EMBO/sd-character-level-ner huggingface dataset:
Overexpression and Tagging of Lam6 Affects the Extent of Contact Sites.Figure 3(A) Fluorescence microscopy demonstrates that the overexpression of Cherry-Lam6 (OE-Cherry-LAM6) results in an expansion of the following three contact sites: ERMES (Mdm34-GFP), NVJ (Nvj1-GFP), and vCLAMP (GFP-Vps39). This suggests that an increase in Lam6 levels in the contact site is sufficient for its expansion. The numbers represent the average contact site size (120 cells per sample). Scale bar represents 5 μm. (B) Immuno-EM verified that GFP-Lam6 overexpression indeed causes an expansion of contact site size. While vCLAMP was hardly visible in WT cells, it could be detected easily in the OE strain (albeit often had ER tubules invading it). The NVJ underwent expansion as well as evoked PMN in OE strains, and the ER-mitochondria contact became large and elongated instead of small and distinct. N, nucleus; M, mitochondria; V, vacuole. Scale bar represents 200 nm (see also Figure S3I).
Figure 2(A) Co-immunoprecipitation of endogenous SLP2, PARL and YME1L in mitochondria isolated from human FITR293T cells using either SLP2- or PARL-specific antibodies. IgG was used as a negative control. In, input (10%). (B, C) Co-immunoprecipitation of endogenous SLP2 and YME1L with PARL-specific antibodies in mitochondria isolated from wild type MEFs (WT) and MEFs lacking YME1L, SLP2 or PARL. In, input (10%). (B, C) Co-immunoprecipitation of endogenous SLP2 and YME1L with PARL-specific antibodies in mitochondria isolated from wild type MEFs (WT) and MEFs lacking YME1L, SLP2 or PARL. In, input (10%). (D) Assembly of SLP2, PARL and YME1L into a high molecular weight complex. Mitochondria isolated from FITR293T cells (Con) and cells inducibly expressing SLP2-FLAG (FLAG) were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, PARL-, YME1L- and PHB2-specific antibodies. In, input (8%); E, eluate (100%). (E) High resolution BN-PAGE analysis of mitochondria lacking PARL, YME1L or SLP2. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-9% gradient gel containing 0-10% glycerol and analyzed using SLP2-specific antibodies. (F, G) Submitochondrial localization of SLP2. (F) Mitochondria were isolated from MEFs and fractionated in the presence or absence of proteinase K as indicated. Fractions were analyzed by SDS-PAGE and immunoblotting. MFN2 served as OM marker, TIMM23, SMAC/DIABLO, and YME1L as IMS markers, and AFG3L1 and AFG3L2 as matrix marker proteins. (F, G) Submitochondrial localization of SLP2. (G) Mitochondrial membranes were extracted with sodium carbonate at the indicated pH and separated into pellet (P) and supernatant (S) fraction by centrifugation. Fractions were analyzed by SDS-PAGE and immunoblotting. (H) Topology of the SLP2-PARL-YME1L (SPY) complex in the IM. IM, inner membrane; IMS, intermembrane space.
H3K9me3 accumulation in Jmjd2a/c KO ESCs impairs gene expression.Figure 6A-B. Independent RT-qPCR validations of genes identified as being (A) down-regulated or (B) up-regulated by expression arrays. Graphs show mean ± SD for technical triplicates and are representative of results obtained with at least 2 different ESCs lines of each genotype in independent experiments. A-B. Independent RT-qPCR validations of genes identified as being (A) down-regulated or (B) up-regulated by expression arrays. Graphs show mean ± SD for technical triplicates and are representative of results obtained with at least 2 different ESCs lines of each genotype in independent experiments. C-D. Density plots showing average ChIP-seq signals for (C) H3K9me3 or (D) H3K36me3 with 95% confidence intervals of the mean indicated in grey. Plots are shown for genes, which are down- or up-regulated according to the microarray analyses (FDR<0.05, absolute fold change log2(|FC|)>0.5) for both 2ac and 2abc KO ESCs (overlaps presented in Appendix Fig S6A), or do not show strong expression changes (log2(|FC|) < 0.2). Genes are further classified as "Bound" if containing binding sites for both Jmjd2a and Jmjd2c within +/- 1kb of a TSS. E. Heat map presenting array data in the form of log2(fold change) values comparing OHT with control treated ESCs. Genes are shown for which TSS regions (+/- 1kb) show the most substantial increase in H3K9me3 levels in OHT treated cells according to the ChIP-seq analyses (defined in Fig EV4).
Figure 5A) Analysis of REC8 distribution in representative pachytene wild-type sex chromosomes. Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3 and REC8. Bars, 10 μm in spreads and 1 μm on insets. Magnified XY pair is indicated by dashed area. Scatterplot showing the number of REC8 foci per X-chromosome. 225 foci identified in 14 X-chromosomes. Horizontal line indicates the mean, error bars indicate s.d. 3.02. B) Top: representative computationally straightened pachytene wild-type X-chromosomes. Nuclear spreads were immunostained for SYCP3 and REC8. Chromosomes were aligned at the PAR, and subdivided into 25 regular intervals. Bar, 1 μm. Bellow: histogram showing the distribution of the percentages of REC8 foci (n=225) among 25 regular intervals along 14 X-chromosomes. Dotted line indicates mean. C) Histogram showing the relative frequency of inter-REC8 distances along X-chromosomes.211 inter-REC8 distances from 14 X-chromosomes. Grey area represents 15% of the chromosome axis length. The respective cumulative distribution function is shown. D) Histogram showing the best fit of the observed inter-REC8 distances to the gamma distribution. Green curve indicates the best fit of the observed inter-REC8 distances.  indicates the value for which the best fit was obtained, with estimated standard error 0.27. E) Graph comparing the median distance of REC8 foci with computationally predicted even and random distributions. Median distances between foci subdivided in three categories (11-14, 15-18 and 19-22 REC8 foci per chromosome). Purple asterisk indicates the median distance of all REC8 foci. F) Scatterplot comparing inter-REC8 distances along on X-chromosomes with the distances between flanking REC8 foci at sites of LSAEs in the Stag3 mutant. 211 inter-REC8 distances measured along X-chromosomes and 10 distances measured between flanking REC8 foci in Stag3 mutant. Grey area represents 15% of the chromosome axis length. Horizontal lines indicate the median. p < 0.0001, obtained with two-tailed Mann-Whitney test.
a–d) Degradation of long-lived proteins in MEFs from wild-type (18Q-htt) and mutant huntingtin knock-in (111Q-htt) mice.figf1(a-d) Degradation of long-lived proteins in MEFs from wild-type (18Q-htt) and mutant huntingtin knock-in (111Q-htt) mice. (a,b) Rates of protein degradation after serum removal (a) (a-d) Degradation of long-lived proteins in MEFs from wild-type (18Q-htt) and mutant huntingtin knock-in (111Q-htt) mice. (a,b) Rates of protein degradation after treatment with rapamycin (rapam) or thapsigargin (thapsig) (b). (c) Lysosomal degradation calculated as percentage of protein degradation sensitive to NH4Cl. (d) Contribution of macroautophagy calculated as percentage of protein degradation sensitive to 3-methyladenine (3-MA). (e) Degradation of long-lived proteins in striatal cells from wild-type (7Q-htt) and mutant huntingtin knock-in mice (111Q-htt) in response to different autophagic stimuli. (f,g) Rates of protein degradation in wild-type and HD MEFs (f) and striatal cells (g) in the absence (control) or presence (Atg7−) of Atg7 RNA interference. (h) Contribution of macroautophagy (macroaut) to the degradation of long-lived proteins in lymphoblasts from unaffected controls (UC) and people with HD. Mean + s.d. of values from two to four different individuals and three or four different experiments. *P 0.05 versus untreated, #P 0.05 versus control.
Figure 5Top: Left: MECP2-KO neurons exhibit aberrant synaptic transcriptomics and synaptic morphology as well as decreased calcium transients and neural network activities. Right: Pharmacological compounds were selected for screening based on the expected ability of their mechanisms of action to counteract the synaptic and neurotransmitter pathologies we identified. Center: With step-wise progression through a pipeline of assays (shown as dark-to-light shades of blue), drugs were eliminated either if they did not reverse the MECP2-KO pathology identified in the assay or if they affected controls. Nefiracetam and PHA 543613 emerged as the two compounds that could reverse MECP2-KO neurocytopathology without affecting controls. Middle: The effects of Nefiracetam and PHA 543613 were validated in two 3D human cell models, MECP2-mosaic neurospheres (Left) and MECP2-KO cortical organoids (Right). Left: One or both drugs reversed calcium transient frequency and amplitude as well as MEA spike frequency in MECP2-mosaic neurospheres, but not their size. Right: One or both drugs reversed organoid size, SYN1 puncta, and MEA spike frequency in MECP2-KO cortical organoids. Bottom: A schematic proposing a hypothetical mechanism by which Nefiracetam and PHA 543613 might influence synaptic function.
Figure 8A qRT-PCR analysis of LDHA mRNA levels in A375 cells treated with BRAF inhibitor (PLX-4032, 1 μM) for 24 hr. Data information: Statistical analysis was performed by unpaired t-test for the comparison of two groups. Data are shown as mean ± SD, n = 3. ns: not significant, **p ≤ 0.01, ****p ≤ 0.0001. GC-MS-based quantification of (B) intracellular 3-PG in A375 cells treated with PLX-4032 (1 μM) for 16 hr. Data information: Statistical analysis was performed by unpaired t-test for the comparison of two groups. Data are shown as mean ± SD, n = 3. ns: not significant, **p ≤ 0.01, ****p ≤ 0.0001. GC-MS-based quantification of (C) lactate levels in A375 cells treated with PLX-4032 (1 μM) for 16 hr. Data information: Statistical analysis was performed by unpaired t-test for the comparison of two groups. Data are shown as mean ± SD, n = 3. ns: not significant, **p ≤ 0.01, ****p ≤ 0.0001. D Immunoblotting of the indicated proteins in A375 cells treated with PLX-4032 (1 μM) for 2, 6, or 24 hr. E, F Heatmap for expression fold-change of ASNS, GPT2, PSAT1, and MITF between (E) post-treatment (BRAF inhibitor, BRAF-i; MEK inhibitor, MEK-i) and pre-treatment, or (F) post-treatment (BRAF-i) and pre-treatment tumor in each of the patients (y-axis). The p-value of paired Wilcoxon test following the adjustment multiple hypothesis correction (FDR) are also displayed; post-treatment expression significantly greater than pre-treatment except in the case of MITF, where post-treatment expression is significantly less than pre-treatment.
Figure 1A Heat map from RNA-Seq data from FACS-isolated muscle stem cells and single myofibers showing relative expression of Pax7, Myf5, MyoD, Myog and Myf6. B Pie chart showing the distribution of Myf6 ChIP-Seq peaks within various genomic features. C Motif analysis using MEME suite (Bailey et al., 2009) identified canonical E-box motif as the most highly enriched sequence among all sequences under Myf6 peaks, followed by the Mef2a DNA binding motif. D Spatial distribution of transcription factor binding motifs within Myf6 ChIP-Seq peaks using CentriMo available via MEME suite (Bailey et al., 2009). E Normalized distribution of E-box motifs that occur within Myf6 peaks compared to their occurrences in the genome normalized per kilo base of DNA (two-tailed t-test). F Gene Ontology (GO Molecular Function) analysis of genes associated with Myf6 peaks based on association by proximity (single gene within 100kb of peaks) analyzed by Genomic Regions Enrichment of Annotations Tool (GREAT) (McLean et al., 2010). G Gene Ontology (GO Biological Process) analysis of genes associated with Myf6 peaks based on proximity similar to the analysis in "f". H Colormap of Myf5, MyoD and Myf6 peaks within 100kb of the Transcription Start Sites (TSS) of myokines (supplemental table EV3) ranging from zero (black) to six peaks (red) occupancy. The onset of differentiation coincides with increased binding of MRFs to the regulatory domains of the myokines genes.
Figure 3(A) RT-qPCR analyses of Hlf, Tef, Dbp and Nfil3 expression monitoring changes induced by acute ERS in MPH (3 to 5 independent experiments) (left panel) or mouse liver (5 mice per group) (right panel). The bar graphs show means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05. (B) Total protein extracts from MPH (left panel) or mouse liver (right panel) were subjected to Western blot with an antibody against NFIL3. ACTB was used as loading control. (C) Chromatin fractions from MPH (upper panel) or mouse liver (lower panel) were subjected to Western blot with an antibody against NFIL3. Histone H3 was used as loading control. (D) Enrichment scores from GSEA performed using LIVER-ID genes repressed by acute ERS in MPH (MPH ERS DOWN) as the gene set and liver transcriptomic changes induced by acute ERS and/or deletion of Nfil3 (NFIL3 KO) as the ranked gene lists were integrated and corrected for multiple testing using the BubbleGUM tool. For the NFIL3 KO ERS vs WT ERS comparison, the Core Enrichment genes (i.e. the subset of genes that contributes most to the enrichment result) were subjected to functional enrichment analyses using the ToppGene Suite. The top ranked KEGG Pathway with its Bonferroni-corrected p-value is shown. (E) Box plots showing mRNA expression for 5 genes from the Core Enrichment from D involved in xenobiotic metabolism issued from the transcriptomic analyses. Shown are Log2 FC relative to the mean normalized expression in the WT Control group (5 mice per group). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance, *P < 0.05. (F) The Integrated Genome Browser (IGB) was used to visualize ChIP-seq profiles for NFIL3 (green) and several LIVER-ID TFs (red) in the mouse liver at the Gsta3 gene locus. Levels of H3K27ac in MPH and cells from the non-parenchymal fraction (NPC) are shown in blue. The grey bar indicates the position of a BRD4 SE.
Figure 5(A) Experimental design for phosphotyrosine analysis of BV-2 cells with stable expression of Siglec and corresponding 2xY->F mutants. Samples were run as three 10-plex experiments: (1) Siglec-F: WT vs. 2xY->F; (2) Siglec-F, CD33, Siglec-5, and Siglec-8: WT vs 2xY->F; (3) Siglec-F: WT vs. 2xY->F ± 1 μM SHP099. Phosphotyrosine peptides were enriched and analyzed by LC-MS/MS. (B-C) Volcano plots showing phosphotyrosine peptides that were (B) increased or (C) decreased after Siglec-F expression. Plots show Siglec-F compared to 2xY->F mutant data integrated across all three runs. Protein names are shown for altered peptides. Labels are only shown for peptides with maximum directional change from each protein. Labels colored according to GO terms: magenta = signal transduction, red = endosome, cyan = cell adhesion, yellow = cytoskeleton, green = metabolism. (D) Heatmap showing phosphosites in CK-p25 which overlapped in their directional change with BV-2 Siglec-F-associated sites. Colors indicate fold change relative to 2xY->F controls. Heatmap showing phosphosites from CD33, Siglec-5, and Siglec-8 overexpression that were increased with Siglec expression and shared their directional change with Siglec-F. Siglec-F column indicates average of all untreated Siglec-F replicates. Color scale is same as (D). Heatmap showing phosphosites from CD33, Siglec-5, and Siglec-8 overexpression that were decreased with Siglec expression and shared their directional change with Siglec-F. Siglec-F column indicates average of all untreated Siglec-F replicates. Color scale is same as (D). (G) Western blot quantification of myc, SHP-2, and SHP-1 from myc co-IP eluates. Co-IP lysates were prepared from BV-2 cells stably expressing Siglec-F and optionally treated with 1 μM SHP099. (H) Heatmap showing phosphosites that were perturbed by SHP099 treatment. Color scale is same as (D).
Mitochondrion labelled with specific antibodies (see results section) was found as a reduced structure, or even absent, in TgAtg3-depleted cell lines (middle series of micrographs, kept for 2 days in the presence of ATc or pre-incubated with ATc for 4 days before invasion, respectively).Figure 10TgAtg3-depleted parasites show a defect in mitochondrion morphology.Mitochondrion labelled with specific antibodies (see results section) was found as a reduced structure, or even absent, in TgAtg3-depleted cell lines (middle series of micrographs, kept for 2 days in the presence of ATc or pre-incubated with ATc for 4 days before invasion, respectively). Parasites left to develop for two days in the presence of ATc to progressively extinguish the expression of TgAtg3 showed an accumulation of mitochondrial marker at the residual body. TgAtg3 depletion was verified by detecting myc-tagged regulatable extra-copy with specific antibody. TgAtg3-expressing cell line imyc-sTgAtg3cultivated in the presence of ATc for 4 days was used as a control for mitochondrial morphology (bottom). DNA was labelled with DAPI.
Autophagy can be efficiently regulated by cytoplasmic (de)acetylation reactions.Figure 8.Autophagy can be efficiently regulated by cytoplasmic (de) acetylation reactions. (A and B) HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and enucleated to obtain cytoplasts. The cytoplasts were treated with either vehicle (Co, control), 100-µM resveratrol (Resv), or 100-µM spermidine (Spd) for 4 h. 1-µM rapamycin (Rapa) was used as a positive control. (A) Representative images of cytoplasts indicative of autophagic activity. Arrows indicate GFP-LC3-transfected cytoplasts, whereas insets show nonenucleated transfected cells. (B) Quantitative data. Bars show the percentages of cells or cytoplasts showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac). (C-E) HCT 116 cells were cotransfected with a plasmid for the expression of RFP-LC3 together with an empty vector (pcDNA3) or plasmids encoding wild-type (WT) SIRT1 or a SIRT1 variant with a mutation in the nuclear localization signal fused to GFP (WT GFP-SIRT1 and mtNLS GFP-SIRT1, respectively) for 12 or 24 h, fixed, and analyzed by fluorescence microscopy. (C) Representative images indicative of 24-h autophagic activity. (D) Quantitative data. Bars depict the percentages of cells exhibiting the accumulation of RFP-LC3 in puncta (RFP-LC3vac). (B and D) Means ± SEM; n = 3; *, P < 0.05. (E) Representative immunoblots for 24-h endogenous LC3 lipidation. The asterisk stands for a nonspecific band. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 3(A) ZR-75-1 cells were treated with 1 M MK-2206 for 5 days and then one hour (1h) prior to cell lysis, cells were further treated with increasing doses of VPS34-IN1. Cell lysates were subjected to immunoblot analysis with indicated antibodies. (B) ZR-75-1 cells cultured in serum in the absence of Akt inhibitor were treated for one hour (1h) with the indicated concentrations of VPS34-IN1. The cell lysates were analysed by immunoblot using the indicated antibodies. (C) ZR-75-1 cells were treated for 5 days with either 1 µM MK-2206, 1 µM AZD5363, 1 µM GDC0941 or 1 µM BKM120. One hour prior to lysis the cells were incubated in the presence or absence of 1 µM VPS34-IN1. SGK3 was immunoprecipitated from lysates and subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM 32PATP in a 30 min 30oC reaction (upper panel). Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) and lysates were analysed by immunoblot with the indicated antibodies. One-hour (1h) treatment with the PDK1 inhibitor GSK2334470 (Najafov et al, 2011) (1 µM) was used as a control for SGK3 inhibition. (D) ZR-75-1 cells were stably transfected with either a control shRNA vector (scrambled) or a shRNA vector that targets Rictor expression (shRictor). The cells were grown in the presence or absence of 1 µM MK-2206 or 1 µM GDC0941 for 5 days. SGK3 was immunoprecipitated from the lysates and subjected to in vitro kinase assay as in (C). Immunoprecipitates (IP) and lysates (lower panel) were also subjected to immunoblot analysis with the indicated antibodies. (E) ZR-75-1 cells were cultured in the absence or presence of 1 µM MK-2206 for 5 days. Cells were then treated in the absence or presence of 0.1 µM AZD8055 or 0.1 µM Rapamycin for 1 hour (1h). SGK3 was immunoprecipitated and subjected to in vitro kinase assay as in (C). The immunoprecipitates (IP) and lysates were analysed with indicated antibodies.
Comparison between different migration modesA: Protrusion and retraction speed for amoeboid, oscillatory and fan-shaped cells, defined as the average of the pixels with the 20% lowest and highest membrane speed.Figure 5A: Protrusion and retraction speed for amoeboid, oscillatory and fan-shaped cells, defined as the average of the pixels with the 20% lowest and highest membrane speed. B: Ratio of the edge velocity, stress, LimE-GFP and GFP-myo intensity in membrane regions identified as protrusions and retractions. C: Average edge velocity in regions of low and high LimE-GFP and GFP-myo fluorescence. High fluorescence was defined as the 20% brightest LimE-GFP and GFP-myo pixels in the kymographs while low fluorescence consisted of the remaining 80% pixels. D: Ratio of the stress in regions of high and low LimE-GFP and GFP-myo fluorescence for the three modes of migration. The ratio was significantly different for all modes and was found to be much smaller in the fan-shaped cells, which have large traction force poles at the back of the cell.
Figure 6A-B Pths increases the translation of RNAs involved in (A) mitochondrial function and (B) metabolic pathways, along with transcription, translation, signal transduction, immune responses as well as redox processes C-F RT-qPCR analysis of mRNA from ribosomal protein fractions for control (black) and pths KD (gray) transcripts from S2R+ cells. RNA was isolated individually from fractions and pooled into five categories: RNP, 40S/60S, monosome, low polysome (di- and trisome), and high polysome (remaining fractions). The data were plotted relative to the amount present in the monosome fraction for each transcript. The mRNA levels of subunits of mitochondrial complexes (C) I, (D) III and (E) V were reduced in low and high polysome fractions in pths KD cells compared to the control cells, while (F) mRNA levels of GAPDH in the same fractions, used as an internal control, were not changed (n=3 independent biological replicates for control and pths KD). For (C,E-F) low polysome fraction for control vs. pths KD: (C) p=0.057, (D) p=0.0039, (E) p=0.09, (F) p=0.5. For (C,E-F) high polysome fraction for control vs. pths KD: (C) p=0.039, (D) p=0.046, (E) p=0.041, (F) p=0.66. G-I Western blots and their quantifications of protein extracts from control and pths KD S2R+ probed with (G) MT-ND1 (for mitochondrial complex I) n=4, p<0.0001), (H) ATPsynt-β (for mitochondrial complex V) n=3 p=0.034 and (I) tubulin β antibodies p=0.56) (n=3). Profilin serves as loading control. N=biological replicates. Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. Unpaired t-tests (C-F, G-I).