EMBO/sd-character-level-ner huggingface dataset:
Figure 7(A) Immunoblot of cytoplasmic phospho-SRSF1/2 after SRPIN340 treatment. GAPDH is used as the loading control. Error bars represent mean +/- SD (n=3 biological replicates). Statistical analysis was performed using Student's t test with Welch's correction. ∗p < 0.05 (B) ICC images of FLAG-tagged SRSF1 after SRPIN340 treatment compared to vehicle (DMSO). Quantification shows the ratio of nuclear and cytoplasmic intensity of SRSF1 signal Error bars indicate mean +/- 95% CI (n= 85-126 cells/condition). Statistical analysis was performed using t test with Welch's correction, ∗∗∗∗p < 0.0001. (C) Nucleocytoplasmic distribution CGG repeat RNA after SRPIN340 treatment compared to vehicle (DMSO) as detected by HCR. Quantification shows the ratio of nuclear and cytoplasmic intensity of CGG RNA signal as parts of whole. Error bars indicate mean +/- 95% CI (n= 124-151 cells/condition). Statistical analysis was performed using t test with Welch's correction, ∗∗∗∗p < 0.0001. (D) Anti-FLAG immunoblot blot of DMSO and SRPIN340 pre-treated HEK293T cells transfected with in vitro transcribed CGG-nLuc-3xFLAG reporter RNA. β-Actin is used as a loading control. Error bars represent mean +/- SD (n=6 biological replicates). Statistical analysis was performed using Student's t test with Welch's correction. ∗p < 0.05 (E) Relative expression of in vitro transcribed CGG-nLuc reporter in HEK293T cells pre-treated with DMSO and SRPIN340. Error bars represent mean +/- SD (n=9 biological replicates). Data information: Statistical analysis was performed using Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01 (F) Expression of in vitro transcribed Aug-nLuc and CGG-nLuc reporters in rabbit reticulocyte lysate (RRL) in vitro translation system following pre-treatment with DMSO or SRPIN340. Error bars represent mean +/- SD (n=9 biological replicates). Data information: Statistical analysis was performed using Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01
Pointed end cargo adaptor interaction sites A.Figure 6Site 1 in the disordered loop of p62 (orange, in clear density), binds to each cargo adaptor (BICDR1 in yellow, Hook3 in purple, BICD2 in green). Density can be seen between p62 and the adaptor at low threshold. BICDR1- and Hook3-containing maps are filtered to 6 Å. Residues on dynactin's pointed end (cartoon) that interact with cargo adaptors are shown as spheres, colored according to which adaptor they bind (yellow denotes binding to BICDR1, purple to Hook3 and green to BICD2). Binding sites 2-4 are shown in boxes. Conservation of the surface residues plotted onto the density from the pointed end map, filtered to 6 Å. Sites 2-4 for cargo adaptor interaction are marked on the front face and end face. Conservation scores are from ConSurf, with lower conservation shown in green, and higher conservation in purple. Pulldowns of pointed end site 1-4 mutants using Strep-Hook3 (D) or Strep-BICD2 (E). Binding is shown relative to the binding of the wildtype pointed end construct (dashed lines). * shows p<0.05. For BICD2, p=0.06 for Site 1 mutant, and p=0.1 for Site 4 mutant. Data presented as mean ±SEM, n=3. Statistical significance calculated using ANOVA with Tukey's test for multiple comparisons.
Figure 7Splenocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-IFNγ and anti-IL17 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (left panel). The percentages of IFNγ- and IL17-secreting CD4+ T cells were compared among Ifih1-/- (n = 4), Cd4cre A1fl/fl Ifih1-/- (n = 5) and Cd4cre A1fl/fl mice (n = 6) (right panel). The same data shown in Fig 4E was used for Cd4cre A1fl/fl mice. No incidence of spontaneous colitis observed in Ifih1-/- (n = 5) and Cd4cre A1fl/fl Ifih1-/- (n = 11) mice. For side-by-side comparison with Cd4cre A1fl/fl mice Representative images of HE staining of colon dissected from an Ifih1-/- and a Cd4cre A1fl/fl Ifih1-/- mouse. Scale bar, 200 μm. Histological scores for the colitis in Ifih1-/- (n = 3) and Cd4cre A1fl/fl Ifih1-/- (n = 4) mice. For side-by-side comparison with Cd4cre A1fl/fl mice,
Patient-derived xenografts display similar resistance mechanisms as the parental metastases.Figure 7Staining for hematoxylin-eosin (H&amp;amp;E) and p-ERK on the PDX samples M032.X1, M032R1.X1, M032R2.X1, M032R4.X1, and M032R5.X1. Stainings showed that p-ERK is higher in the PDX derived from the resistant metastases. Scale bar represents 100 μm. Immunoblotting for components of the MAPK pathway confirmed reactivation of p-ERK in the PDX derived from the vemurafenib-resistant metastases, although the p-ERK signal is heterogeneous in the pre-treatment PDX. Validation of the BRAF amplification was performed by qPCR on gDNA derived from the PDX, using primers for BRAF and CRAF and normalized on LINE expression. Bars represent the mean of three replicates, error bars indicate standard deviation. The results confirmed that BRAF was amplified in PDX derived from M032R2 and M032R5, but not from M032R1. Presence of the MEK1T55delinsRT was analyzed by PCR using insertion-specific primers. As a control, primers amplifying exon 2 of MEK1WT were used. The insertion was found in M032R1.X1 and M032R4.X1.
a) Smo siRNA-treated cells were transfected with HA-HttQ74 for the last 24 h and the percentage of transfected cells with aggregates scored in HA-positive cells by immunofluorescence.f3(a) Smo siRNA-treated cells were transfected with HA-HttQ74 for the last 24 h and the percentage of transfected cells with aggregates scored in HA-positive cells by immunofluorescence. Graphs show mean values and error bars represent s.d. of a representative experiment performed in triplicate. P-values were calculated by odds ratio. (b) siRNA-targeting Smo was transfected into HeLa cells stably expressing mRFP-GFP-LC3. Representative confocal microscopy images are shown. Bars represent 26 μm. The number of vesicles was analyzed in a Cellomics array scan and percentage was calculated relative to control siRNA-transfected cells. Statistical tests were performed on data from three independent experiments in triplicate. *P0.05; ***P0.001.
Figure 6(A) Schematic representation of the Renilla luciferase (Rluc) reporter mRNAs containing let-7-binding sites (RL-6xB) or mutated seed sites (RL-6xBMUT). (B) PABPDHFR cells were transfected with plasmids encoding RL-6xB or RL-6xBMUT. 24 hours after transfections, the cells were split and cultured for an additional 12 hours in the presence or absence of TMP. The stabilities of reporter mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. First order exponential decay trend lines were generated by non-linear regression analysis.
A limits spurious long-distant contacts in ground-state ESCs A.Figure 6A. Hi-C contact matrices for chromosome 1 of ESC+serum, ESC+2i/siRNA-control and ESC+2i/siRNA-Baz2a showing the presence of strong long-distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC+serum. Cells were subjected to two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Eigenvector values are shown. Increase in far-cis contacts in ESC+2i upon BAZ2A-KD are highlighted with a rectangle. B. Relative contact frequencies over genomic distances in ESCs treated with siRNA-control and siRNA-BAZ2A. Statistical significance (P-values) was calculated using the Kolmogorov-Smirnov test. C. Boxplot indicating differences in eigenvector values at boundaries and centers of A and B compartments. Differences in eigenvector values were calculated from HiC data sets from ESC+2i treated with siRNA-Baz2a and siRNA-control. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (*** < 0.001, ns: non significant). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.
IPSCs generated from patient fibroblasts carrying homozygous and heterozygous POLG mutations.Figure 1A: Morphology on phase contrast microscopy for parental fibroblast lines (upper panel) and iPSCs (lower panel) from control Detroit 551, WS5A and CP2A POLG patients (scale bars, 50 µm). B: Immunofluorescence staining of stem cell markers POU5F1 (green) and SSEA4 (red): upper panel - Detroit 551 control iPSCs, middle panel - WS5A iPSCs and lower panel - CP2A iPSCs (Scale bar, 100 µm). Nuclei are stained with DAPI (blue). C: RT-qPCR quantification of gene expression for LIN28A, NANOG, and POU5F1 for all iPSCs from control Detroit 551, WS5A and CP2A POLG patients (n=7, technical replicates per line for ESCs; n=4, technical replicates per clone for control, WS5A and CP2A iPSCs). The gene expression of the individual clones is assessed with fold change using the comparative ΔΔCt method by normalizing iPSCs to ESC1. D, E: Flow cytometric quantification of expression level of SSEA4 (D, n=9, technical replicates per line for ESCs; n=5, technical replicates per clone for control iPSCs; n=3, technical replicates per clone for WS5A iPSCs, n=8, technical replicates per clone for CP2A iPSCs) and POU5F1 (E, n=9, technical replicates per line for ESCs; n=5, technical replicates per clone for control iPSCs; n=3, technical replicates per clone for WS5A iPSCs, n=8, technical replicates per clone for CP2A iPSCs) for ESCs and iPSCs for both ESC control lines and iPSCs generated from Detroit 551 control, WS5A and CP2A fibroblasts.
Figure 1A. HeLa cells released from double thymidine synchronization in early S phase were treated with 5-azadC for 30 min, washed and collected at the indicated times. Soluble and chromatin-enriched fractions were immunoblotted with indicated antibodies. B. U2OS cells treated as in (A) were pre-extracted and immunostained with DNMT1 antibody. DNMT1 foci formation was analyzed by quantitative image-based cytometry (QIBC) (red bars, mean; >1500 cells analyzed per condition). Data are representative of three independent experiments. C. As in (B), except that cells were pre-treated with the proteasome inhibitor MG132 and/or SUMOi for 30 and 15 min, respectively, before exposure to 5-azadC (red bars, mean; >1900 cells analyzed per condition). Data are representative of three independent experiments. D. HeLa cells stably expressing GFP-DNMT1 were treated or not with 5-azadC for 30 min, collected and subjected to GFP immunoprecipitation under denaturing conditions, and immunoblotted with indicated antibodies. E. HeLa/GFP-DNMT1 cells transfected with indicated siRNAs were processed as in (D). F. Immunoblot analysis of soluble and chromatin-enriched fractions of HeLa cells transfected with indicated siRNAs, left untreated or exposed to 5-azadC for 30 min, and collected at the indicated times. G. U2OS cells transfected with indicated siRNAs were treated with 5-azadC for 30 min and processed for QIBC analysis of DNMT1 foci counts as in (B) (red bars, mean; >5600 cells analyzed per condition). Data are representative of three independent experiments. H. Representative images of U2OS cells transfected with indicated siRNAs followed by mCherry-RNF4 expression plasmid. Cells were treated with 5-azadC in the presence or absence of SUMOi, fixed 2 h later, pre-extracted and co-immunostained with DNMT1 and SUMO2/3 antibodies. Scale bar, 5 μm. I. Immunoblot analysis of soluble and chromatin-enriched fractions of HeLa cells treated with 5-azadC and/or SUMOi as indicated. J. GFP-tagged DNMT1 from extracts of HeLa/GFP-DNMT1 cells treated or not with 5-azadC was immobilized on GFP-Trap agarose, subjected to stringent washing to remove proteins non-covalently bound to GFP-DNMT1 and incubated with recombinant HA-ubiquitin, E1 and E2 (UbcH5a) enzymes and RNF4 proteins (STUbL reaction) at 37 °C for 1 h. Samples were then subjected to immunoblotting to assay for RNF4-dependent STUbL activity towards GFP-DNMT1.
Angiogenesis quenches HIF activity in the developing cortex.Figure 4A-F, Staining for Isolectin B4 (A-C) and oxidized thiols (D-F) in WT cortices at E10.5 (A,D), E11.5 (B,E) and E12.5 (C,F). Panels D-F are pseudocolor conversions of staining intensity (from low [purple] to high [red]). Arrowheads denote the position of the vascular front. G-I, Immunostaining for HIF-1α (red) and DAPI (blue) in WT cortices at E10.5 (G-G''), E11.5 (H-H'') and E12.5 (I-I''). Panels G',G'',H',H'',I' and I'' are magnifications of the boxed areas in their respective original panels. Arrows point to the border of lateral and dorsal cortex. J-L, Immunostaining for Glut1 of WT cortices at E10.0 (J), E11.5 (K) and E12.5 (L). Dashed lines indicate the apical border of the cortex. Scale bar: 100 µm (D,F,G,H,I, J-L) or 50 µm (I''), respectively.
Figure 5Forebrain slices from WT mice were treated with vehicle (water; lanes 1,5; numbers on bottom) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(A) β2AR was IPed before IB for α11.2 (top part of IB), GluA1 (middle part of IB), and β2AR (bottom part of IB), as indicated.(B,C) For quantification, α11.2 and GluA1 IB signals were normalized to β2AR (**p<0.01, ANOVA).ISO-induced displacement of the β2AR from α11.2 (A, lanes 2-6) lasted at least 3 min but not 20 min (compare lanes 6 and 7). Forebrain slices from WT mice were treated with vehicle (water; lanes 1,5; numbers on bottom) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(D) α11.2 and GluA1 were concurrently IPed from same samples as in (A) by simultaneous addition of anti-α11.2 and -GluA1 antibodies before probing and stripping/re-probing upper part of IB for pS1928, pS1700, and total α11.2 (top three panels) and middle part for pS845, pS831, and total GluA1 (bottom three panels).(E-H) For quantification, pS1928 and pS1700 IB signals were normalized to total α11.2 and pS845 and pS831 signals to total GluA1 (**p<0.01, ANOVA).ISO-induced displacement of the β2AR from α11.2 (see A) rendered α11.2 (but not GluA1) refractory to re-phosphorylation of S1928 and S1700 upon a second ISO application of α11.2 (D, compare lanes 5 and 6).
The activity of endogenous Atg4 is inhibited under nutrient starvation.Figure 4(A) CHO cells (for mammalian Atg8s) or cells of S. cerevisiae (for ScAtg8) were incubated in starvation medium (EBSS or SD‐N, respectively) for different time periods as indicated before isolation of RNA by the Tri‐reagent as explained in Materials and methods. For each gene, data obtained from non‐starved cells were set to an arbitrary value of 1, and results from the corresponding starved cells were normalized accordingly. Results represent the means±s.d. of three separate experiments. (B) HEK 293 cells were grown in control medium or starved for 2 h in EBSS in the absence or presence of 100 nM bafilomycin A1. Lysates (100 μg) obtained in Ripa buffer were run on 12% SDS-PAGE and analyzed by Western blot, using anti‐GATE‐16 or anti‐tubulin antibodies. (C) CHO cells were grown in a control medium in the presence or absence of 1 mM H2O2 for 1 h, or starved for 3 or 13 h. Lysates (10 μg) obtained in Ripa buffer were incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer (25 mM Tris, pH 7.4, 50 mM KCl) at 30°C for 45 min, in the presence or absence of 1 mM DTT. The reaction was stopped by addition of sample buffer and boiling, after which the samples were resolved on 15% SDS-PAGE and subsequently analyzed by Western blot, using anti‐His monoclonal antibodies. The experiment was repeated six times; a representative blot is shown. (D) Left panel: HeLa cells transfected with Myc‐GATE‐16‐HA or with an empty vector as control were labeled with [35S]methionine for 10 min and lysed immediately or chased for 1 h before lysis in Ripa buffer. Lysates were immunoprecipitated using anti‐Myc antibodies and the immunoprecipitates were resolved on 15% SDS-PAGE. Middle panel: HeLa cells transfected with Myc‐GATE‐16‐HA were kept in control medium or starved for 30 min or 13 h in EBSS before labeling with [35S]methionine for 10 min, immediate lysis, analysis by SDS-PAGE and quantification (right panel) using NIH image program. Values are presented as the average percentage of unprimed form out of the total GATE‐16. (*) in all sections of this figure indicates non‐cleaved His6‐GATE‐16‐HA or Myc‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16 or Myc‐GATE‐16.
The effect of folate deficiency on the tumorigenicity of oncogene-expressing cells in vitro and in vivo.Figure 5A, B Cyclin E- and Ras (H-RasV12)-expressing 3T3 cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium. Control cells were grown in a normal medium for the whole period. (A) Examples of anchorage-independent growth in soft agar of 3T3 cells. (B) Average number of colonies per soft agar plate of 3T3 cells. The number of colonies per plate is expressed as the average ± SEM from three independent experiments. C-E Ras (H-RasV12)-expressing MCF10A cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium. Control cells were grown in a normal medium for the whole period. (C) Examples of anchorage-independent growth in soft agar of MCF10A cells. (D) Average number of colonies per soft agar plate of MCF10A cells. The number of colonies per plate is expressed as the average ± SEM from three independent experiments. (E) Percentage of tumor-free flanks at the indicated time points after cell injection. Ten mice were injected in both sides in each group.
Figure 2Immunoblot of Vero cells infected with BTV-8 for 16 h, and probed for total and phosphorylated STAT1/2 levels. Vero cells infected with BTV-8 were fixed at 16hpi, and stained with anti-BTV-VP7 antibody. STAT1 phosphorylation in IFN-I-treated Vero cells infected with BTV-8 detected by (C) flow cytometry and (D) confocal microscopy (Scale bar = 30 µm). Means ± SD of P-STAT1 mean fluorescence intensity (MFI) in 3 experiments are shown. STAT2 expression in BTV-8-infected Vero cells detected by (E) flow cytometry and (F) confocal microscopy (Scale bar = 30 µm). Means ± SD of STAT2 MFI in 3 experiments are shown.
Purification and stability analysis of Mpc proteins.Figure 1Strategy for purification of the Mpc1/Mpc3 hetero-complex by nickel-affinity chromatography. Expression of Mpc proteins in mitochondria assessed by SDS-PAGE and immunoblot analysis of crude mitochondrial preparations. The individual untagged Mpc1 (Mpc1), histidine-tagged Mpc1 (Mpc1his), histidine-tagged Mpc3 (Mpc3his) or the Mpc1/Mpc3 hetero-complex (Mpc1/Mpc3his) were detected with antibodies raised against Mpc1 (left panel) or Mpc3 (right panel) and are shown with dashed arrows. Five micrograms of each affinity-purified Mpc protein were analysed by SDS-PAGE and the bands were visualised by Coomassie Blue stain. Peptide mass finger printing was used to identify the major protein bands The stability of the purified proteins was assessed via thermal denaturation by fluorescent CPM-adduct formation. The thermal denaturation profiles (left) were used to calculate the first derivative (right), which provides the apparent melting temperature, indicated with the same colour coding. The stability of the same samples, as in panel (D), was assessed by NanoDSF. The changes in the 330 nm/350 nm ratio with temperature (left) were used to calculate the first derivative (right).
Cytosolic and nuclear misfolded proteins share INQ (JUNQ) as a common deposition siteS.Figure 1A S. cerevisiae wt or hsp42Δ cells expressing mCherry‐VHL (red) and GFP‐luciferase‐DM‐NLS (green) were grown at 30°C and shifted to 37°C for 90 min. MG132 was added prior to temperature upshift. Changes in protein localizations were recorded. DNA was stained by DAPI (blue). B S. cerevisiae wt cells or hsp42Δ cells expressing GFP‐VHL (green) were treated as in (A). Nuclear membranes were visualized by Nsp1immunofluorescence microscopy labeling (red). C S. cerevisiae wt cells or hsp42Δ cells expressing mCherry‐VHL (red) and GFP‐Nup49 (green) were treated as in (A). Three‐dimensional reconstructions of respective cells are given. D Time‐lapse microscopy of wt cells expressing mCherry‐VHL (green) and GFP‐Nup49 (red) upon temperature upshift from 30°C to 37°C. * indicates collision of peripheral foci leading to fusion, and an arrow indicates apparent encounters of peripheral foci and nuclear INQ, followed by foci separation.
Figure 1(A Schematic illustration of a constricting cell undergoing a cycle of ratcheted pulsatile constriction. Upregulation of myosin-II (blue) causes coalescence of actin fibers (red) in a radially polarized manner. Contraction of medio-apical actomyosin fibers, which are anchored to the plasma membrane (black) through adherens junctions (purple dots) and are mechanically coupled to a junctional actomyosin belt (red), causes constriction of the cell surface. Disassembly of medial actomyosin is accompanied by surface relaxation, which is contained by a ratcheting mechanism that stabilizes the constricted state. Repeated cycles of actmyosin-mediated ratcheted contraction result in an incremental shrinkage of the cell surface. (B) Schematic illustrating medio-apical (cyan) and junctional (purple) actomyosin fibers. (C) Confocal image of a constricting cell during ventral furrow formation showing an overlay between the myosin-II probe Sqh::GFP and the plasma membrane marker GAP43::mCherry. Scale bar, 5 μm (D) Movie (still frames) of a gastrulating Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin imaged in a cross section using two-photon microscopy. βH-spectrin is enriched at the apical surface of ventral mesodermal cells during tissue invagination (arrowheads). Scale bar, 50 μm. (E) Z-Projections of confocal image stacks showing the apical cell surface of a Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin (green) and the plasma membrane marker GAP43::mCherry at 5 min (top), 7 min (middle) and 9 min (bottom) after initial medio-apical accumulation of βH-spectrin. Scale bar, 20 μm (F, G) Immunostaining of βH-spectrin (green) visualized by STED nanoscopy revealed medio-apical βH-spectrin supra-cellular fibers in mesodermal cells (F). In ectodermal cells, βH-spectrin localizes to apical cell junctions (G). Note, in (F) the junctional βH-spectrin signal is shown in magenta as a proxy for cell membranes. Scale bars, 2.5 μm. (H-J) Confocal images of the ventral surface of a Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin (H), the myosin-II marker Sqh::mCherry (I) and a merge of the two (J) with βH-spectrin in green and Sqh::mCherry in magenta. Scale bars: 20 μm. (K-L) Co-staining of phalloidin (F-actin reporter; magenta) and βH-spectrin (green) of the apical surface of a mesodermal cell at the onset of ventral furrow formation (K) and at a later time point (L) visualized by confocal microscopy. White dashed lines indicate the cell boundaries segmented based on the phalloidin-staining of sub-apical confocal sections. Scale bars: 5 μm.