cells were transfected with an expression vector containing the murine PD-1, CD28, BTLA, CTLA-4, or ICOS gene and screened for cells stably expressing each gene. The transfected cells were stained with antibodies against the respective PD-1, CD28, BTLA, CTLA-4, or ICOS protein (open histograms) or isotype Ab (shaded histograms). Representative flow cytometric profiles showing the expression of each receptor. PD-1, CD28, BTLA, CTLA-4, or ICOS gene-transfected HEK-293 cells were stained with biotinylated TAPBPL-Ig and control Ig, followed by streptavidin-PE. Representative flow cytometric showing the binding of TAPBPL-Ig (open histograms) or control Ig protein (shaded histogram) to the transfected cells. The data are representative of 3 independent experiments.
EGFR signaling in the lamina glia is dependent on Spitz from the R1-6 photoreceptors.Fig 6(A, A') Anti-Spitz detected Spitz (magenta) colocalizing (arrowhead) with epithelialgliacytoplasm marked by anti-Black (green). Scale bar: 5 μm. (B-B') EGFR reporter pointed-lacZ (pnt-lacZ) exhibited expression in the epithelial (arrow) and marginal glia (arrowhead). (C, C') Dominant-negative form of EGFR (DERDN) expressed in glia at 28°C for 3 days inhibited pnt-lacZ expression. (D, D') pnt-laZ expression was lost in Shits-expressing glia at 28°C for 3 days. (E, E') The knockdown of Spi in R1-6 photoreceptors at 28°C for 12 days also inhibited pnt-lacZ expression in the glia. The penetrances of (B-E) are shown in the upper right corner of each panel. DAPI: nuclei (white in B'-E'). Scale bar: 20 μm.
c and induces oligomerization of the complex in a concentration dependent manner A, B: 5 µM, 7.Figure 5A, B: 5 µM, 7.5 µM or 10 µM Dam1WTc alone or in combination with respective concentrations of Bim1-Bik1 complex were analyzed by size exclusion chromatography. SDS-PAGE analysis of elution fractions is shown in B. High molecular weight species of Dam1WTc were eluted after 0.9 - 1.0 ml (see chromatogram in A). C: Chromatogram of size exclusion chromatography of Dam1c with Bim1 and Bik1 and SDS-PAGE analysis of elution fractions. The elution buffer contained 100 mM NaCl in contrast to 200 mM in previous experiments. Please note that under these conditions the equilibrium is shifted towards the high oligomeric complex. Dashed boxes indicate elution fractions used for EM analysis shown in D. D, E: EM images of negatively stained Dam1c in the presence of Bim1 and Bik1 at low salt conditions (10mM NaCl). The final concentrations of Dam1c, Bim1 and Bik1 were 0.3 µM, 0.1 µM, and 0.25 µM, respectively. The Dam1c-Bim1-Bik1 complex could be further purified by gel filtration in presence of 100 mM NaCl (20 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM MgCl2, and 0.1 mM TCEP) prior to the final dilution to low salt conditions for EM (D). The scale bars are 100 nm, boxes mark representative complexes shown as magnified inserts.
Figure 1A: Myelin basic protein (MBP) or albumin (Alb) were added to liposomes that contained purified tafazzin (TAZ). Acyl transfer reactions were measured by mass spectrometry. B: CL formation was measured in the presence of 60 nmol MLCL(18:2)3, 40 nmol PC(18:2)2, 4 µg TAZ, and 0.2 mg MBP or 0.2 mg Alb. C: CL formation was measured as in B with different amounts of MBP or Alb. D: PC16:0/18:3 formation was measured in the presence of 85 nmol LPC16:0, 15 nmol PC18:3/18:3, 4 µg TAZ, and MBP. E, F: CL formation was measured in the presence of 60 nmol MLCL(18:2)3, 40 nmol PC(18:3)2, 4 µg TAZ, and 0.2 mg MBP (2 independent measurements per time point). G: CL formation was measured in the presence of 40 nmol PC(18:2)2, 10 nmol MLCL(18:2)3, 8 µg TAZ, 1250 nmol Triton X-100 (TX), and 5.6 nmol bacteriorhodopsin (BR). H: PC formation was measured in the presence of 180 nmol CL(18:2)4, 20 nmol LPC16:0, 8 µg TAZ, 20 mM Ca2+, and 5.6 nmol BR. I: Transacylations were measured in mixtures containing 90 nmol CL(18:1)4, 90 nmol CL(18:2)4, 10nmol LPG18:1, 10 nmol LPE18:1, 8 µg TAZ, 20 mM Ca2+, and different amounts of BR (2 independent measurements per BR/Lipid ratio). J: Transacylations were measured as in I in the presence of 0.9 g BR/ g lipid. The ratio of total linoleoyl transfer over total oleoyl transfer was calculated.
A Whole-Genome siRNA Screen to Identify Regulators of Endosome-to-Golgi Retrieval.Figure 2(A) Plate layout for genome-wide loss-of-function screen.(B) Whole-genome screen hit selection.(C) Scatterplot of the normalized TGN retrieval ratios for the two replicates of the whole-genome screen. Each data point represents a single siRNA pool. Only valid measurements (18,465 siRNA pools) are included. Negative (cyan square) and positive (SNX1 siRNA, red data points) controls are shown. Green data points indicate siRNA pools with TGN retrieval ratio SSMD ≤ (−3) (very strong hits). Blue star data points indicate the three hits characterized further in Figures 3, 4, 5, and 6. (D) Plate layout for the ON TARGETplus validation screen.(E) Replicate normalized TGN retrieval ratios for the ON TARGETplus smartpool validation screen (360 pools). The colors used are as in (C), with additional magenta data points indicating siRNA pools with TGN retrieval ratio SSMD between (−2) and (−3) (strong hits). (F) Table listing the 88 genes that were very strong or strong hits in the validation screen. Red-bordered cells indicate kinase and phosphatase genes, green-bordered cells contain genes encoding membrane proteins, and blue-bordered cells contain genes encoding cytoskeletal proteins. More information about these genes is provided in Table S4.
Laboratory evolution method schematic and the growth trajectory of D-lyxose experiments.Figure 1schematic of the two-part adaptive laboratory evolution (ALE) experiments. The 'weaning/dynamic environment' stage involved growing cells in supplemented flasks containing the non-native substrate (blue) and growth-promoting supplement (red). As cultures were serially passed, they were split into another supplemented flask as well as an 'non-native substrate test flask' containing only the non-native nutrient (no supplement) to test for the desired evolved growth phenotype. The 'static environment' stage consisted of selecting for the fastest growing cells and passing in mid log phase. Growth rate trajectories for duplicate experiments (n=2 evolution experiments per substrate condition) (green and purple) for the example case of D-lyxose. Population growth rates are plotted against cumulative cell divisions. Clones were isolated for whole genome sequencing at notable growth-rate plateaus as indicated by the arrows. Mutations gained at each plateau are highlighted beside the arrows (mutations arising earlier along the trajectory persisted in later sequenced clones).
Figure 6Only a few nucleosomes are shown to illustrate the main dimensions and the interdigitation of two layers. Our results suggest that the two turns of the nucleosomal DNA are oriented slightly tilted with respect to the axis normal to the plate surface, but they may have diverse orientations (not represented in this scheme) with respect to the other two axes of the plate. The thickness of single- and double-layer plates (~7.5 and ~13 nm, respectively) obtained from cryo-tomograms is indicated in blue. The main scattering peak at ~6 nm observed in SAXS experiments with condensed chromosomes under metaphase ionic conditions is probably due to the repetitive distances between nucleosomes (face-to-face interactions) and between stacked layers; these distances (~6 nm) are indicated in red.
activation mediates hTau-induced STAT1 activation.Figure 6(A, B) Overexpression of hTau in HEK293 cells for 48 h increased the activity-dependent phosphorylation of JAK2, JNK1 and ERK1 compared with the empty vector control (Ctrl) measured by Western blotting (n=3, student's t test). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT. Pharmacological inhibition of ERK1 (C, D) for 24 h did not significantly affect the hTau-induced STAT1 phosphorylation at pY-STAT1 (Tyr701) in total extracts (C and the nuclear fraction (D measured by Western blotting (n=3). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT. Pharmacological inhibition of JNK1 (E, F) for 24 h did not significantly affect the hTau-induced STAT1 phosphorylation at pY-STAT1 (Tyr701) in total extracts E) and the nuclear fraction F) measured by Western blotting (n=3). The alteration of pS-STAT1 (Ser727) confirms the efficacy of JNK1 inhibitors. Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT. Pharmacological inhibition of JAK2 (G, H) abolished hTau-induced STAT1 phosphorylation at Tyr701 in total extracts (G and the nuclear fraction (H (n=3). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT. knockdown JAK2 by siRNA (I, J) abolished hTau-induced STAT1 phosphorylation at Tyr701 in total extracts I) and the nuclear fraction J) (n=3). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT. (K, L) The phosphorylated JAK2 level increased in the hippocampus of 12-month-old hTau transgenic mice (K), and the hippocampus of C57 mice infected with AAV-hTau (1.13×1013 v.g./ml) (L) (n=4, Mann-Whitney test). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.
Subretinal MPs accumulate on the retinal pigment epithelium in AMD.Figure 1Confocal microscopy of IBA-1 (green staining) immunohistochemistry of RPE flatmounts (RPE autofluorescence visible as orange due to its autofluorescence in the red and green channel) from a healthy donor (A), a geographic atrophy lesion (B), and large drusen (C and D). (A, B, D): orthogonal Z-stack projection; (C): oblique Z-stack projection and dissecting microscope appearance of postmortem large drusen after removal of the overlaying retina (inset). Double-labeling on the subretinal side of the retina (to avoid masking by RPE autofluorescence) of IBA-1+ (E, green fluorescence) and CD18 (F, red fluorescence; G, merge). Orthogonal and lateral Z-stack of a subretinal IBA-1+ (green fluorescence) MPs adjacent to the RPE (orange autofluorescence) in the vicinity of a large drusen.
Figure 1A 3-D reconstructions of propidium iodide (PI)-stained cell walls (red) and BCECF-stained vacuoles (green) of epidermal atrichoblasts in the early and late meristem and in the early and late elongation zone. Scale bars: 5 µm. B Boxplots showing vacuolar occupancy of cells in the defined zones (n=7-11). Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values. c) Graph depicts absolute values of cell volume and vacuolar volume in the depicted root zones (data points display mean, error bars depict s.e.m., n=7-11). "cytosol calc." is approximated as the difference between cell volume and vacuolar volume.
CAR induces non-typical immunological synapses.Figure 2T cells expressing GFP-CAR were plated on CD19 and ICAM-1 coated SLBs. TIRF microscopy was implemented to visualize CAR localization. A. CAR microclusters underwent centripetal movement to form cSMAC (upper panel) or remained separated (lower panel). B. Quantification of the categories regarding the formation of CAR cSMAC. Unstable cSMACs are defined by a cSMAC duration below 12 min. Data are shown as mean ± SD. n = 3 independent experiments. 37, 38, and 47 cells were quantified in individual experiments. C. Quantification of time from cell landing to the start of retrograde flow. Data are shown as mean ± SD. n = 33 (stable), 31 (unstable), 10 (kinapse), and 41 (no cSMAC) cells. Results are pooled from three independent experiments. D. Localization of CAR and CD45 in the immunological synapse. CAR T cells were fixed 20 min after being plated on the SLB, followed by staining with an anti-CD45 antibody. E. Line scan of CAR and CD45 across the long axis of the immunological synapse. Shown are the normalized fluorescence intensity.
Figure 2A-D Adult wing phenotype of knock-down of Gapdh (C765>GapdhRNAi), over-expression of full-length Ecd (C765>ecd), over-expression of C-terminally truncated form of Ecd (C765>ecdDN), and knock-down of ecd (C765>ecdRNAi) in the whole wing disc (see Appendix Fig S3A for the expression pattern of C765-Gal4). Images of adult wings of control (A), C765>GapdhRNAi (A'), C765>ecd (B), C765>ecdDN (C), and C765>ecdRNAi (D) male flies. (A'') Overlay of the wings shown in A and A'. (C') Overlay of control wing and wing shown in C and C'. Loss of Ecd throughout the wing discs produces adults with vestigal wings (D). In contrast, the effects of ecdDN over-expression or Gapdh knock-down are less severe on tissue survival, giving rise to smaller wings with altered proportions. Images consist of a series of tiles that were stitched together by the microscope software. Scale bar= 500 μm. E, F Tukey box-and-whiskers plot showing quantifications of the area (E) and shape (aspect ratio: major/minor axis, F) of control, C765>GapdhRNAi, C765>ecd, and C765>ecdDN wings. Lower and upper hinges correspond to the first and third quartiles, vertical lines extend to ± 1.5 times the interquartile range. Sample size is indicated for each perturbation. Loss of Gapdh or over-expression of EcdDN leads to smaller wings that are increased along the anterior-posterior (A/P) axis compared to the proximal-distal (P/D) axis. Statistical analysis was performed using one-way ANOVA, followed by Dunnett's multiple comparisons test. ***p≤0.001.
/6 hexamer with the Nse5/6 dimer.Figure 3A. Pulldown ('pd') assays using immobilized Smc5/Smc6-Twin-Strep ('TS') hexamers and soluble input material ('in') of Nse5/6 (left), of Nse5/Nse6(177-C) (middle) and of Nse6(1-179)-CPD-His (right). Control pulldowns ('-') were performed by omitting the pre-binding of Smc5/Smc6-Twin-Strep to the beads. Fractions were analysed by SDS-Page and Coomassie staining. B. Salt stability of Smc5/Smc6-Twin-Strep interactions with Nse5/6 and Nse6(1-179). Immobilized Smc5/Smc6-Twin-Strep was mixed with Nse5/6 or Nse6(1-179)-CPD-His ('in'). Beads were washed with buffers containing the indicated salt concentrations. Bead fractions were analysed by SDS-Page and Coomassie staining. Pulldown efficiencies were estimated from the intensity of Coomassie gel bands. C. XL-MS cross-links detected between Nse5 and Nse6 proteins and subunits of the Smc5/6 hexamer in schematic representation (middle panel). The positions of lysine residues on the Smc6 head and the Smc5 head, left and right panels, respectively, with cross-links to Nse5 are denoted on Phyre2-generated homology models. D. Analysis of spore viability by yeast tetrad dissection. Diploid strains heterozygous for alleles of Nse6 (wt, 86-C, 177-C and 179-C) were sporulated. Isolated spores were grown on YPD plates. Viable clones were tested for the marker cassette conferring resistance to G418 (marked by circles in green colours). Dead spores were marked by circles in red colours.
Figure 3(A) Number of characterized substrates with significant increases of NTA yields. The NTA yields of all retrieved N-termini upon a given GNAT expression was divided into four categories and each column is the result of the sum of all categories: 50-100%, dark green; 20-50%, light green; 10-20%, orange; 5-10%, red. The number of protein occurrence with a yield higher than 30% is also displayed to visualize the most significant acetylation status of each GNAT. The respective overexpression level of each GNAT as assessed from gel electrophoresis (++++ is for very high expression, +++ for high, ++ for medium, + for intermediate). (B) Overview of GNATs substrate specificities. The number of NTAed subtrates with each of the 6 major active GNATs is displayed in a pie chart according to the N-terminal pattern retrieved. The dataset is identical to that of panel A. N-terminal residues of proteins starting with Met is displayed in dark and light orange while all the other likely resulting from NME are colored with a blue pallet. Very similar pictures are obtained if only N-termini with NTA yields over 50% are figured. The starting, acetylated amino acid is indicated and labeled with the cytosolic Nat-type substrate specificity (i.e., NatA-F) according to the classic distribution
Figure 7A, PTPN3, PTPN3 (D811A) and PTPN3 (L232R) are similarly expressed in Huh7 stable cell lines. Expression levels of indicated proteins were detected with appropriate antibodies in Western blotting. B, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), attenuates tumorigenesis. Luciferase-harboring Huh7 tumor cells (2 × 106 cells/mouse) expressing PTPN3, PTPN3 (D811A) or PTPN3 (L232R) or empty vector (served as negative control) were subcutaneously injected into 5-week-old nude mice. Twenty days after injection, mice were analyzed by bioluminescence using Xenogen IVIS imaging system. Tumors expressing luciferase is indicated by radiance bar (p/sec/cm2/sr). C, Quantitation of bioluminescence with data from (B). Data are shown as mean ± SEM; n = 5. D, Depletion of PTPN3 accelerates tumorigenesis. HepG2 cells stably expressing shPTPN3 or shControl were subcutaneously injected into nude mice. Fifty days after cell implantation, tumors were dissected and photographed. E, Depletion of PTPN3 increased tumor sizes. Tumor volume was measured at indicated days post-implantation. Data are shown as mean ± SEM; n = 6 for each group. F, Depletion of PTPN3 increased tumor weight. Weight from all tumors was recorded from both control and shPTPN3 groups. Data are shown as mean ± SEM; n = 6 for each group. , PTPN3 depletion impairs TGF-β signaling in tumors. Levels of PTPN3, GAPDH and TGF-β signaling associated proteins, such as TβRI, P-Smad2, P-Smad3, Smad2/3, Smad4, PAI-1, c-Myc and p15 in tumors were analyzed by Western blotting analysis. H, Clinical significance of PTPN3 expression in LIHC based on TCGA database. In the boxplots, Y-axis represents the expression level of PTPN3 in normal and tumor samples. The boxes show the median (horizontal line in the box) ± 1 quartile; the upper and lower box limits represent the upper and lower quartiles, respectively. The whiskers (vertical lines) extend from the hinge to the smallest or larget value within 1.5 interquartile range from the box boundaries.
The Spectrin cytoskeleton restricts tissue growth in the Drosophila eye and wing.Figure 1A-O UAS.RNAi lines were driven with eyeless.Gal4gmr.Gal4 for expression during eye development or nubbin-Gal4 for expression during wing development. (A, B) Control adult Drosophilaeye (A) and wing (B). (C, D) α-spectrin RNAi results in overgrowth of the eye (C) and wing (D). (E, F) βH-spectrin/karst RNAi results in overgrowth of the eye (E) and wing (F). (G, H) β-spectrin RNAi does not affect eye size (G) or wing size (H). (I, J) kibra RNAi results in overgrowth of the eye (I) and wing (J). (K, L) α-spectrin, kibra double RNAi results in stronger overgrowth of the eye (K) and wing (L). (M, N) βH-spectrin/wing, kibra double RNAi results in stronger overgrowth of the eye (M) and wing (N). (O) Quantification of female wing sizes by pixel area, 5 wings per genotype were measured. Error bars show standard deviation.P-RThe eyeless FLP MARCM system was used to generate clonally mutant flyeyes. eyes mutant eyes (Q) overgrow slightly compared to controls (P), while α-spectrin mutant eyes expressing α-spectrin RNAi (R) overgrow strongly compared to controls (P).Data information: Scale bars, 250 μm.