EMBO/sd-character-level-ner huggingface dataset:
a) Pseudocolour-coded images of EPACI–camps FRET from real-time imaging of wild-type MEFs transfected with EPACI–camps.figf3(a) Pseudocolour-coded images of EPACI-camps FRET from real-time imaging of wild-type MEFs transfected with EPACI-camps. Where indicated, cells were perfused with the starvation solution for the indicated times (in min) or with 25 μM forskolin (FRSK). Scale bar, 20 μm. Colour scale indicates F1/F0 ratio. See also Supplementary Movie S3. (b) Quantitative analysis of CFP/YFP FRET ratio. Experiments were carried out as in a. Where indicated, cells were perfused with the starvation solution or with 25 μM forskolin. Data represent mean ± s.e.m. of 13 independent experiments. (c) Lysate samples (50 μg) from MEFs of the indicated genotypes were analysed by SDS-PAGE and immunoblotting using the indicated antibodies. Where indicated, MEFs were starved, or treated with 25 μM forskolin. Where indicated, 20 μM H89 was added during starvation. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8. (d) Representative images of the effect of H89 on mitochondrial morphology on starvation. Wild-type and Mfn2−/−MEFs were transfected with mtYFP, and after 24 h confocal micrographs were acquired. Where indicated, cells were starved for 2.5 h and 20 μM H89 was added. Scale bar, 20 μm. (e) Morphometric analysis. Experiments were carried out as in d. Data represent mean ± s.e.m. of five independent experiments (n=100 cells per condition). (f) Starvation-induced mitochondrial elongation depends on Ser 637 of DRP1. Representative confocal micrographs of mitochondrial morphology of wild-type MEFs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. (g) Morphometric analysis of mitochondrial shape. Experiments were carried out as in f. Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition). (h) Representative confocal micrographs of mitochondrial morphology of Drp1−/− MEFs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. (i) Morphometric analysis of mitochondrial shape. Experiments were carried out as in h. Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition).
Blood velocity profiling in FLNAΔECS and wt mice.Figure 7(A) Graph showing the individual blood velocity profile determined by MRI over time in 4 wt mice (shades of grey) and 5 FLNAΔECS mice (shades of red). The mouse shown in dotted purple line is clearly an outlier within the FLNAΔECS cohort. Aortic velocity waveform represents velocity during different stages of cardiac cycle. A significant reduction of velocity was observed at 40ms in the systolic cycle. (B) Representative wt (top) and FLNAΔECS (below) mouse showing the blood flow just above the aortic valve. Note: Reduced blood flow in FLNAΔECS mouse as depicted by pseudocoloring. The red circle represents the time window at which the images are shown.
Lnc-SMaRT functional characterization.Figure 1A Schematic representation of the lnc-SMaRT genomic locus. Genomic coordinates and exon/intron lengths are indicated. qPCR primers location is indicated by blue arrows while the regions corresponding to the siRNAs target sites are indicated by red lines. B RT-PCR on cytoplasmic (C) and nuclear (N) extracts showing the subcellular localization of lnc-SMaRT. RNA was isolated from C2C12 cells after two days of differentiation. GAPDH mRNA and pre-mRNA (pre-GAPDH) were used respectively as cytoplasmic and nuclear controls. Representative results from three independent experiments are shown. C qPCR analysis of lnc-SMaRT RNA expression in C2C12 cells undergoing differentiation at the indicated time points. The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots). D qPCR on RNA extracts from C2C12 myoblasts treated with either control siRNAs (si-SCR) or with two different siRNAs against lnc-SMaRT (si-SMaRT-1, si-SMaRT-2) and induced to differentiate for two days. The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots). Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. ***P < 0.001. E Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) C2C12 murine myoblasts transfected with either control (si-SCR) or lnc-SMaRT siRNAs (si-SMaRT-1, si-SMaRT-2) fixed after at two days of differentiation. Histograms represent MHC-positive mononucleated cells/total MHC-positive cells ratio and fusion index quantification (F.I.). At least 5 randomly chosen microscope fields of two independent biological samples were analyzed (n>600 cells for each field). Data are presented as the mean. of the two biological replicates (dots).
Figure 6Chromosome parameters obtained for laggard-producing chromosomes are shown in A-D on the vertical axis, time in min on the horizontal axis relative to the anaphase II onset. Light blue lines indicate 12 laggard-producing chromosomes with correct (equational) chromatid segregation; darker line highlights one chromosome with chromatid non-disjunction. On the schemes near the charts chromosomes are red with green centromeres; spindle equator in A and spindle axis in B-C are indicated by black dashed lines. Changes in the distance to the equator plane (shown by a black arrowed line on the scheme) for 13 laggard-producing chromosomes. All laggard-producing chromosomes are positioned above the equator at the start of analysis period. Black dotted lines indicate the average thickness of the metaphase plate. Changes in the distance to spindle axis (shown by a black arrowed line on the scheme) for 13 laggard-producing chromosomes. Black dotted line indicates the average radius of the metaphase plate. Changes in the angle to the spindle axis (highlighted in black on the scheme) for 13 laggard-producing chromosomes. Dashed line on the chart indicates the mean angle for normally segregating chromosomes. Changes in the inter-centromere distances (shown by black arrowed line on the scheme) for 13 laggard-producing chromosomes. Dashed line indicates the mean inter-centromere distance for normally segregating chromosomes.
A putative model of Dln1 pore formation.Figure 7The lectin module, aerolysin module and receptor are colored in yellow, hot pink and green, respectively. The membrane was shown in gray sphere. The β-barrel is adopted from that of γ-hemolysin (PDB 3B07).
Crosslinking and docking analysis suggest that the flap domain binds to pRab8a.Figure 2(A) SDS-PAGE analysis of the PPM1H(D288A): pRab8a complex in the presence of DSBU crosslinker. Control migration of proteins are on the left. NP, non-phosphorylated Rab8a; P, phosphorylated Rab8a. The migration of control and crosslinked proteins is marked on the right. (B) Crosslinked peptides from PPM1H and pRab8a are mapped onto the sequence. The flap domain forms extensive crosslinks with pRab8a. In addition, two flexible loops (104-142, 188-226) also have multiple crosslinks with the substrate. (C) Ribbon model of pRab8a (left) and the switch 2 phosphopeptide (right) docked onto the active site of PPM1H. The crosslinks shown between PPM1H and pRab8a are within accepted distance constraints (32 Å) for DSBU (He et al., 2015). (D) Kinetics of phosphate hydrolysis. 25 nM recombinant wild-type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a (GTPγS or GDP) as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of released phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 phosphorylated Rab8a[GTP bound conformation] (blue) and pThr72 phosphorylated Rab8a[GDP bound conformation] (red). The experiments were repeated twice, and both data points are shown in curves. Line fittings for the left and middle panel were performed using the mean of the two values. Kinetic constants (Kcat, Vmax, Km) were obtained using GraphPad software, and their uncertainties (±) correspond to the SE of mean. (E) Kinetic analysis of PPM1H as in (D) using 50 nM PPM1H and phosphopeptide substrates, as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 Rab8a phospho-peptide (blue) and Rab10 pThr73 phospho-peptide (red). Each experiment was performed twice (individual data points shown). (F) Side by side comparison of the catalytic activity against protein and peptide by in vitro malachite green time course analysis. 50 nM recombinant wild-type PPM1H was incubated with 16 μM Rab8a GTPγS pThr72 phosphorylated protein (blue) or Rab8a pThr72 phosphorylated peptide (red) for indicated times and analysed as described in Materials and Methods. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates.
SEZ6 extracellular domain in the secretory pathway rescues GluK2/3 maturation in SEZ6KO neurons and binds to GluK2/3.Figure 8A) Schematic representation of the constructs used: SEZ6 full length (SEZ6FL), SEZ6 lacking the C-terminal domain (SEZ6Δcyto), SEZ6 ectodomain generated by BACE1 cleavage (SEZ6ecto), SEZ6 C-terminal fragment generated by BACE1 cleavage (SEZ6CTF) and SEZ6 lacking the C-terminal domain but fused to an ER retention signal (SEZ6ΔcytoER). B, SEZ6 constitutive KO neurons were transduced with SEZ6 constructs. SEZ6FL and SEZ6Δcyto were detectable as two bands corresponding to mature (higher) and immature (lower) forms of SEZ6. No difference in the apparent molecular weight was detected because the high percentage of the gel used (12%) and the small C-terminal deletion (39 amino acids). SEZ6ecto was present as the immature band in the neuronal lysates SEZ6CTF (with HA and Flag-tags) was detected at the expected low molecular weight. Additionally, the C-terminal fragment generated from SEZ6FL by BACE1 cleavage was detected. Due to its lack of an HA-tag, it had a lower molecular weight than SEZ6CTF. The C-terminal fragment generated from SEZ6Δcyto upon BACE1 cleavage was not visible because of its low molecular weight. GluK2/3 glycosylation was rescued when SEZ6FL, SEZ6Δcyto and SEZ6ecto were expressed in the neurons, but SEZ6CTF was not able to rescue the phenotype. Color coding for asterisks is the same as in figure 4A. Representative blot of 3 independent experiments. C) SEZ6ecto was present as the mature, secreted band in the conditioned medium in panel C. D) Sez6 constitutive KO neurons were transduced with SEZ6ΔcytoER and SEZ6Δcyto. SEZ6ΔcytoER presented only the band corresponding to the immature form of SEZ6, as expected for a protein retained in the ER. SEZ6Δcyto, but not SEZ6ΔcytoER, was able to rescue GluK2/3 glycosylation, showing that SEZ6 localized to the ER is not sufficient to rescue the GluK2/3 glycosylation (n=5). Color coding for asterisks is the same as in figure 4A. E) HEK293T cells were co-transfected with a GluK2a plasmid and the SEZ6 constructs. Expression of SEZ6 constructs was analyzed in the total lysates ("Input") and revealed a similar expression of the different proteins. GluK2a was immunoprecipitated with GluK2/3 antibody and the SEZ6 mutants were detected with the HA.11 antibody ("IP"). SEZ6FL, SEZ6Δcyto and SEZ6ecto, but not SEZ6CTF were co-immunoprecipitated with GluK2a, suggesting that SEZ6 extracellular domain is necessary for GluK2a binding. Representative blot of 2 independent experiments. * indicates the light chain of the antibody used for immunoprecipitation.
Although FLIP can interact directly with FADD in a caspase-8-independent manner, its DISC recruitment is highly caspase-8-dependent.Figure 3(A) Western Blot analysis of Caspase 8, Caspase 10, FLIP and FADD recruitment to the TRAIL-R2 DISC in procaspase 8 (WT) or procaspase 8 deficient (Null) A549 cells incubated with AMG655 beads (4x) for either 30, 60 or 180 minutes. (B) Western blot analysis of the recruitment of FLIP, Caspase 8, Caspase 10 and FADD to the TRAIL-R2 DISC in HCT116 Parental, caspase 8 null (CASP8), caspase 10 null (CASP10) or CASP 8/10 cells treated with 4x AMG655 conjugated beads for 90 minutes. (C) AlphaScreen® assessment of the interaction of recombinant FLIP DED1/2 and recombinant FADD DED. (D) AlphaScreen® assessment of the impact on the FLIP-FADD interaction of unstapled and stapled peptides corresponding to the FADD α1 helix. (E) NanoBiT® assay to quantify FLIP/FADD interactions in a U2OS cell line stably expressing the NanoBiT® constructs, FLIP (LgBiT) and FADD (smBiT) following 48 hour silencing of FLIP, FADD, or caspase 8. Western blot analysis was used to confirm successful knockdown of target proteins by siRNA. (F) NanoBiT® assay of HCT116 and U20S wild type (WT) and procaspase 8 null (Null) cells transiently co-transfected with FLIP (LgBiT) and FADD (SmBiT) NanoBiT® constructs for 24 hours. Construct expression was analysed by Western blotting.
Loss of peripheral RAB13 RNA localization disrupts cell migration and phenocopies acute RAB13 protein loss.Figure 5A. Transwell migration of MDA-MB-231 cells treated with control PMOs or RAB13 PMOs (191+230). Cells reaching the bottom surface after 4hrs were counted. n=25 fields of view in each of 6 independent experiments. Bars: mean ± s.e.m.. B. Cells expressing Cherry-NLS, and treated with the indicated PMOs, were tracked every 5min for 10hrs to derive average migration speed. n=65 cells. Bars: mean ± s.e.m.. C. PMO-treated cells were induced to invade through a Matrigel plug. Cell staining intensity in arbitrary units (a.u.) was used to quantify relative invasion from n=4 independent experiments. Bars: mean ± s.e.m.. D. Lifeact-GFP-expressing cells were treated with the indicated PMOs and imaged every minute over 1 hr. Sequential image frames, from a control cell, highlight edge retraction (red arrowheads) or protrusion (yellow arrowheads). Corresponding edge velocity is shown, with negative values indicating retraction and positive values indicating extension. Average protrusion and retraction speeds were calculated from n=11-13 cells. Bars: mean ± s.e.m..See also movies EV2 and EV3. E. Cells treated with the indicated PMOs or siRNAs were analyzed by western blot (upper panels) to detect RAB13 and GAPDH protein levels. Migration speed was assessed as in (B) from n=55-78 cells (bottom graph). Bars: mean ± s.e.m.. F. Protrusion and retraction speed of cells treated with the indicated PMOs or siRNAs was assessed as in (D). Graphs show average normalized values from n=10-13 cells imaged for 1 hr. Bars: mean ± s.e.m.. G. Migration speed of RAB13 knockdown cells re-expressing GFP-RAB13 or GFP-RAB13 with a frameshift (fs) mutation at the beginning of the RAB13 coding region. 28-52 cells were analyzed. Bars: mean ± s.e.m.. Similar results were obtained in two additional independent experiments. Re-expressing cells were identified through GFP fluorescence and cells with similar, low GFP signal were tracked. All cells are expressing cherry-NLS for accurate tracking.
a) A COS-7 cell expressing EBFP2-Ub (red), AcGFP-sec61β (green) and mcherry-parkin (cyan) was treated with 10 μM CCCP for 1 h and imaged.f4(a) A COS-7 cell expressing EBFP2-Ub (red), AcGFP-sec61β (green) and mcherry-parkin (cyan) was treated with 10 μM CCCP for 1 h and imaged. Impaired mitochondria (parkin) displayed preferentially ubiquitinated foci (white arrows) at selected regions that overlap with the ER (sec61β). (b) HeLa cells expressing KR-dMito (red), EGFP-LC3 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted square regions, generating parkin-labelled mitochondria (bottom right, magnified view of the light-activated regions). (c) Magnified view of the photo-inactivated region in b 80 min after illumination (post-stained with anti-ubiquitin antibody). LC3 and ubiquitin colocalized at discrete sites along parkin-labelled mitochondria (white arrows). (d) A MG-132 treated HeLa cell expressing KR-dMito, EBFP2-parkin (green) and EGFP-LC3B (red) was 559 nm illuminated, leading to the appearance of parkin-labelled mitochondria (white arrows one to three indicated structures, top panels). The parkin-labelled structures did not fragment nor became engulfed by LC3 structures (bottom panels) in the presence MG-132. Scale bars (all panels), 5 μm.
Figure 4Fis1 knockdown abolishes DFP-induced LC3-II. Fis1 siRNA was introduced into HeLa cells for 48 h, and the cells were treated with DFP for further 24 h (Nsi, non-specific siRNA; Fis1i, Fis1 siRNA; siRNA concentration, 20nM; DFP, 1mM). Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=3, biological replicates; **, p<0.01; paired t-test). Fis1 knockout abolishes DFP-induced LC3-II. Fis1+/+ or Fis1-/- cells were treated with DFP for 24 h. Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=6, biological replicates; *, p<0.05; paired t-test). Expressing exogenous Fis1 restores LC3-II induction by DFP in Fis1 KO cells. WT or Fis1 KO HeLa cells transfected with pcDNA3 or Flag-Fis1 for 48 h were treated with DFP for a further 24 h. Whole cell lysate samples were blotted as indicated. Fis1 knockdown prevents DFP-induced mitophagic autolysosomes. Nsi or Fis1i together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), which were analysed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Blue: Fis1; Scale bar, 10 μm; n=25 cells per condition from three individual experiments; *** p<0.001; unpaired-t test).
Figure 2A. Average ribosome occupancy and their respective codon bias-derived occupancy scores across the CDS of transcript (in 25 bins). Ribosome occupancy for 16,423 transcripts and their respective codon bias-derived occupancy were firstly binned into 25 bins and the mean occupancy was calculated for each bin. B. Cumulative distribution plots showing the distributions of correlations between ribosome occupancy and codon bias-derived occupancy. Correlations obtained from the ribosome occupancy and scrambled codon bias-derived occupancy served as the control. A Kolmogorov-Smirnov test was performed between the codon bias-derived occupancy and the control group. Data information: error bars represent the 95% confidence intervals. C. Comparison of average transcript translation efficiencies (TEs) across their respective GC3-content ranges. Number of transcripts within each gene optimality range is indicated above their respective points. Data information: error bars represent the 95% confidence intervals. D, E. Principal component analysis of CDS codon frequencies of protein-coding genes derived from a +1 frameshift (D) and a -1 frameshift (E). Shaded ellipses indicate codons which are GC-rich (orange) and AT-rich (blue).
Figure 4A Model prediction and experimental validation for pAKT, ppERK and pS6 dynamics of 32D-EpoR cells in response to 50 U/ml Epo. Simulations are based on measured, cell-type-specific protein abundance of 32D-EpoR cells and global kinetic rates estimated from mCFUE and BaF3-EpoR cells. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model. B Sensitivity analysis for integrated pS6 in mCFU-E, 32D-EpoR and BaF3-EpoR cells. Measured, cell-type-specific protein abundance and estimated global kinetic rates were taken into account. Proteins were grouped according to the network modules. Integrated pS6 exhibited high sensitivity towards RSK in 32D-EpoR and BaF3-EpoR cells but not in mCFU-E cells. C Model prediction and experimental validation for RSK, AKT, ERK and S6 activation upon RSK overexpression (oe) and 5 U/ml Epo stimulation in 32DEpoR cells. Simulations are based on measured, cell-type-specific protein abundance of 32D-EpoR cells and global kinetic rates estimated from mCFUE and BaF3-EpoR cells. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model. Integrated pS6 was significantly higher upon RSK overexpression as compared to 32D-EpoR wild-type (wt) cells, N=4 (lower right panel). Two-Sample t-Test, *** p<0.005. D Impact of AKT, Ras or PTEN overexpression on BaF3-induced pS6 dynamics in EpoR and BaF3-PTEN cells. Quantitative AKT upon overexpression of PTEN, Ras, a constitutive active BaF3 protein, or the empty vector control in EpoR cells or BaF3-mCFU-E cells. BaF3-factor deprived mCFU-E cells (5x106 cells per condition) and Epo-immunoblotting cells (1x107 cells per condition) were stimulated with 5 U/ml Epo for indicated time points. Cellular lysates were analyzed by S6 employing sequential reprobing anti-pAKT, anti-ppERK, anti-pS6, anti-Epo and to ensure equal loading with anti-beta-actin antibodies. Detection was performed with chemiluminescence using a CCD camera device (ImageQuant). Quantification of pS6 on the right is depicted as fold change to wild-type samples at 30 minutes after Epo stimulation. Error bars represent standard deviation. oe: overexpression. N=3. Welch Modified Two-Sample t-Test, n.s. = not significant, * p < 0.05