EMBO/sd-character-level-ner huggingface dataset:
Defect in early endocytic steps and lysosomal trafficking caused lamina vacuolization.Fig 10(A) repots>Rab5S43N. (B) repots>Rab7T22N. (C) repots>Rab11S25N. (D-H) MARCM clones labeled by RFP or GFP (green) of control (D), Rab52 (E), α-Ada3 (F), HrsD28 (G), Rab7KO (H), dor8 (I) and carΔ146 (J). The penetrance (number of samples with vacuole over the number of samples examined) is indicated in each panel. Adults of all genotypes were incubated at 28°C for 14 days. DAPI: nuclei (white in A-J). Scale bar: 20 μm. (K) The percentage of the vacuole area in (A-C) was summarized. Adults of these genotypes were incubated at 28°C for 12 days. P-values were calculated using Kruskal-Wallis with Dunn's post-tests.
Figure 2A. TNF-α-stimulation affects a smaller fraction of ubiquitylation sites compared to phosphorylation sites. The plot shows the fraction of ubiquitylation and phosphorylation sites that were affected upon TNF-α-stimulation. Site-specific changes in ubiquitylation and phosphorylation were analyzed as described in Figure 1A. B. Gene Ontology cellular component (GOCC) term enrichment analysis of proteins with upregulated ubiquitylation sites. The bar plot indicates terms that showed a significant enrichment (p<0.001) C. Comparative Gene Ontology biological process (GOBP) term enrichment analysis of proteins with TNF-α-upregulated ubiquitylation and phosphorylation sites. The heatmap shows the relative enrichment of significantly enriched (p<0.01) terms. D. Functional interaction network of proteins with TNF-α-upregulated ubiquitylation sites. The network shows functional interactions among proteins that showed increased ubiquitylation after 5 or 15 minutes of TNF-α-stimulation. Blue nodes show proteins that contained TNF-α-upregulated phosphorylation and ubiquitylation in our dataset, and the TNF-RSC components are marked in red type.
Figure 2A. Lysates from HEK293 cells expressing GFP-TBC1D31 were subjected to pull down assay with GST and GST-praja2531-631 polypeptides. B. MST signal (normalized fluorescence) of P1 (red curve), P2 (green curve) and P3 (cyan curve) plotted against TBC1D31, at increasing concentrations of peptides. The threading modelled structure of the overlapping binding segment of praja2 (praja2550-570) is shown. C. Co-immunoprecipitation of GFP-TBC1D31 and flag-praja2 ring mutant (flag-praja2rm) or praja2Δ550-570. D. Threading modelled structure of TBC1D31, with a zoom of its C-terminus. Mutated residues are highlighted in stick colored by atom type. E. MD derived binding mode of praja2530-570 (green cartoon) to the C-terminal region of TBC1D31 (red cartoon). F. MST signal of P1 plotted against increasing concentrations of TBC1D31 peptides: wild-type (red curve), R948A-R951A (violet curve) and R957-R959D-H960A (orange curve).
Figure 4Bacterial growth assessed by color change due to medium acidification for both the undefined ATCC 1161 and semi-defined CSY media with or without sucrose, 4% and 1% respectively. (+), inoculated wells; (-), non-inoculated control. The picture was taken after a 24-hour incubation period. Impact of horse serum (HS) and yeast extract (YE) dilution on M. florum doubling time. The green and red dotted lines indicate HS and YE concentrations found in ATCC 1161 medium (20% HS, 1.35% YE) and CSY medium (0.313% HS, 0.02% YE), respectively. Doubling times were measured in a CMRL-1066 base medium using colorimetric assays. Dots and error bars indicate the mean and standard deviation calculated from three biological replicates. The maximal biomass concentration observed for M. florum cultures growing in CSY medium with varying initial concentrations of sucrose. Biomass was measured using colony forming units (CFU/ml; left axis) and converted to grams of dry weight (gDW/L; right axis). A rectangular hyperbola fit is shown (yellow), and the dotted line represents the maximal biomass value predicted by the fit (1e8 x 5.95 CFU/ml; 0.013 gDW/L). Relationship between varying initial sucrose concentration and growth rate in CSY medium. Growth rates were determined by fitting a simple exponential growth model to CFU/ml data from time-course experiments and error bars indicate the standard deviation associated with each value. CFU/ml quantifications were performed in technical duplicate as described in Appendix Figures S4 and S5. A rectangular hyperbola fit is shown, and the predicted maximal growth rate (0.44 hr-1) is indicated by the dotted line. Sucrose specific uptake rate (e) and combined lactate/acetate specific secretion rates (f) at varying initial sucrose concentrations in CSY medium. Boxplots represent the median and interquartile range of uptake or secretion rate values calculated at different time intervals during the exponential growth phase of M. florum cultures. Whiskers indicate minimal and maximal values. Dotted lines indicate the selected uptake (-5.26 mmol∙gDW-1∙hr-1) and secretion rate (8.69 mmol∙gDW-1∙hr-1) values used for modeling. Sucrose quantifications were performed in technical duplicate, whereas lactate and acetate quantifications were performed in single replicates. See Appendix Figure S5 for further details.
Figure 5A. Primary data - polyacrylamide gel - a representative result. For description of bands/fragments see next panel legend. Asterisks indicate STUBS (16-19 nt). B. A schematic representation of quantitation of fragments. OUTSIDE (16-30 nt) are RNA fragments that originated by digestion of the full length RNA by RNases that were stopped either before reaching or at the point of reaching RNAP; STUBS (16-19 nt) are RNA fragments that originated by digestion of the full length RNA that were stopped at the point of reaching RNAP; INSIDE (< 16 nt) are RNA fragments (oligonucleotides) that could be only generated after the disassembly of the complex by the torpedo mechanism. The red color indicates parts of RNA that were digested. C. Quantitation of three independent experiments. "Full length" indicates the remaining undigested RNA. "OUTSIDE", "INSIDE" are fragments as explained in (B) and indicated in (A). The bars represent 100 % (all fragments). The black-grey-white boxes indicate the percentage of each fragment group (in %). The error bars indicate ± SEM for each group of fragments calculated from three biological replicates.
Figure 3(A) Immunoblot of p-SMAD2/3, p-SMAD1/5, SMAD6, SMAD2/3 and SMAD1 proteins in DNFB- versus acetone-treated WT skin (n=4 in each group). Data information: GAPDH is used as the control housekeeping protein in (A Graphs show mean + SD, NS: not significant, unpaired t-test. (B) Quantitative PCR (qPCR) of Il33 and Smad6 mRNA in Pam212 cells treated with poly (I:C) or PBS (carrier control, n=4 in each group). (C) ChIP-qPCR assay for Smad6 in the presence of poly (I:C) or PBS using an anti-IL-33 antibody. After Pam212 cells were transfected with poly (I:C) for 24 hours, cell lysates were subjected to chromatin-immunoprecipitation with anti-IL-33 antibody and eluted IL-33-bound chromatin was used for qPCR with Smad6 regulatory peak region (63,968k) primers and Smad6 no peak region (63,955k) primers (n=4 in each group). (D) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β and poly (I:C) treatment. Pam212 cells were treated with poly (I:C) or PBS followed by incubation with and without TGF-β (5nM). Data represent three independent experiments with similar results. (E) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β + poly (I:C) after knocking down of IL-33. Pam212 cells were transfected with siIl33 knockdown construct or siRNA control (siCon) for 30 hours followed by incubation with poly (I:C) and TGF-β. Data represent three independent experiments with similar results. (F) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins upon the expression of IL-33 full length or cytokine domain. Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours followed by incubation with and without TGF-β. Data represent three independent experiments with similar results. Data information: GAPDH is used as the control housekeeping protein in D-F). Graphs show mean + SD, NS: not significant, unpaired t-test. (G) Impact of IL-33 knockdown on cell proliferation in response to TGF-β. Pam212 cells were treated with siIl33 or siCon followed by TGF-β treatment (n=7 in each group). (H) Impact of IL-33 knockdown and SB431542 treatment on cell proliferation. Pam212 cells were treated with siIl33 or siCon in combination with SB431542 or DMSO (carrier control, n=7 in each group). (I) Representative images of H&E- and Ki67-stained back skin of IL-33KO and WT mice treated with 22-day DNFB protocol in combination with SB431542 or acetone (carrier control). Note the epidermal thickness, keratinocytes proliferation and dermal inflammation in each group (scale bars: 100 μm). (J) The epidermal thickness of DNFB-treated IL-33KO and WT skin after treatment with SB431542 or acetone. Each dot represents the average of three measurements in an HPF image. 10 random HPF images per skin sample are included (n=10 for WT with acetone, n=8 for WT with SB431542, n=6 for IL-33KO with acetone, n=5 for IL-33KO with SB431542 per group).
Figure 1(A) Western Blot shows developmental expression of CDC50A in isolated synaptosomes from mouse brains. The quantification was normalized to the CDC50A expression of P7. GAPDH was used as an internal control. P1, postnatal day 1; 1M, 1 month; 1Y, 1 year. N = 4 for technical replicates. (B) IHC for CDC50A and RIM1 on cultured neurons at DIV21. The left panel shows CDC50A is expressed in both neuronal soma and processes. The right panel exhibits CDC50A colocalize to pre-synapses alongside neuronal processes. MAP2, Microtubule-associated protein 2, was used to identify neuronal cells and trace dendritic processes. RIM1 is an active zone protein, which marks pre-synapses. Left scale bar = 50 µm; right scale bar = 2 µm. (C) Quantification of the percentage of RIM1+ pre-synapses colocalizing to CDC50A. N = 3 for biological replicates. (D) Single cell RNAseq in the retina revealed the percentage of RGCs that express the members of flippase family and their chaperon gene Cdc50a in mice (Macosko et al., 2015). (E) IHC for CDC50A and vGluT2 in the developmental dLGN at P14, P21 and P30. The left panels show the overview of immunostaining in the dLGN, scale bar = 200 µm. The right panels are the zoomed-in images, scale bar = 5 µm. Arrows indicate the colocalized CDC50A and vGluT2. (F) Quantification of the percentage of CDC50A+/vGlut2+ synapses. The result shows that a significantly higher percentage of CDC50A+/vGlut2+ synapses at P21 and P30 than P14. N = 6 for biological replicates.
Figure 4(A) ROCKI is recruited to the MARCKS promoter in Pam3-stimulated THP1-derived macrophages. ChIRP analysis of THP1-derived macrophages treated with Pam3 for 8 h. ChIRP assays were performed using a non-specific lacZ probe or probes specific for lnc-MARCKS. Specificity of ROCKI probes (left), and enrichment at the MARCKS promoter (right). (B) Mass spectrometry identifies APEX1 as a candidate ROCKI-binding protein in THP1-derived macrophages. APEX1 MS/MS count represents the ratio between the number of APEX1 peptides and all peptides detected. Mean of n = 2 samples. ROCKI and ROCKI-AS represent ROCKI and antisense strand of ROCKI transcripts, respectively. (C) ROCKI inhibition of MARCKS expression is dependent on APEX1. RT-qPCR analysis of MARCKS, ROCKI, and APEX1 expression in THP1-derived macrophages overexpressing ROCKI and/or depleted of APEX1. (D) APEX1 specially binds to ROCKI. Western blot analysis of APEX1 in lacZ, or ROCKI pull-down fractions. Vimentin, which non-specifically binds to all RNA probes, is shown as a loading control. (E) ROCKI associates with APEX1. Left: RT-qPCR analysis of ROCKI present in anti-APEX1 antibody or control IgG immunoprecipitates from nuclear lysates of Pam3-treated THP1-derived macrophages. Right: APEX1 western blot of the same immunoprecipitates.
EC8042 leads to durable tumor regression in a rhabdoid tumor xenograft model.Figure 7A: Immunohistochemistry analysis recapitulates the biochemistry described in vitro for mithramycin. G401 tumor sections at 20X magnification stained with H&E, cleaved caspase 3 (CC3; apoptosis) or H3K27me3. A marked increase in CC3 that correlates with H3K27me3 staining is seen only in mice treated with the continuous infusion schedule but not vehicle. Scale bar (lower left): 50μm. B: Prolonged durable response and cure of mice bearing G401 xenografts treated with 30mg/kg EC8042 administered continuously over 72-hours. Treatment duration indicated by gray shaded box. Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text). C: Kaplan-Meier survival curves indicating extended survival for mice bearing established G401 xenografts treated with the 3-day continuous infusions of EC8042 in (B). The shaded box indicates the duration of treatment. Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text). D: 20X image of section of G401 treated tumors stained for H3K27me3, cleaved caspase 3, and γH2AX. The sections compare vehicle to treatment started on day 1 with 30 mg/kg of EC8042 administered continuously for 72-hours (3-day pump). H3K27me3 increases and correlates with apoptosis (CC3), however induction of CC3 is modest. γH2AX staining does not increase with treatment indicating DNA damage is not responsible for these effects. Scale bar (lower left): 50μm.
Figure 4Schematic of hnRNPLL knockout mouse, in which its exon two is deleted. Birth rate of the different hnRNPLL genotypes, the birth rate of hnRNPLL-/- mice was significantly lower than that of WT hnRNPLL+/+ mice. The data represent the means ± SD. T-test was used to determine the significance. Representative images of hnRNPLL+/+ and hnRNPLL-/- littermates at 3 weeks. Body weight growth curve of mice at different postnatal days. The data represent the means ± SD (n=5-10). ***P＜0.001. T-test was used to determine the significance. Survival curve analysis for hnRNPLL+/+, hnRNPLL+/-and hnRNPLL-/-mice (ten mice in hnRNPLL+/+/+/-group and seven mice in hnRNPLL-/-group). Genotypes and representative images of hnRNPLL+/+ embryo and retarded hnRNPLL-/- embryos at E11.5 which generated from hnRNPLL+/- intercrosses. Scale bar, 1mm.
Figure 4(A) Simulated changes in the free energy of K+ binding to GltPh induced by different mutations. Values are calculated as differences of free energy changes during alchemical transformations of amino acid side-chains with the protein either in the K1 bound or in the apo state (Mean ± SD of 1000 bootstrap samples). (B) Representative WT and mutant whole-cell current responses to voltage jumps before and after removal of external K+. Cells were dialyzed with a KNO3-based pipette solution and the external solution contained either 140 mM CholineNO3 or 140 mM KNO3. (C) Relative changes in fluorescence for WT, D312N and D405N GltPh upon a temperature jump in MST experiments as a function of [K+]. Solid lines represent non-parametric fits, and shaded area show bootstrapped 99% confidence intervals for those fits. Non-overlapping confidence intervals indicate significant difference between WT and mutant binding curves. Values are given as mean ± SD (n≥3). (D) Representative whole-cell current recordings from cells internally dialyzed with NaNO3 and glutamate in choline-NO3-based external solutions or in NaNO3-based solutions supplemented with 1 mM L-glutamate. Glutamate-induced changes in anion current amplitudes indicate that D399N and D486N EAAT2 (GltPh D312 and D405) are K+-independent, but are functionally expressed in the plasma membrane. (E) Current-voltage relationships of steady-state currents from cells dialyzed with a KNO3-based pipette solution. Currents were measured at external 140 mM KNO3 and normalized to the current at -170 mV from a consecutive recording of the same cell in K+-free external solutions. Values are given as mean ± SD (n≥3)
a) Autophagosomes were visualized in U2OS cells stably expressing a GFP-LC3 reporter construct.f2(a) Autophagosomes were visualized in U2OS cells stably expressing a GFP-LC3 reporter construct. GFP-labelled puncta were examined after 24 h in the presence (+Q) or absence (−Q) of glutamine, with or without co-addition of 100 nM CCI-779 (CCI) and 400 nM BafA1. Scale bar, 10 μm. (b) Graphical summary of experiments performed as described in a. Percentage of cells with >10 puncta per cell from three independent experiments is depicted. Bars represent mean±s.d. from three independent experiments (>50 cells per experiment). The statistical significance (P value) was determined by a two-tailed, paired Student's t-test. *P0.05. (c) Immunoblot analysis of U2OS cells subjected to glutamine deprivation with or without 400 nM BafA1. Cells were pretreated with BafA1 for 1 h before and during exposure to glutamine-deficient medium. Autophagic activity was monitored by detection of p62 and LC3-II proteins. d) U2OS mCherry-GFP-LC3 cells were cultured in the presence (+Q) or absence (−Q) of glutamine for 18 h. Red vesicles denote autolysosomes, whereas yellow vesicles represent autophagosomes. Bars indicate numbers of yellow vesicles (autophagosomes) or red vesicles (autolysosomes) per cell±s.d. (e) Images of U2OS mCherry-GFP-LC3 cells cultured for 18 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm. (f) Phagophore formation in mCherry-ULK1. Scale bar, 10 μm. (g) mCherry-ATG5-expressing U2OS cells after 24 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm.
Figure 6A, B. 293T cells were transfected with the indicated expression plasmids. At 1 day post-transfection, whole cell lysates were subjected to dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with an anti-FLAG antibody. C. A luciferase assay was performed using 293T cells transfected with the IFN-βluciferase promoter and TK-Renilla together with 2 ng of RIG-I wild-type, RIG-I 858Q, RIG-I 858R, RIG-I K909Q, or RIG-I K909R mutant, followed by transfection with poly(I:C) (1 μg/ml) for another 12 h. D. Fluorescence microscopy, green fluorescence absorbance, and virus replication at 24 hpi in RIG-I-/- MEFs. Cells were transiently transfected with empty vector, RIG-I wild-type, RIG-I K909Q, or RIG-I K909R mutant for 24 h, followed infection with VSV-GFP (MOI = 1). bar, 100µm E. HDAC6-/- MEFs were transfected with the indicated expression plasmids. At 1 day post-transfection, whole cell lysates were prepared and used in dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with anti-RIG-I and anti-β-actin antibodies. F. 293T cells were transfected with 2 μg of FLAG-tagged RIG-I and 1, 5, or 10 μg of V5-tagged HDAC6. At 36 h post-transfection, whole cell lysates were prepared for co-immunoprecipitation analysis of V5-tagged HDAC6 and acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody. G. 293T cells were transfected with 2 μg of FLAG-tagged RIG-I. At 24 h post-transfection, cells were infected with VSV-GFP (MOI = 0.1). At 2 and 4 h post-infection, whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody. Intensity of ac-909K normalized to Flag-RIG-I was quantified. H. whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody. Intensity of ac-909K normalized to RIG-I was quantified.
Figure 4Triglyceride content of cell cultures after six days of differentiation using the protocol described in Methods. Data are shown as mean ± SEM of 3-7 samples, and the entire experiment was repeated twice. [3H]-labeled 2-deoxyglucose uptake in preadipocyte cell lines in the basal state and after pretreatment for 20 minutes with 100 nM insulin. Uptake was measured for one hour, and data are shown as mean ± SEM of 3-7 samples and repeated twice. [3H]-labeled 2-deoxyglucose uptake in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin. Uptake was measured for one hour, and data are shown as mean ± SEM of 3-7 samples. The experiment was repeated twice. [14C] D-glucose incorporation into lipids in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin. [14C] D-glucose incorporation was measured after three hours, and data are shown as mean ± SEM of 3-7 samples. The experiment was repeated twice. Lipolysis rates after stimulation with 1 nM isoproterenol as measured by glycerol release from adipocytes after six days of adipogenic differentiation. Data are shown as mean ± SEM of 3-7 replicates and normalized by lipid content of the cells. The entire experiment was repeated twice. Extracellular acidification rate (ECAR) of preadipocytes and adipocytes after 4 days of in vitro differentiation was determined by calculating the area under the curve (AUC) during measurements of basal respiration. The whole experiment was repeated three times. Data are shown as mean ± SEM of 3-7 cell lines per group. Basal respiration of preadipocytes and adipocytes after 4 days of in vitro differentiation was determined by calculating the area under the curve (AUC) during measurements of oxygen consumption rate (OCR). The whole experiment was repeated three times. Data are shown as mean ± SEM of 3-7 cell lines per group. Ratio of oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) of preadipocytes and adipocytes after 4 days of in vitro differentiation. Data are shown as mean ± SEM of 3-7 cell lines per group. Representative Western blot analysis of three independent adipocyte cell lines after 6 days of in vitro differentiation for: pJnk T183/Y185 and total Jnk after treatment with 10 nM TNFα for 15 minutes (Inflammation); pAkt S473 and total Akt after treatment with 100 nM insulin for 15 minutes (Insulin Signaling); pStat5 Y694 and total Stat5 after treatment with 500 nM GH for 20 minutes (Growth Hormone).