EMBO/sd-character-level-ner huggingface dataset:
Binding of m152 to both ER luminal loop regions of murine STING is a prerequisite for its antagonistic activity (A) 293T cells were co-transfected with expression plasmids for IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and indicated expression plasmids together with murine Cherry-STING (left panel) or human STING (right panel).Figure 4(A) 293T cells were co-transfected with expression plasmids for IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and indicated expression plasmids together with murine Cherry-STING (left panel) or human STING (right panel). Lysates were analyzed by dual-luciferase assay. Data is combined from two out of three independent experiments and shown as mean ± SD. (B) Schematic representation of the predicted m152 (red) and STING (grey) topology in the ER membrane. (1) and (2) specify the ER luminal loop regions of STING with the sequence alignments from murine and human STING shown below. In murine STING, N41 in loop (1) was mutated to E41 and loop (2) was exchanged with loop (2) of human STING (hL2). The resultant constructs were designated mSTING N41E, mSTING hL2 and mSTING N41E-hL2. In human STING, mutations were introduced vice versa, resulting in hSTING E41N, hSTING mL2 and hSTING E41N-mL2. (C) IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and either LacZ or m152 together with the indicated murine STING mutants described in (B) were transiently expressed in 293T cells. Luciferase activity was measured as described in (A). Data information: Data in (C) is normalized to LacZ, combined from three independent experiments and shown as mean ± SD. (D) 293T cells were co-transfected with expression plasmids for either LacZ or m152 together with the indicated murine STING mutants described in (B). An anti-V5 IP was performed and samples were analyzed by IB with the indicated antibodies. IB shown in (D) representative of three independent experiments. (E-F) Luciferase assay (E) were performed as described in (C) and (D), respectively, using the indicated human STING mutants in place of the murine STING mutants described in (B). Data information: Data in (E) is normalized to LacZ, combined from three independent experiments and shown as mean ± SD. (E-F) co-IP (F) were performed as described in (C) and (D), respectively, using the indicated human STING mutants in place of the murine STING mutants described in (B). Data information: IB shown in (F) are representative of three independent experiments.
Atg1/ULK1 localize to autophagosomes in yeast and mammals.Figure 4(A) Exponentially growing ypt7Δ cells were starved in SD‐N medium for 4 h, lysed and the extract separated into a cytoplasmic (S) and a 5000 g membrane pellet fraction. The pellet was treated with (+) or without (−) TX‐100, centrifuged and the supernatant (S) and pellet (P) fractions analysed by western blotting with anti‐Atg1, anti‐Pep12 and anti‐Pgk1 antibodies. (B) Exponentially growing ypt7Δatg1Δ cells expressing wild‐type Atg1 or the Atg1‐VE mutant protein were starved in SD‐N for 4 h, lysed and the extract separated into cytoplasmic (S) and membrane (P) fractions. The fractions were analysed by western blotting with anti‐Atg1, anti‐Pgk1 and anti‐Ape1 antibodies, and the ratio of Atg1 to Ape1 in the pellet fractions was quantified and indicated as a ratio compared to wild‐type cells. (C) HEK293 cells were co‐transfected with GFP‐GABARAP and HA‐ULK1, starved for 2 h, and subsequently immunostained for HA (red) and WIPI2 (white). The arrows mark putative autophagosomes that stain positive for all three markers. Bar=10 μm. (D) HEK293 cells stably expressing GFP‐LC3 were transfected with wild‐type HA‐ULK1 or the DFP mutant, and immunostained for HA and WIPI2 after 2 h starvation. HA‐ULK1 and WIPI2 spot numbers were counted using Imaris software in over 70 cells (from one out of two experiments) and plotted as the ratio of ULK1 to WIPI2 puncta. ***Indicates a statistically significant P‐value of 0.0001. One of the two independent experiments is shown. (E) Quantification of HA‐ULK1 and WIPI2 spot numbers in HEK293 cells expressing wild‐type HA‐ULK1 or the DFP mutant. Cells were treated as above, and the spot number counted using Imaris software. A representative immunofluorescence image is shown in Supplementary Figure S3F. *** and * indicate P‐values of 0.0001 or 0.0158, respectively. Figure source data can be found with the Supplementary data.
Figure 1Occupancies of Pol II at the myogenin locus in C2C12 myoblasts (MB, shown in blue) and myotubes (MT, shown in red). Myoparr is located immediately upstream from the myogenin gene. Schematic representation of the upstream region of myogenin and amplified regions by RT-PCR (top). RT-PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes (bottom). The presence or absence of reverse transcriptase (RT) is shown by (+) or (-), respectively. The templates (cDNA or genomic DNA) are indicated by C or G, respectively. The primers used for RT-PCR (top). Strand-specific RT-PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes using total RNA (middle and bottom) and poly(A)+ RNA (bottom). Relative expression of indicated RNAs in differentiating C2C12 cells. The presence or absence of RT is shown by (+) or (-), respectively. PCR products were verified by sequencing. n = 3, mean ± SD. The nuclear/cytoplasmic fractionation is confirmed by the expression of H2B and tubulin, respectively. Chromatin pellet extract (CPE)/soluble nuclear extract (SNE) ratio of SRA1, Malat1, and Myoparr in C2C12 myotubes on a log scale. n = 3, mean ± SD.
Intestinal lymphatic vessel regression leads to impaired lipid absorption and resistance to diet-induced obesity.Figure 3Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Body weight change during seven weeks of HFD, expressed as average fold change in comparison with the starting weight. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.Body weight comparisons at seven weeks of HFD. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT. *P = 0.004; **P = 0.003. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26. Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Glucose tolerance test (GTT) after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.014; **P = 0.041. n = 5, WT; n = 6, VCiΔR26. Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Total fatweight, fat percentage from body composition measurements after six weeks of HFD, and weights of visceral fat (VF) and subcutaneous fat (SF) at the time of necropsy. Significant differences were determined using unpaired two-tailed t-test. *P = 0.006; **P = 0.001; #P = 0.008; §P = 0.006. n = 4 in each group. Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Food consumption during the fifth week of HFD. n = 9, WT; n = 10, VCiΔR26. Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Whole-mount immunofluorescence staining of blood (PECAM1, red) and lymphatic vessels (LYVE1, green) in intestinal villi and intestinal wall.Quantification of the lacteal and villus length (solid and striped color bars, respectively) and the intestinal wallLYVE1+ area percentage from images represented in (F). Significant differences were determined using unpaired two-tailed t-test. *P = 0.0002; **P = 0.00007. n = 5, WT; n = 6, VCiΔR26. Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Free fatty acid (FFA) and cholesterol measurements from the feces after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.001; **P = 0.007. n = 5, WT; n = 6, VCiΔR26.
Figure 4A. Schematic of the question addressed for the computational simulation. B. Population frequency of the phase separation-prone variant over time (in number of generations with respect to wild-type strain at 30ºC, 2.7 hours) in different non-oscillating conditions with a positive measured selection coefficient (S) for protein phase separation. A dashed line indicates the point where the mutant is present in 50% of the population. The corresponding value in the x-axis for each profile denotes the time required to reach this point. C. Population frequency of the phase separation-prone variant over time in different environmental conditions with positive and negative S. The dashed line indicates the initial fraction of mutant cells in the population and the colored lines indicated the fraction of mutant cells in the population at different time points (generations of WT is used as the unit of reference). D. Diagram showing how the frequency of the mutant cells can change in fluctuating environments (environment, E and time in environment, t). The sequence of environments (history) experienced by a mutant determines the overall selection coefficient (Sh). E. Population frequency of the variant over time in two different alternating environments. Black line, profile of population frequency of the variant, where, at the moment in which the first mutant cell appears, the environment positively selects it; however, in the subsequent environment the selection is negative. Grey line, profile of population frequency of the variant, where, at the moment in which the first mutant cell appears, the environment negatively selects it; however, in the subsequent environment the selection is positive. The area of the plot is divided in grey (E1) and white (E2) sections to indicate the fluctuating environment with positive and negative S. F. History of environments resulting from any possible combination of two environments (e.g. "environment 1" 25ºC and "environment 2" 0.5M Proline), from those experimentally tested in this work
Isolation of zebrafish thyroid gland (A , B) A brightfield image showing the zebrafish thyroid gland along with surrounding tissue.Figure 1(A , B) A brightfield image showing the zebrafish thyroid gland along with surrounding tissue. The thyroid follicles reside in the soft tissue surrounding the ventral aorta, which extends from the outflow tract of the heart into the gills towards the basibranchial cartilage in the lower jaw. The thyroid follicular cells, or thyrocytes, are labeled in green in the Tg(tg:nls-mVenus-NTR) transgenic line (B'). Scale bars: 500 µm (C) Maximum intensity projection of 3D confocal stack obtained from the dissected thyroid gland of Tg(tg:nls-EGFP) animal and labeled with DAPI. (D) Confocal scan of a transverse section across the dissected thyroid gland from Tg(tg:nls-EGFP) animal at 3 mpf. Sections were stained with DAPI to visualize cells surrounding thyroid follicles. (E) Boxplot depicting the proportion of thyrocytes present in transverse sections obtained from three Tg(tg:nls-EGFP) animals at 3 mpf. Each dot represents a transverse section. The Tukey boxplot marks the 25th percentile, median and 75th percentile with whiskers extending from smallest to largest values. (F , G) Representative FACS plot of single cells from Tg(tg:nls-mVenus-T2A-NTR) animals at 2 mpf (F) and 8 mpf (G). Calcein (Pacific Blue) labels live cells, while green fluorescence (FITC) labels thyrocytes. Percentage values represent proportion of calcein+ thyrocytes within total calcein+ cells.
Cool temperatures enhance dimerization/oligomerization of GC‐G but not GC‐AHEK‐293T cells were co‐transfected with plasmids encoding two differential epitope‐tagged (FLAG or Myc) versions of GC‐G or GC‐A, respectively.Figure 4A HEK‐293T cells were co‐transfected with plasmids encoding two differential epitope‐tagged (FLAG or Myc) versions of GC‐G or GC‐A, respectively. After 2 days, transfected cells were lysed with detergent and lysates were subjected to immunoprecipitation (IP) for 2 h with anti‐Myc antibody at two different temperatures (15 or 25°C), followed by Western blotting (WB) using anti‐FLAG antibody to determine the amount of dimerized/oligomerized GC proteins (upper panel). These approaches demonstrated that in contrast to GC‐A, homomeric protein/protein association of GC‐G seems to be enhanced by cool temperatures (upper panel). Cell lysates were also precipitated and immunoblotted with the anti‐Myc antibody, demonstrating that cooling does not interfere with immunoprecipitation of Myc‐tagged GC‐G via the anti‐Myc antibody (middle panel). Likewise, precipitation and immunoblotting with the anti‐FLAG antibody yielded bands of similar intensity for 15 and 25°C, respectively (lower panel).B Relative quantification of homomeric protein/protein association of GC‐G and GC‐A was performed by densitometric scanning. Each intensity value from the upper panel was subsequently normalized to the total protein levels as determined in the middle and the lower panel of Fig A (intensity of the immunoreactive bands obtained with the anti‐Myc or anti‐FLAG antibody, respectively). Relative dimerization level at 15°C was further calculated by normalizing to values obtained at 25°C; the latter were set as 1. Results are means ± SD of three experiments, **P 0.01 (compared to 25°C).
Size characterization of schistosomal-EVs.Figure 2EVs were extracted from the schistosomal-growing medium and were subjected to cryo-TEM analysis: images were taken as described previously [90]. The black arrows indicate EVs at the size of ~100nm. EVs were extracted from the schistosomal-growing medium and were subjected AFM analysis: images were taken from the schistosomal-growing medium (I) or from 1/10 dilution of this medium (II). The small images magnify the arrow-indicated EVs. (III) Image of the extract deriving from the unused schistosomal-medium. EVs were extracted from the schistosomal-growing medium and were subjected NTA size fractionation of particles analysis; the graph represents average of 5 different measurements.
Fig.Figure 6A. Wild type and GAL-Mia40 cells, and cells expressing a cytosolic version of Mia40 under GAL1 control were dropped on the indicated plates; ev, empty vector. Note, that the mitochondrial version of Mia40 suppressed polyQ toxicity, whereas the cytosolic version did not. B. In wild type or GAL-Mia40 cells, Q25-GFP or Q97-GFP was expressed for 4.5 h. For control, wild type cells were used which expressed also for 4.5 h the 'clogger' protein cytochrome b2-DHFR, a strong competitive inhibitor of protein import. Samples were analyzed by Western blotting. The presence of precursor proteins (labeled as p) in addition to the mature forms (labeled as m) is indicative of a strong import defect. C. ∆ura3 cells expressing the Oxa1-Ura3 reporter for the cytosolic accumulation of mitochondrial precursor proteins (Hansen et al., 2018) were transformed with plasmids expressing the proteins TDP-43, the TDP-43Q331K mutant, Q25-GFP, Q97-GFP, α-synuclein, FUS or GFP from a constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. Note that the expression of all aggregation-prone proteins (indicated by red arrows) allows cells to grow on uracil-deficient plates indicating the cytosolic accumulation of the Oxa1-Ura3 fusion protein. Wild type cells were transformed with plasmids for the expression of the indicated proteins. Q97-GFPnF is a non-fluorescent version of Q97-GFP. Close proximity of the two split GFP parts results in fluorescence which was quantified in a spectrometer E. Wild type cells were transformed with plasmids for the expression of the indicated proteins. Q97-GFPnF is a non-fluorescent version of Q97-GFP. Close proximity of the two split GFP parts results in fluorescence which was visualized by microscopy. Shown are mean values and standard deviations of three replicates. Bar, 5 µm.
a) Representative images of a J774 macrophage expressing GFP–LC3 incubated in media containing red dextran.figf5(a) Representative images of a J774 macrophage expressing GFP-LC3 incubated in media containing red dextran. The arrow marks LC3 recruitment to a dextran-containing macropinosome. Scale bar, 2 μm. (b) Quantification of LC3 recruitment to macropinosomes in control and Atg5 shRNA J774 macrophages; n, number of macropinosomes analysed; ***P0.0001, chi-squared test. See Supplementary Movie S9. (c) Representative image of LC3 recruitment to macropinosomes (arrows) in MCF10A cells expressing GFP-LC3 incubated with red dextran. Scale bar, 2 μm. (d) Quantification of LC3 recruitment to macropinosomes in MCF10A cells treated with siRNA against FIP200 or ATG5; n, number of macropinosomes analysed; ***P0.0001, chi-squared test. (e) J774 GFP-LC3 macrophages were incubated with 3 μm uncoated latex beads and imaged by confocal microscopy for 1 h, followed by the addition of LysoTracker red to mark acidified compartments. A macropinosome recruited LC3 (arrow) whereas engulfed latex beads did not (arrowheads). Scale bar, 5 μm. See Supplementary Movie S10.
α-hydroxyVLCFA-GM3 species in adipose tissue showed increased abundance in obesity.Figure 7(A) GM3 molecular species of 6-wk-old control C57/BL6 mice and ob/ob mice were analyzed respectively by LC-MS/MS (n=3). (B) GM3 molecular species of normal-diet (ND) and high-fat-diet (HFD) C57/BL6 mice were analyzed by LC-MS/MS (n=4). (C) TLC analysis of acidic GSL fraction (equivalent to 0.1 mg protein) from epididymal fat pads of C3H/HeN (A) and C3H/HeJ mice (B) on ND or HFD for 8 wk. (D) GM3 molecular species of C3H/HeN mice (ND, HFD) and C3H/HeJ mice (ND, HFD) were analyzed by LC-MS/MS (n=4). Comparison of increased GM3 species in human serum and mouse adipose tissue. Feedback loop mediated by TLR4 and GM3 species, promoting disease progression (schematic).
Figure 8(A-B) Representative confocal images and quantification of IBA-1 in the Cortex, Hippocampus, and Cerebellum brain section derived from WT or CLN7Δex2 mice injected with the vehicle or Tamoxifen. (***/**/* vs WT, °°°/°°/° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, */o: P ≤ 0.01, **/oo: P ≤ 0.001, ***: P ≤ 0.0001, as determined by ANOVA (N≥3 biological replicas). Scale bars: 50 µm. (C) Plots show the quantification latency to fall from the rotarod. Data are presented as mean ± SD, *: P ≤ 0.01, as determined by ANOVA (N≥3 biological replicas). (D) Representative Images of hindlimb clasping test in mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen.
Figure 6A) Left: comparison of the number of transposon insertions per gene (dots) for libraries with PE synthesis targeted to the MIM and PC synthesis targeted to peroxisomes, in KennedyON and OFF conditions. CSF1 and VPS13 (highlighted in red) are more required in KennedyOFF conditions. Right: Transposon insertion profile displayed in the UCSC genome browser for the indicated libraries for the genomic region of CSF1. Bars correspond to transposons, rows to libraries. The boundaries of the full-length Csf1 protein, the truncated Csf1ΔC protein, the Chorein-N motif and the transmembrane (TM) domain are indicated. B) Five-fold serial dilutions of strains of the indicated genotypes grown at 30°C on SD medium supplemented with 10 mM ethanolamine and/or 10 mM choline. Growth assays are representative images of biological replicates. C) PS and PE transfer assay. Rewired strains were pulse labeled with 15N-serine. Lipids were extracted at the indicated time-points and measured by mass spectrometry. Graphs depict quantification of the appearance of labeled species of the indicated phospholipids normalized by the amount of its precursor lipid.
Spinal cord and intestine show sexual dimorphism in Ub-K48 inked proteins and proteasome assembly.Figure 2A-D Western blot analysis for K48Ub-linked proteins in spinal cord (A) and intestine (C) lysates from male and female mice. (B,D) Quantification of panels A and C, respectively. Values were normalized to actin as a loading control. Each central line and error bar indicate mean +/- SEM of a biological replicates. Data information: * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples. E) Curve of correlation between chymotrypsin-like activity and Ub-K48 linked proteins in the intestine, where variability between mice is observed. F) Native gel followed by western of β5 subunit to distinguish unassembled catalytic core (CP) or assembled with regulatory core (RP) in male (n=5) and females (n=5) mice in intestine. G) Native gel followed by western blot of β5 subunit to distinguish unassembled catalytic core (CP) or assembled with regulatory core (RP) in male (n=5) and female (n=5) spinal cord. H) Quantification of the ratio of assembled (RP-CP) versus unassembled (CP) proteasome in the intestine of males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 per group). I) Quantification of the ratio of RP-CP versus CP proteasome in the spinal cord of males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 per group). Data information: * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples. J) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) intestine. K) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) spinal cord.
Fig.Figure 8A and B, Efficiency of AAV9-mediated gene delivery. AA9 viruses expressing the reporter GFP gene were stereotaxically injected into the SN of mice, and GFP expression in astrocytes, microglia, and neuronal cells was quantified. Data are represented as mean ± SEM. n=3 mice (>5 randomly selected microscopic fields per animal were counted). Significantly different at p=0.006*, 5.1E-09#, 5.36E-09+ in graph B. (C) Schematic of the experimental procedure. SGK1 knockdown effect in vivo by treatment with shSgk1-AAV9. Total SGK1Cell type-specific SGK1 knockdown was estimated by counting SGK1-immuonoreactivity in neuron (MAP2+), astrocyte (GFAP+), and microglia (Iba1+) populations (E). Data are represented as mean ± SEM. n=4 mice in each group. Significantly different at p=0.0004# in graph D and p=0.049*, 0.007**, 0.002*** in graph E. (F-H) Behavior of the PD mice assessed by pole (F), beam (G) at 6-12 weeks, and locomotor activity (H) tests at 12 weeks post- SNCA injection. Significantly different at p=0.0003#, 0.0428*, 7.46E-06##, 0.0012** in graph F and p=8.04E-08#, 0.004*, 1.62E-09##, 3.3E-06** in graph G and p=0.0002#, 0.0194* (total distance), 9.54E-06#, 0.0047* (average speed) in graph H. Histologic assessment of p129-SNCA immunoreactivity ( I,J), TH+ mDA neuron counts (I,K), neurite lengths (L), and cell body sizes (M) in the SN Significantly different at p=0.0001* in graph J, p=2.35E-25#, 1.09E-16##, 4.36E-08* in graph K and p=1.5E-40#, 1.08E-24##, 2.32E-07* in graph L and p=9.07E-20#, 2.59E-16##, 0.05* in graph M and p=4.62E-22#, 5.11E-12##, 3.49E-06* (TH), n= 7 sh-SGK1-AAV9-treated PD mice, 7 sh-cont-AAV9-treated PD mice, and 8 SNCA-untreated mice. Data are represented as mean ± SEM. One-way ANOVA. Scale bar, 100µm. Histologic assessment of TH+ and DA transporter (DAT) fiber intensities in the striatum n= 7 sh-SGK1-AAV9-treated PD mice, 7 sh-cont-AAV9-treated PD mice, and 8 SNCA-untreated mice. Data are represented as mean ± SEM. One-way ANOVA. Scale bar, 100µm.
Membrane tethering is Atg16 dependent.Figure 6(A) Widefield images of rhodamine‐PE labelled GUVs 30 min GUVs cluster only in the presence of the Atg5-Atg12/Atg16 and Atg5/Atg16 complexes. (B) SUV aggregation assay using Folch liposomes and the indicated proteins. The proteins were added after a short delay. The symbols above, below the images or next to the curves represent Atg5 (blue oval), Atg12 (red circle) and Atg16 (green Y). The overall shape of the Atg5-Atg12/Atg16 complex is according to the structural model of the complex as proposed in Fujioka et al (2010). Scale bars: 10 μm.