EMBO/sd-character-level-ner huggingface dataset:
LUZP1 knockout (LUZP1 KO) cells display apical constriction defects with a significant reduction in di-phosphorylated myosin light chain (ppMLC) levels within circumferential rings (CRs).Figure 2Representative confocal micrographs of co-cultures of Venus-LUZP1-expressing LUZP1 KO (REV) and LUZP1 KO Eph4 cells in the apical and basal plane. Scale bar, 10 μm. Box plots with dot density plots showing that the apical area/basal area ratio was significantly smaller in REV cells than in LUZP1 KO cells [0.65 ± 0.16 (REV) vs. 1.30 ± 0.17 (LUZP1 KO)]. n = 10. **p < 0.01 (unpaired t-test). The value < 1.0 indicates apical constriction. The solid lines represent the medians and the boxes represent the inter-quartile ranges. The error bars extending from the box represents the data within 1.5 times of the interquartile range. Representative micrographs of co-cultures of wild-type (WT) and LUZP1 KO Eph4 cells. ppMLC levels within CRs were clearly reduced in LUZP1 KO cells compared to those in WT cells. Scale bar, 10 μm. Bar plots with dot density plots showing that apical areas of WT cells were significantly smaller than those of LUZP1 KO cells [118.34 ± 57.84 μm2 (WT) vs. 185.32 ± 76.69 μm2 (LUZP1 KO)]. n = 3. **p < 0.01 (Mann-Whitney U test). Bars and error bars represent the mean ± standard deviation (SD). Bar plots with dot density plots showing that ppMLC levels within CRs were significantly reduced in LUZP1 KO cells compared to those in WT cells [19.19 ± 10.02 arbitrary units (a.u.) (WT) vs. 2.92 ± 1.81 a.u. (LUZP1 KO)]. n = 3. **p < 0.01 (Mann-Whitney U test). Bars and error bars represent the mean ± SD. A scatter plot showing that mean fluorescence intensities of ppMLC and those of LUZP1 significantly correlated within CRs [Pearson's correlation coefficients (r) = 0.75, p < 0.01]. Representative confocal micrographs of co-cultures of wild-type (WT) and ZO-1/-2 double knockout (DKO) Eph4 cells. ppMLC levels within CRs were clearly reduced in ZO-1/-2 DKO cells compared to those in WT cells. Scale bar, 10 μm.   Bar plots with dot density plots showing that apical areas of WT cells were significantly smaller than those of ZO-1/-2 DKO cells [119.62 ± 31.62 μm2 (WT) vs. 199.96 ± 94.22 μm2 (LUZP1 KO)] (See also Fig 1I). n = 3. **p < 0.01 (unpaired t test). Bars and error bars represent the mean ± SD.   Representative confocal micrographs of co-cultures of WT and LUZP1 KO MTD-1A cells. ppMLC levels within CRs were clearly reduced in LUZP1 KO MTD-1A cells compared to those in WT MTD-1A cells. Scale bar, 10 μm. Representative confocal micrographs of co-cultures of WT and LUZP1 KO CSG120/7 cells. ppMLC levels within CRs were clearly reduced in LUZP1 KO CSG120/7 cells compared to those in WT CSG120/7 cells. Scale bar, 10 μm.
Metabolomic fingerprints of inclusion body myositis, muscle disorders of non-mitochondrial origin, and MIRAS carriers A - C Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of IBM (A), NMD patients (B), and MIRAS carriers (C).Figure 2Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of IBM (A) Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3; CDCA, chenodeoxycholic acid; OH-Kyn, 3-hydroxy-DL-kynurenine SDMA, symmetric dimethylarginine. Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of NMD patients (B) Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3; CDCA, chenodeoxycholic acid Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of MIRAS carriers (C). Data information Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. cAMP, cyclic AMP; C3, component 3; GABA, γ-aminobutyric acid HIAA, 5-Hydroxyindole-3-acetic acid; IMP, inosine monophosphate; OH-Kyn, 3-hydroxy-DL-kynurenine; OH-Trp, hydroxytryptophan; SDMA, symmetric dimethylarginine.
Figure 4A Representative cell pellets of untreated or b-estradiol-treated (48 hours) WT clone 1 and double-mutant clone 1, incubated with or without 5-ALA. B Concentration of heme per cell expressed in femtomolar (fM) quantities. The concentration of heme from oxyhemoglobin was quantitated using known extinction coefficients for human hemoglobin monomers. The total heme concentration was quantified using the pyridine hemochrome assay (n = 2; bars show the mean; individual data points are shown; each data point was based on two measurements).C Visible absorption spectra of cleared cytosolic fraction (upper panel) and the pyridine hemochromogen (lower panel) of β-estradiol-treated WT clone 1 or double-mutant clone 1, incubated with or without 5-ALA.
Figure 7A Cumulative population doubling analysis of the proliferation of representative scramble and CD38 knockdown DC fibroblasts or DC fibroblasts treated with vehicle or 3 mM NR. Each data point is represented as mean ± SD of three replicates. B, C Representative images of BrdU (red) and DAPI (blue) staining of DC and age-matched healthy control fibroblasts treated with vehicle or NR. Percentage of BrdU -positive cells per condition. Each dot represents the percentage of cells with BrdU staining per image. ~400 cells were counted per condition. All values are presented as mean ± SD. D, E Representative images of SPiDER-β-gal (green) and DAPI (blue) staining of DC and age-matched healthy control cells treated with vehicle or 3 mM NR. Percentage of SA-β-gal-positive cells per condition. Each dot represents the percentage of cells with SA-β-gal staining per image. ~400 cells were counted per condition. All values are presented as mean ± SD. F The levels of IL-6, IL-8, and MCP-1 in both supernatant and cell lysate of indicated DC fibroblasts treated with vehicle or 3 mM NR. All values are represented as mean ± SD of three replicates. G, H Representative immunoblots of p21 and p16 in DC fibroblasts treated with vehicle or 3 mM NR. Quantification of the indicated proteins are presented as mean ± SD from three immunoblots.
| Kugeln do not interact with macrophages.Figure 6The possible interaction of macrophages with kugeln was studied in the transgenic Tg(kdrl:HRAS-mCherry)s916, Tg(fms:GAL4.VP16)i186, Tg(UAS-E1b:nfsB.mCherry)il149, visualizing macrophages and the vasculature simultaneously. Coloured depth-coding indicates the different anatomical depths of kugeln and macrophages (ventral purple, dorsal white). Maximum intensity projections of z-stacks acquired every 10 minutes during a time-lapse showed no interaction of macrophages (unfilled arrowhead) with kugeln (filled arrowhead; n=21 kugeln in 8 3dpf embryos). Macrophages are all white, indicating they are on a different z-plane to the vessels seen.
Identification of known cell death–related genes in autophagy regulation in l(2)mbn cells using RNAi.Figure 2.(A) The percentage of LTGhigh cells was reduced by hid-RNAi (P = 0.006) but not by rpr, grim, and skl-RNAi. (B) Knockdown of Ras, phl, and rl expression by RNAi resulted in an increase in the percentage of LTGhigh cells. (Ras, P = 0.003; phl, P = 0.001; and rl, P = 0.028). (C) th-RNAi treatment (24 h) had no significant effect on LTG levels; in contrast, RNAi of Bruce resulted in an increase in LTG fluorescence levels (P = 0.01). (D) Reduction of debcl, Buffy, or p53 expression by RNAi resulted in a decrease in LTG fluorescence levels. (debcl, P = 0.018; Buffy, P = 0.006; and p53, P = 0.004). (E) RNAi of effector caspase Dcp-1 resulted in a significant decrease in the LTGhigh population (P = 0.001). (F) Representative images of GFP-LC3 puncta in cells treated with the indicated RNAi after a 2-h starvation treatment. Bar, 10 μm. (G) Quantification of cells with GFP-LC3 puncta after RNAi treatment. Cells with more than three GFP-LC3 punctate dots were considered to be GFP-LC3-positive cells. Cells treated with the RNAi indicated here all showed a significant difference (P < 0.05) in the percentage of GFP-LC3-positive cells compared with the human (Hs) RNAi control. (Pten, P = 0.006; Tor, P = 0.034; Buffy, P = 0.005; debcl, P = 0.003; Bruce, P = 0.003; Dcp-1, P = 0.007; hid, P = 0.002; Ras, P = 0.006; and phl, P = 0.050). Results represent the mean value ± SD from three independent experiments.
Fig.Figure 1. MEFs from δWD and littermate control mice were incubated with HBSS, chloroquine or monensin for 2 hours as indicated. Cells were fixed and permeabilized and stained for LC3. Arrows indicate LC3 vacuoles where a ring of LC3 signal surrounds vacuoles ranging between 2 and 8 μm diameter. B. Numbers of fluorescent LC3 puncta and LC3 vacuoles in each cell were quantified by fluorescence microscopy. LC3 puncta were identified using spot function software to locate puncta ranging from 0.5-1.0mm diameter. LC3 vacuoles were identified by eye as rings of fluorescence ranging between 2 and 8 μm diameter. MEFs from δWD and littermate control mice were incubated with HBSS, chloroquine or monensin for 2 h as indicated and cell lysates analysed by western blot for ATG16L1, δWD and LC3 as indicated. Control MEFS express α and β isoforms of ATG16L1 at 70 kDa, and MEFs from δWD mice express a truncated ATG16L1 at 30kDa. (D) shows the level of conversion of LC3 to LC3II estimated by densitometry from a mean (±SD) of three replicate blots. E) fluorescence images of phagosomes following engulfment of Zymosan (red) by bone marrow-derived macrophages (BMDM) from control and δWD mice. White line indicates track used for line profile analysis to compare the LC3 signal on the limiting membrane of the phagosome with the center. F shows examples of line profile analysis G, H. (G) shows the percentage of LC3 positive phagosomes per cell and (H) shows line profile analysis of data from 10 cells, bars represent the mean ± SD and were compared by Student's t-test (****P<0.0001).
a) HCT116 cells or stable FoxO1-RNAi HCT116 cells were treated with a non-specific RNAi control or SIRT2-RNAi, and LC3-II accumulation or p62 degradation was detected in the presence or absence of E64 and pepstatin A.figf6(a) HCT116 cells or stable FoxO1-RNAi HCT116 cells were treated with a non-specific RNAi control or SIRT2-RNAi, and LC3-II accumulation or p62 degradation was detected in the presence or absence of E64 and pepstatin A. (b) HCT116 cells or stable FoxO1-RNAi HCT116 cells were treated with 10 μM AGK2 for 24 h, in the presence or absence of E64 and pepstatin A. Changes in FoxO1, p62 and LC3-II were determined by western blotting (upper panels), or the formation of LC3 punctate signals was observed (lower panels). HCT116 cells were treated with 10 μM AGK2 for 24 h and the formation of LC3 punctate signals was observed. Quantification of the ratio of LC3 punctate cells to overall HCT116 cells is shown in the lower right panel. Data are means ± s.d. (n = 3). (c) H1299 cells were transfected with Flag-FoxO1(WT), SIRT2(WT) or SIRT2(168A). At 24 h after transfection, the cell lysate was extracted and immunoblotted with anti-Flag, anti-SIRT2, anti-LC3, anti-p62 or anti-β-actin.. (d) HCT116 cells were transfected with a control plasmid, a SIRT2(WT)-expressing plasmid or a SIRT2(168A)-expressing plasmid. At 24 h after transfection, the cells were subjected to serum starvation for 24 h. The cell lysate was extracted and immunoblotted with anti-SIRT2, anti-LC3, anti-p62 or anti-β-actin. (e) H1299 cells were transfected with Myc-Atg7, Flag-FoxO1(WT) or mutated Flag-FoxO1(KR). At 24 h after transfection, the cytosolic lysate was extracted for co-immunoprecipitation with anti-Myc, followed by probing with anti-Flag. (f) H1299 cells were transfected with a control plasmid, Flag-FoxO1(WT), Flag-FoxO1(K262R), Flag-FoxO1(K265R), Flag-FoxO1(K274R) or Flag-FoxO1(KR). At 24h after transfection in the presence or absence of E64 and pepstatin A and immunoblotted, the cell lysate was extracted and immunoblotted with anti-Flag, anti-LC3, anti-p62 or anti-β-actin. (g) Left: H1299 cells were co-transfected with GFP-LC3, Flag-FoxO1(WT) or Flag-FoxO1(KR). The formation of GFP-LC3-labelled punctate signals was observed 24 h after transfection. Right: quantification of the ratio of GFP-LC3-labelled punctate cells to overall survived H1299 cells. Data in g are means ± s.d. (n = 3). Scale bars, 25 μm (b), 10 μm (g).
Noc recruits NBS DNA to the cell membrane A–C Representative images of TetR‐mCherry in cells lacking (A) or containing (B and C) the NBS plasmid pDWA117.Figure 5A-C Representative images of TetR‐mCherry in cells lacking (A) or containing (B and C) the NBS plasmid pDWA117. Strains DWA427 (A) and 429 (B and C) (both strains contain Pspac‐noc) were examined after growth for 2 h in the absence (A and B) and presence (C) of 1 mM IPTG. DNA was stained with DAPI. D-G Representative images showing the localisation of (D) Noc‐mYFP (DWA519), (E) NocNΔ10‐mYFP (DWA522), (F) NocQ68R‐mYFP (DWA520) and (G) NocG86S‐mYFP (DWA521), in the presence of the NBS plasmid pSG4929.
Loss of SEZ6 impairs glycosylation and maturation of GluK2/3.Figure 4A) In WT neurons, GluK2/3 was seen as two closely comigrating bands, whereas the upper one of lower intensity was missing or running even more closely to the lower band of main intensity in SEZ6KO neurons (schematic representation on the right). When the total lysates of SEZ6KO and WT neurons were digested with endoglycosidase H (EndoH) - which removes immature, but not mature sugars - the two closely comigrating bands were converted to three bands of a lower apparent molecular weight, consistent with full deglycosylation of the lowest band and a partial deglycosylation of the upper two bands (marked in light blue, red and green in the right panel). In SEZ6KO neurons the uppermost (light blue) band was missing and a new band of lower apparent molecular weight was seen that was overlapping with the red labeled band and was indicated with the purple asterisk in the SEZ6KO. No difference in the glycosylation of GluA2 was detectable, pointing to a specific effect of SEZ6 on GluK2/3. B) The running pattern of GluK2/3 in SEZ6KO and WT brains was compared upon EndoH digestion. Similar to primary neurons, GluK2/3 displayed an immature glycosylation pattern in SEZ6KO brains. C) Total GluK2/3 levels were quantified and showed a significant but moderate reduction in SEZ6KO compared to WT neuronal lysates (plot shows mean ± S.E.M., 9 replicates in 3 independent biological experiments, Mann-Whitney **p-value=0.0012). D) No significant change in GluK2/3 total levels was detectable in SEZ6KO and WT adult brains (plot shows mean ± S.E.M., 6 replicates, Mann-Whitney test was used). E) Synaptosomes from knock-out (KO) brains of single SEZ6 family members (SEZ6, SEZ6L, SEZ6L2), triple knock-out (TKO) and respective controls (WT and TWT - triple WT) were digested with EndoH. GluK2/3 displayed the immature glycosylation in SEZ6KO and TKO brains, but not in SEZ6L and SEZ6L2 brains, demonstrating non-redundant functions for the SEZ6 family members. (n=4) F) Brain homogenates from SEZ6KO and WT mice at different ages were digested with EndoH. GluK2/3 displayed the immature glycosylation at all developmental stages of SEZ6KO mice, suggesting that no compensation effect is occurring with aging.
Figure 2A Pie chart showing the distribution of 12492 H3K9cr peaks in annotated genomic regions in the E13.5 forebrain. B Sequence logos corresponding to enriched elements identified by de novo motif analysis of two clusters of H3K9cr peaks located in promoter regions in the E13.5 forebrain. C Top panel: Venn diagram of a combined comparison of genes under the regulation of H3K9cr or annotated in TCF4 peak regions. Bottom panel: Representative terms (P-adjust < 0.05) for KEGG pathway enrichment analysis of 452 genes that were co-marked by H3K9cr and TCF4. D KEGG pathway enrichment analysis of genes under regulation of H3K9cr in the E13.5 forebrain.
Fluorescence and electron microscope images showing various stages during microautophagic degradation of peroxisomes.Figure 3Fluorescence and electron microscope images showing various stages during microautophagic degradation of peroxisomes. The three panels in each figure are as follows (a-e, left) FM4-64; (center) GFP-SKL; (right) superimposed FM4-64 and GFP-SKL signals. (a, f) stage 0; (b, g) early stage 1; (c, h) late stage 1; (d, i) stage 2; (e) stage 3. (d, arrowhead) During the capture of these fluorescence images, this peroxisome-containing vesicle had just pinched off from the outer vacuolar membrane. Therefore, the FM4-64-stained vesicle membrane and GFP-SKL could not be superimposed in this picture.
Figure 4(A) AAV2 constructs containing 5'-inverted terminal repeats (ITR), U6 promoter, shRNA sequences, CMV promoter, hChR2-mCherry and 3'-ITR. Cdc50a shRNA was used to knock down Cdc50a. Scrambled shRNA was used as controls. (B) An experimental diagram showing 1-month old mice were intravitreally injected with AAV2 control and shRNA viruses and collected 17 days later. (C) Confocal images of dLGN after AAV2 injection. The dLGN was outlined with the dotted line. MCherry reveals axons and synapses of AAV2-infected RGCs. Left scale bar, 50 µm. Right scale bar, 5 µm. (D) Representative IHC images of CDC50A expression at synapses. Scale bar, 5 µm. Arrows indicate VGluT2+ RGC inputs. (E) Relative CDC50A expression at mCherry+ synapses in Cdc50a RNAi mice and controls. N = 3 for biological replicates, mean ± SD, * p < 0.05 by Student's t-test. (F) PSIVA staining in the dLGN. Exposed PS was detected by pSIVA on RGC inputs in Cdc50a-RNAi mice. Scale bar, 5 µm. Arrows indicate pSIVA+/mCherry+ RGC inputs. (G) Quantification of the percentage of pSIVA+/mCherry+ RGC inputs in total mCherry+ RGC inputs. N = 4 for biological replicates, mean ± SEM, **p < 0.01 by Student's t-test.
a) Neutral lipids in MEFs from 18Q-htt and 111Q-htt mice stained with BODIPY 493/503.figf7(a) Neutral lipids in MEFs from 18Q-htt and 111Q-htt mice stained with BODIPY 493/503. (b) Electron micrographs of livers from 18Q-htt and 111Q-htt mice. Right: number of lipid droplets (LD) per cell profile (prof.), mean area of LDs and percentage of cellular area occupied by LDs. Mean + s.d.; n = 3. (c-e) Neutral lipids in striatal cells from 7Q-htt and 111Q-httmice (c), grown on a monolayer of their own astrocytes (c-e) Neutral lipids in primary striatal neurons from 18Q-htt and 111Q-httmice grown on a monolayer of their own astrocytes (d) (c-e) Neutral lipids in lymphoblasts from a control (UC) and HD-affected human (e) stained with BODIPY 493/503. MAP2 staining highlights neurons. Extended study in Supplementary Figure 15b. (f) Electron micrographs of a lymphoblast from an individual with HD (with 78 and 15 polyglutamine repeats in the two alleles). Green arrows, LDs. (g) Fraction of cellular cytosol occupied by LDs quantified in lymphoblasts from three UC and four HD subjects. Numbers of polyglutamine repeats are shown below. Mean + s.d. of >100 cell profiles. Dotted red line, mean value of the respective samples. *P 0.05. (h) Oil Red O staining of striatal tissue from brain of two different unaffected control individuals (top) and two individuals with HD (bottom). Nuclei are highlighted with hematoxylin. Lipid droplets are visible as red puncta. Subject identifiers are shown in each panel. (i) The percentage of total cellular area occupied by lipid droplets (left) and the average number of lipid droplets per cell (right) was calculated for each of the samples by quantification of eight or nine different fields. *P 0.001; analysis of variance, P 0.01 for each person with HD versus each control individual by Tukey post hoc test. Subject identifiers are as in h.
Immuno-EM of autophagosomes.Figure 8Immuno-EM of autophagosomes. A and B, Mature autophagosome. C and D, Premature autophagosome. Arrows show expanded regions of the intramembrane space of the premature autophagosomes. TK116 cells were incubated in SD(−N) medium for one hour (D), two hours (B and C), or three hours (A). The localization of 3 × HA-Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A and D) or 10-nm gold- (B and C) conjugated goat anti-mouse IgG. AP, autophagosome; N, nucleus; V, vacuole.
Figure 2A Ribbon diagram of module 3 showing a tandem of double-Ca2+-binding motifs with loop-helix-loop (LHL) topology (LHL1 (limegreen) and LHL2 (limon)). Residues involved in Ca2+ coordination are highlighted in stick representation. The Ca2+ ions and Ca2+-bound water are shown in magenta and red spheres, respectively. B Superimposition of the LHL1 and LHL2 motifs together with the Ca2+ ions, with a backbone RMSD of ~ 0.3 Å. C Ribbon diagram of a canonical EF-hand (PDB: 2NXQ) and details of its Ca2+-binding sites. D Sequence alignment of module 3 orthologs using ESPript 3.0. NCBI accession numbers for the respective bacteria are given in parentheses. The positions of secondary structure elements are indicated above the sequences (α represents α-helix). E Amino acid composition comparison of the LHL motifs and a canonical EF-hand (PDB: 1J7O). F-G The interaction networks between modules 3 and 2 (F) as well as between modules 3 and 4 (G) H Schematic diagram of LHL1m, LHL2m and LHL12m mutants, which have D1N, D3A, E12N mutations in LHL1 and/or LHL2.
Overall results of the screenSchematic diagram illustrating number of dsRNAs analyzed, numbers of primary positives, negatives and other classes, and the results of rescreening.Figure 2Schematic diagram illustrating number of dsRNAs analyzed, numbers of primary positives, negatives and other classes, and the results of rescreening.
The spectrum of endocytic compartment morphologies: properties of 17 mutant phenotypes.Figure 2Representative images of wild-type and mutant cells organized by marker and phenotype (labels on the left of each panel). For each phenotype, three cells labelled for the training set (labelled single cells) and cells identified by the 2NN classifier (identified single cells) are shown. The table to the right of the images shows (from left to right): [1] the occurrence of each phenotype in a wild-type population (% in WT); [2] the number of specific phenotype mutants (all) and stringent specific phenotype mutants (str) for each of the 17 mutant morphologies; [3] the most significantly enriched GO Slim biological process and; [4] the most significantly enriched protein complex. # denotes term below statistical significance.