EMBO/sd-character-level-ner huggingface dataset:
Figure 1A) Schematic representation of the Y48G1C.1 / pid-2 gene and its mutant alleles (xf23 and tm1614). The line plot displays the predicted disorder of the Y48G1C.1 / PID-2 protein, as obtained from PONDR, using the algorithms VL3 and VL-XT. B) Expression of the 21U sensor (left) and DAPI staining (right) of gonad arms in the indicated genetic backgrounds. Gonads are outlined by a dashed line. The mCherry signal is represented in pseudo-colours [LUT fire (ImageJ)] to reflect differences in the intensity of the signal. Number of animals analysed and with indicated phenotype are given in the panel. Animals not showing the activation of the 21U sensor(+) were still silenced, and animals that did not show the silenced 21U sensor(RNAe) state were expressing weakly, comparable to the 21U sensor(+). Scale bar: 25 μm. C) Tc1 reversion assay in different genetic backgrounds. All the strains tested carried the unc-22::Tc1(st136) allele. Tc1 excision can result in restoration of unc-22 function, which can be scored visually. Negative control = unc-22::Tc1(st136) in a wild type background; positive control = wago-1/-2/-3. Two independent experiments per strain are represented. D) Crossing scheme to address the re-initiation of silencing of the 21U sensor. A mut-7 mutant male expressing the 21U sensor is crossed with either a wild type (left), prg-1 (middle) or pid-2 (right) mutant hermaphrodite. Their F1 offspring was scored for expression of the 21U sensor by microscopy. Three states of expression were scored, and represented in a pie chart. The three expression states are exemplified by representative images at the bottom: OFF (left), FAINT (right) or ON (middle). DIC images are shown above the fluorescence panels. Gonads are outlined by a dashed line. Scale bar: 25 µm. E) Crossing scheme to address if maternal 21U RNAs are sufficient to re-initiate the silencing of the 21U sensor. A pid-1 mutant male expressing the 21U sensor is crossed with a hermaphrodite, heterozygous for the same mutation. All their F1 offspring inherit a pool of 21U RNAs from the hermaphrodite, but in 50% of the F1, which is pid-1 homozygous mutant, no zygotic PID-1 is present, hence no zygotic 21U RNAs can be made. The silencing or expression of the 21U sensor in the pid-1 homozygous mutant F1 has been scored by microscopy, and depicted in a pie chart. At the bottom, a representative image of an animal carrying a silenced 21U sensor (lower: mCherry signal; upper: DAPI staining) in pid-1 mutant offspring. Gonads are outlined by a dashed line. Scale bar: 25 μm. F) Crossing scheme to test the role of PID-2 in re-initiating the silencing of the 21U sensor, mediated by maternally provided 21U RNAs only. The expression of the 21U sensor in the F1 has been scored by microscopy, and depicted in a pie chart. White arrow heads indicate the many arrayed oocytes, typical of a feminized germline. DIC and fluorescence image of a representative animal are shown at the bottom. Gonads are outlined by a dashed line. Scale bar: 25 μm.
The combination of γ-radiation and inducible deletion of BCL-XL causes severe kidney damage.Figure 3Bcl-xfl/fl;RosaCreERT2+/Ki (n=7) or as controls, Bcl-xfl/fl (n=8) and RosaCreERT2+/Ki (n=16) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=5), Bcl-xfl/fl;GFP-Chimeras (n=8) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles.(B) Picture showing both kidneys from age-matched healthy control wild-type mice (left) and sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (right). (C) Histological analysis of H&E-stained sections of the kidneys of age-matched healthy control mice or sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras as indicated, showing from left to right, shrunken scarred kidney, segmental chronic tubulo-interstitial disease, accumulation of cellular debris and amorphous material within the collecting ducts of the papilla. The higher magnification shows tubular epithelial degeneration, apoptosis and segmental secondary glomerular sclerosis. Pictures are representative of at least 5 mice for each genotype and treatment. (D) Multiphoton analysis of fixed sections of the kidneys of age-matched healthy RosaCreERT2+/Ki;GFP-Chimeras (left) or sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (right). Pictures are representative of at least 3 mice for each genotype and treatment. green=second harmony, blue=short wavelength autofluorescence.
Figure 7Figure 7. Schematic representations of mechanisms that may underlie brain maldevelopment in ASD. In control brains (top panel) both smaller and bigger brains develop and function normally. At the cellular level, there are no significant alterations of cell division phases and the number of cells produced is within normal variation. At the molecular level, there are no genetic alterations and changes in gene expression are within normal variation with no significant alterations of network structure and function. In ASD brains (bottom panel), brain development is abnormal and smaller and bigger brains represent two different anatomical and functional outcomes. In smaller ASD brains, the cellular characteristics are currently less clear compared to bigger brains. We hypothesize that G1/S phase transitions may be longer and/or checkpoints may stall/delay the timing of cell divisions leading to a reduced number of cells. Alternatively, there are an increased number of apoptotic cells. These cellular phenotypes may be related to genetic mutations (red lightning bolt) of Hc-genes that, for instance, regulate chromatin modification as to the case of CHD8 (blue). The mutation leads to altered regulation (red arrows) of downstream transcription factors or regulatory elements (yellow, cyan, green diamond shapes) that in turn regulate the expression of brain-relevant genes (purple square). Mutated CHD8 can also alter directly the expression brain-relevant genes, as to the case of CCNE2. Gene expression and networks functions are altered, but closer to normal brains. In bigger brains, cellular and molecular phenotypes are more pronounced compared to smaller ASD brains. Cellular evidence suggests that the increased number of neurons may be due to the shortening of G1/S phases. At the molecular level, this may be related to mutations and changes in gene expression that lead to a reorganization of networks controlling neuroprogenitor cell divisions. In addition to the downstream effects of Hc mutations (i.e. CHD8, red arrows), pronounced gene expression changes cause a substantial reorganization of the network with the activation of new regulatory genes (i.e. TOP2A, TYMS, triangle shapes) and new interactions (green arrows), thus leading to altered or different network functions.
MERTK is a predictive marker for inhibitors targeting MEK1/2 in CRC cell lines.Figure 5(A) Effect-size heat maps of two drugs (one from two different drug sensitivity screens) targeting MEK1/2 show consistent association of high MERTK expression with drug resistance. The colour scheme is the same as in Figure 4. (B) Bar chart visualising the top kinases recurrently associated (absolute effect-size>0) with drug resistance (top seven bars) and sensitivity (bottom seven bars) in the GDSC and CCLE drug sensitivity datasets. (C) Dose-response curves of two drugs for which high MERTK (left panels) or ACVR2A (right panels) expression was predicted to confer drug resistance. For each drug, three cell lines predicted to be sensitive (dark blue) and three cell lines predicted to be resistant (yellow) were assayed for viability. The experimental data validated that cell lines predicted to be more sensitive to a drug indeed showed this phenotype (data represents the average of three technical replicates; see Appendix for details). (D) Boxplots summarizing the data shown in panel (C) using the area under the curve (AUC) as a measure for drug sensitivity. High AUC values indicate drug resistance. Again, it is evident that cell lines predicted to be more resistant to a certain drug were in fact significantly (p≤0.05, one-sided Mann-Whitney test) more resistant than cell lines predicted to be more sensitive. (E) Western blot of and cells, visualising successful knockout by CRISPR/Cas9. (F) Dose-response curve of RDEA119 (MEK1/2 inhibitor) in and cells. The knockout is more sensitive to MEK1/2 inhibition than the wild type. See also Figure EV6.
Figure 4Percentages of macrophage populations in wild type and Sl/Sl E14.5 brain, skin (dissected from the back), lungs and limb buds, as determined by flow cytometry. Cells were gated as Ter119- CD45+ and further gated into F4/80hi CD11blo or F4/80lo CD11bhi populations. Representative dot plots of skin analysis are shown in Appendix Figure S1A. Data are the mean (±SD) of 4 biological replicates for either genotype, with each replicate consisting of individual or pooled samples (from up to 3 embryos). A total of 8 wild type and 5 Sl/Sl embryos were analyzed over 2 independent experiments. Percentage and absolute numbers of tissue macrophages per E14.5 FL. Data are mean (±SD). Number of embryos analyzed: as in (A). Representative flow cytometry analysis of macrophages in wild type and Sl/Sl E10.5 AGM+VU. Cells were gated as shown, with percentages in individual gates as indicated. Data are the mean (±SD) of 4 wild type (32-35 sp) or 3 Sl/Sl (34-35 sp) biological replicates, analyzed over 2 independent experiments. Embryos were analyzed individually. Quantification of flow cytometry analysis in (C), showing percentage of F4/80+CD11b+ macrophages within live cells. E11.5 data are the mean (±SD) of 4 wild type (10-13 tail somites) or 3 Sl/Sl (10-13 tail somites) biological replicates, analyzed over 3 independent experiments. Embryos were analyzed individually. Confocal whole-mount immunofluorescence analysis of macrophages in wild type and Sl/Sl E10.5 AGM+VU. 3D panels show maximum intensity projections from 400μm-thick Z stacks. Slice panels show single 2.5μm-thick Z slices of clusters in the dorsal aorta (da). A total of 4 wild type (31-34 sp) and 3 Sl/Sl (31-33 sp) embryos were analyzed. Scale bars: 50μm (3D), 10μm (slice). Quantification of whole-mount immunofluorescence in (E), showing absolute numbers (mean ± SD) of F4/80+ macrophages within hematopoietic clusters in the dorsal aorta.
EHD1 localizes at REs in an ATP8A1 activity‐dependent manner and is required for fission of TfnR‐positive membrane carriers A COS‐1 cells were treated with EHD1 siRNAs for 72 h.Figure 3A COS‐1 cells were treated with EHD1 siRNAs for 72 h. Cell lysates were prepared and then immunoblotted with EHD1 antibody. α‐tubulin was used as a loading control. B, C Cells were treated with EHD1 siRNA1 for 72 h. Cells were pulsed with Alexa 488‐Tfn and chased in medium with unlabeled Tfn. During the chase, cells were imaged every 3 min for 57 min. In (B), representative images of Alexa 488‐Tfn in control or EHD1‐depleted cells are shown. In (C), the fluorescence intensity of Alexa 488‐Tfn is shown as percentage of that at 0 min (mean ± SEM, n > 3 cells from three independent experiments). ***P 0.001; two‐tailed Student's t‐test. Scale bars, 10 μm. B, C Cells were treated with EHD1 siRNA1 for 72 h. Cells were pulsed with Alexa 488‐Tfn and chased in medium with unlabeled Tfn. During the chase, cells were imaged every 3 min for 57 min. In (B), representative images of Alexa 488‐Tfn in control or EHD1‐depleted cells are shown. In (C), the fluorescence intensity of Alexa 488‐Tfn is shown as percentage of that at 0 min (mean ± SEM, n > 3 cells from three independent experiments). ***P 0.001; two‐tailed Student's t‐test. Scale bars, 10 μm. D COS‐1 cells were treated with EHD1 siRNA1 for 72 h. Cells were pulsed with Alexa 594‐CTxB for 5 min and chased for 60 min. Cells were then fixed and stained for GM130. The Pearson's coefficient between CTxB and GM130 is shown (mean ± SD, n > 30 cells from two independent experiments). ***P 0.001; two‐tailed Student's t‐test. See Supplementary Fig S7A for the representative images. E COS‐1 cells were treated with EHD1 siRNA1 for 72 h and stained for TfnR. The image represents the maximum intensity z‐projection of four confocal slices. Scale bar, 10 μm. F COS‐1 cells co‐transfected with GFP‐ATP8A1 and myc‐CDC50A were stained for EHD1. Scale bar, 10 μm. G Cells were treated with ATP8A1 siRNA1 for 72 h and stained for EHD1. For the rescue experiments, siRNA‐resistant GFP‐ATP8A1 (WT or E191Q) and myc‐CDC50A were transfected 48 h after siRNA transfection. Asterisks indicate cells with GFP‐ATP8A1 expression. The graph represents percentage of cells with perinuclear localization of EHD1 as mean ± SD of three independent experiments (at least 141 cells were counted for each condition). Scale bars, 10 μm.
Nucleosome dynamics of human pluripotent stem cell and differentiated cells.Figure 1A Genome-wide nucleosome maps were generated from the frequency distribution of the read midpoint positions of pair-end sequenced MNase-resistant fragments in the size range 138-161 bp (spanning the nucleosome footprint). Figure shows a section of a nucleosome map taken from chromosome 12 derived from pluripotent iPSC (pl-iPSC) to show examples of nucleosomes selected as highly positioned, marked *. B The distribution of positioned nucleosomes (derived 138-161 bp size class) and sub-nucleosome fragments (derived 112-137 bp size class) for pl-iPSC and iPSC-derived NPC (NPC). Actual calculated numbers are presented above each column. C Venn diagram showing the overlap in the genomic location of positioned nucleosomes in pl-iPSC and in the same cell line differentiated into neural progenitor cells (NPC). D Average frequency distribution of nucleosome positions relative to each other. Data was aligned to each mapped nucleosome in the genome and plotted for a 600 bp window for pl-iPSC, NPC and the chronic myelogenous leukaemia (Cml) derived cell line, K562. In all cases, a prominent single nucleosome is presented with only minor flanking peaks, indicating that the majority of nucleosomes do not occur as evenly spaced arrays in human cells. E Distribution of nucleosome array sizes for pl-iPSC, NPC and K562, calculated as the number of nucleosomes within a distance of 150-200 bp of each other. Bars show the number of nucleosomes arrayed as singletons (1), pairs (2), triplets (3) and greater than 3 (>3), displayed as log10 values and the percentage distribution within a cell line is shown above the column.
Figure 4(A). Serum AKG concentration-time profile obtained from male C57BL/6 mice (10 weeks) fed with normal chow before or after i.p AKG (10 mg/kg body weight). The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after injection (n = 8 per group). (B-C). Representative images (B) and quantification (C) of BAT thermogenesis after 6 hrs cold exposure at 4℃. Male C57BL/6 mice (10 weeks) were i.p. injected with 10 mg/kg AKG or saline and immediately exposed to cold stress at 4℃ (n = 8 per group). (D). Immunoblots and quantification of p-HSL and ATGL in the gWAT of male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of 10 mg/kg AKG or saline (n = 3 per group). (E). Serum E level in AKG treated male mice 3 hrs after i.p. injection (n = 8 per group). Oxygen consumption (F-G) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day. RER (H-I) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day. (J-N). Body weight gain (J), cumulative food intake (K), body composition (L), fat weight (M) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks (n = 8 per group). serum NEFA (N) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks (n = 8 per group). (O-P). Representative images (O) and quantification (P) of BAT thermogenesis after 6 hrs cold exposure at 4℃ in sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 8 per group). (Q). The mRNA expression of themogenic genes in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 6 per group). (R-S). Immunoblots (R) and quantification (S) of p-HSL and ATGL protein in the gWAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group). (T-U). Immunoblots (T) and quantification (U) of UCP1 protein in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group).
Figure 1A Indicated tail-anchored (TA) proteins were in vitro translated in rabbit reticulocyte lysate (RRL) using RNAs containing a stop codon. Translations were carried out in the presence of 2 μM HisTrx or HisTrx-SGTA (HT-SGTA), followed by incubation with HisPur Cobalt resin Bound proteins were eluted at high imidazole concentration, samples resolved by SDS-PAGE and results visualised by phosphorimaging. Sec61β 3R carries three Arg residues within Sec61β transmembrane domain (TMD) which abolish its hydrophobic character and was used as a negative control for SGTA binding. A schematic representation of a TA-protein is also shown. Open circles indicate TA-proteins selectively bound by HisTrx-SGTA. RAMP4 - stress-associated endoplasmic reticulum protein 1; Syb2 - synaptobrevin 2; Synt5 - syntaxin 5. B As for (A) but total translation products (input) were resolved by SDS-PAGE and results visualised by phosphorimaging. When total products are analysed high amounts of haemoglobin in the RRL distort the migration of Sec61β and its variant. C A schematic representation of model polypeptides derived from amyloid precursor protein (APP) that were used in the study. The C-terminal 99 amino acids of APP (C99) were fused in frame with the signal sequence derived either from preprolactin (PPL) or APP. Lysine and cysteine residues and amino acids within the preprolactin signal sequence mutated in the PPLssKO-C99 construct are indicated whilst the TMD and signal sequences are shown in dark and light grey, respectively. RNAs coding for C99 variants and containing a stop codon introduced either after the complete coding sequence or before the predicted TMD were used. E As for (D) but total translation products were subjected to immunoprecipation with either anti-C99 antibody (for the full-length variants) or with anti-β-amyloid antibody (for products lacking the TMD). A filled square indicates inefficiently translated APP-C99-TMD immunoprecipitated with anti-β-amyloid antibody.
Deterministic and semi‐deterministic modeling of sustained and pulsed GF stimulation.Figure 5AModel topology scheme. Asterisks indicate active proteins. Blue/red colors indicate wiring in response to EGF/NGF stimulation. &amp;amp;: feedback loop integration, ø: synthesis and degradation, NFB/PFB: protein mediating negative/positive feedback. BDeterministic modeling of sustained and 3′ pulse GF stimulation. Normalized, averaged ER trajectory from EKAR2G ERK activity measurements with means (dots) and StDev (error bars) are shown in red. Simulated ERK activity (ERK*) curves are shown in blue. Black line represents GF stimulation input. The model was simulated with a single parameter set (Appendix Table 1). Sustained and 3′ pulse GF stimulation ERK activity trajectories are from Fig 2 and Fig 3A and C, respectively. C, DAn ensemble of cells was modeled by randomly distributing the parameter values for all kinase, feedback protein, and phosphatase concentrations according to a lognormal distribution shown in (C). Each individual cell is characterized by slightly different parameter values as illustrated in (D), where each column corresponds to one cell. E, FEnsemble modeling of ERK activation dynamics in response to 3′/10′ pulsed or sustained, high‐ and low‐dosage GF stimulation. An ordinary differential equation (ODE) model was run 1,000 times with total signaling component concentrations drawn from the log‐normal distribution shown in (C). (E) EGF, (F) NGF.
Figure 6(A) Expression of 12 neuropsychiatric disorder-related genes in saline- and poly(I:C)-treated WT neurons. (B) Expression of 9 neuropsychiatric disorder-related genes in poly(I:C)-treated Tlr3-/- neurons. Relative expression levels compared with vehicle control are shown. In (B-D), dashed lines indicate the level of saline control. (C, D) Expression of Auts2, Disc1, Fmr1, Pten and Ube3a in poly(I:C)-treated Myd88-/- neurons (C) and poly(I:C)-treated P5 WT mouse brains (D). In (B-D), dashed lines indicate the level of saline control. (E) DIV 5 WT neurons were treated with poly(I:C) for 24 h. The total neuronal lysates were subjected to immunoblotting with the indicated antibodies. VCP and β-actin were used as loading controls. The protein levels were normalized with β-actin. Data were analyzed by unpaired t-test. Numbers of experimental repeats (N) are shown in each column. * P < 0.05, ** P < 0.001, *** P < 0.0001.
Figure 6A. HBV-transgenic mice (5 in mock group, 6 in all others) were injected intravenously with 1x1011 particles of the shown AAV8 vectors, and HBsAg was measured from serum at various time points post-injection. Fifteen days post-treatment, mRNA expression in livers was analysed using microarrays. Significance was calculated using student t-test. B. Genes significantly dysregulated compared to mock treated animals (false discovery rate <0.25; compared to mock). C. Transcripts of genes significantly up- or down-regulated by shHBV7 were analysed for the presence of shHBV7 sense or antisense 2-7 nt seed matches and compared to the background frequency within all genes on the microarray. Significance was calculated using Chi-square test with Yates' correction. D. All genes on the microarray were grouped according to the presence of 3'UTR shHBV7 sense or antisense seed matches. Shown is their relative expression compared to the empty vector. Significance was calculated using Mann-Whitney test.
a) Western blot analysis of the chase experiment in 60Q Neuro2a cells.figf3(a) Western blot analysis of the chase experiment in 60Q Neuro2a cells. Full-length western blots are presented in Supplementary Figure 13. (b) RHQ enhanced the tNHTT-60Q-EGFP degradation during the chase period. 5 μM MG132, 10 mM 3-methyladenine and 15 mM ammonium chloride (NH4Cl) were used to inhibit proteasome, macroautophagy and lysosomal degradation, respectively. NH4Cl had no effect on proteasome activity and did not affect cell viability (Supplementary Fig. 4a,b). Bars in b represent relative mean values ± s.e.m. from three independent experiments. Protein levels at time 0 represent the control condition (arbitrarily set as 1).
Figure 1(A) Schematics of Rango-3 sensor. (B) Representative images of importin β cargo gradients detected by Rango-3 FLIM/FRET imaging. Scale bars, 20µm. (C) Line scans of Rango-3 FRET efficiency (E) in 10 µm-wide areas (D-F) Quantification of the Rango-3 E gradients (top panels) and average cellular Rango-3 E (bottom) in involving untreated oocytes (controls) or oocytes injected with RanT24N mRNA, importin β(71-876) protein or treated with 20 µM IPZ. Data for each panel are from at least 2 separate experiments. Means ± SDs, t-test, oocyte numbers indicated in brackets. (G) Nonlinear single-exponential regression was used to fit the average of radial Rango-3 τdonor line scans in control oocyte with the one-phase decay model. The dashed line corresponds to the average of spindle pole distance from chromosomes in the same cell. (H) Immunoblotting in biochemical pull-downs with recombinant Ran proteins added to human DL D1 cell lysates. A representative of at least 5 repeats.