EMBO/sd-character-level-ner huggingface dataset:
Figure 2A In silico neural network modeling using our previous synaptic puncta values for untreated (top, knockdown (KD) [6/16] = 0.375*control) and treated (bottom, KD [24/16] = 1.5*control) RTT neurons (Marchetto et al, 2010) suggested that isolated increase of synaptic knockdown sufficiently increases neural activity (untreated, mean = 0.665, range = [0.050,1.002]; treated, mean = 1.542, range = [0.997,8.395]). For excitatory-inhibitory (E-I) balance, "0" is fully excitatory, "1" is fully inhibitory. B List of compounds for the phenotypic screening. The compounds were selected for mechanisms of action that counteracted the neurotransmitter deficiencies observed in Figure 1. The final concentration was determined based on previous studies. C Schematic of drug treatment workflow. Briefly, 28-day-old MECP2-KO neurons were treated for 2 more weeks prior to performing phenotypic reversal experiments. D, E Western blot quantification showed decreased presynaptic Synapsin1 (D) and postsynaptic PSD-95 (E) in untreated 6-week MECP2-KO neurons that can be increased by drug treatment (Kruskal-Wallis test, *P < 0.05, **P < 0.01, ***P < 0.001; full Western blot, Fig EV4A) (WT83/Q83X cell lines were used; N = 3 clones from each genotype). F, G 6-week MECP2-KO neurons (F) showed a pharmacologically rescuable reduction of co-localized synaptic puncta (G; one-way ANOVA, F16,118 = 9.148, *P < 0.05; Dunnet's multiple comparisons test vs untreated KO (WT83/Q83X cell lines were used; N = 7-8 neurons/condition). *P < 0.05, **P < 0.01, ***P < 0.001. Z scores relative to KO untreated: Control = 3.903; Nefiracetam = 3.560; Carbamoylcholine = 2.705; Pirenzepine = 0.0448; PHA543613 = 2.121; Acamprosate = 3.339; Baclofen = 0.5672; GR73632 = 2.237; Hyperforin = 3.016, and IGF-1 = 3.791. Scale bar = 5 µm. H Drug treatment increased calcium transient frequency in 6-week neurons. Fluorescence intensity changes reflecting intracellular calcium transients in neurons in different regions of interest (one-way ANOVA, F4,52 = 20.28, Dunnett's multiple comparisons test vs untreated KO: Nefiracetam, **P < 0.01 and Z = 2.364; PHA 543613, ***P < 0.001 and Z = 3.391; Acamprosate, ***P < 0.001 and Z = 3.153; WT83/Q83X cell lines were used; N = 10-14 neurons/condition) and the percentage of active neurons (one-way ANOVA, F4,52 = 23.11; Dunnett's multiple comparison test vs untreated KO: Nefiracetam, ***P < 0.001 and Z = 3.144; PHA 543613, **P < 0.01 and Z = 2.567; **Acamprosate, P < 0.01 and Z = 2.285; N = 10-14 neurons/condition). I Treatment with either Nefiracetam or PHA 543613 increased network spiking activity in MECP2-KO neurons on MEA (two-sided unpaired Student's t-test compared to KO untreated: Nefiracetam, *P = 0.016 and Z = 2.418; PHA 543613, *P = 0.027 and Z = 2.147; Acamprosate, P = 0.39 and Z = 0.477; WT83/Q83X and WT82/K82fs cell lines were used; N = 4-11 MEA wells/condition).
Figure 1A-B: Percentage of pDCs (A) and mDCs (B) from total PBMCs collected at D1 was quantified by flow cytometry and grouped to self-reported duration of symptoms prior to D1 (A-B; 0-4 n= 22; 5-8 n=39; 9-12 n= 39; ≥13 n=16). Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Kruskal-Wallis test *<p0.05, **<p0.01 ***<p0.001, ns= not significant. PBMCs and plasma samples were collected from SARS-CoV-2 infected individuals at time of hospitalization (D1) and after 5 days (D5). C-D: Percentage of pDCs (C) and mDCs (D) at D1 and D5 matched samples from all patients independent of symptom duration (n=93). Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test *<p0.05, **<p0.01 ***<p0.001, ns= not significant. PBMCs and plasma samples were collected from SARS-CoV-2 infected individuals at time of hospitalization (D1) and after 5 days (D5). E-J: Matched D1 and D5 plasma samples were available from 55 donors and protein levels were determined for IFNα2a (E, n=30), IL-3 (F, n=38), IL-8 (G, n=44), IL-6 (H, n=51), TNFα (I, n=42) and CXCL10 (J, n=46), note that some samples could not be quantified and were excluded from the analysis. Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test *<p0.05, **<p0.01 ***<p0.001, ns= not significant. K-L: Percentage of pDCs grouped by COVID-19 severity, as determined by oxygen supplementation (no oxygen n=43, nasal oxygen n=65 and high flow oxygen n=5). Percentage of pDCs (K) was correlated to COVID-19 disease severity (L) using simple linear regression. Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges mean values with the standard error of the mean Statistical significance was determined using the Kruskal-Wallis test and simple linear regression *<p0.05, **<p0.01 ***<p0.001, ns= not significant.
Figure 1(A) Kinetics of cell numbers in Lineage-EPCR+CD150+CD48- (L-ESLAM) HSCs, Lineage-EPCR+CD150+CD48+ (CD150+CD48+) progenitor cells, and Lineage-EPCR+CD150-CD48+ (CD150-CD48+) progenitor cells after the administration of 5-FU (250 mg/kg, i.v.). The small graph is an enlargement of 0 to 8 days after the administration of 5-FU. (* or **) in the small graph represents the statistical analysis of HSC between 3 and 4 or 7 days, respectively. (B) The enrichment plots of genes related to cell cycle in the comparison of the steady state with the early (upper) or late phase (lower). NES and q values represent normalized enrichment scores and FDR, respectively. (C) The frequency of EdC+ cells within L-ESLAM HSCs 24h after the administration of EdC (150 mg/kg, i.p.). (D) A principal component analysis (PCA) using the transcriptome data of L-ESLAM HSCs derived from untreated (steady state) or 5-FU-teated mice at 6 (early phase) or 10 days (late phase). (E) Comparison of gene expression patterns in HSCs between the early (6 days) and late phases (10 days) on GSEA. The graph shows normalized enrichment scores (NES). Bold and italic mean gene sets show (p<0.05, FDR< 0.05) or (p<0.05), respectively. (F) The frequencies of phenotypic HSCs (Orange gate) and CD48+ progenitor cells (blue gate) after a 4-day culture of HSCs derived from 5-FU-treated mice (at 6 to 9 days).
RNF26 promotes polyubiquitination of MITA at K150.Figure 3(A) RNF26 mediated polyubiquitination of MITA at K150. The 293 cells (5×106) were transfected with HA-Ub (1 µg) and RNF26 (1 µg) together with Flag-MITA or the indicated mutants (5 µg each). Twenty-four hours after transfection, cells were subjected IP under denatured conditions with anti-Flag and immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated. B) RNF26 targeted MITA for polyubiquitination at K150 in vitro. RNF26, MITA and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and RNF26 were incubated with MITA or its mutants. The ubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels). All experiments were repeated for at least three times with similar results.
Figure 5Maximal cytoplasmic [Ca2+] in single, freshly sorted NKT1 and NKT2 cells following TCR stimulation in the presence or absence of BTP2 (1uM); Percentage of iNKT cells in total live cells in FTOC with or without BTP2 (1uM) addition at the start of culture (day 0); BTP2 (1uM) was added 48h after the start of culture, and the expression of Tbet in total iNKT cells is shown. Data are representative from one of three independent experiments. Graphs represent mean ± SD with symbols representing individual cells (a), or individual thymic lobes (b-f). **p < 0.01; ****p < 0.0001; n.s. not significant (unpaired two-tailed Student's t test). BTP2 (1uM) was added 48h after the start of culture, and the expression of CXCR3 (d), PLZF (e), and PD1 (f) in total iNKT cells is shown. Data are representative from one of three independent experiments. Graphs represent mean ± SD with symbols representing individual cells (a), or individual thymic lobes (b-f). **p < 0.01; ****p < 0.0001; n.s. not significant (unpaired two-tailed Student's t test).
a) Immunoblot for htt or p62 from total APHs (T) and their corresponding membranes (Mbr) and matrices (Mtx) isolated from livers of wild-type htt (18Q) and 111Q-htt mutant huntingtin knock-in mice (111Q).figf6(a) Immunoblot for htt or p62 from total APHs (T) and their corresponding membranes (Mbr) and matrices (Mtx) isolated from livers of wild-type htt (18Q) and 111Q-htt mutant huntingtin knock-in mice (111Q). Left, representative immunoblots. Right, distribution of htt and p62 between Mbr and Mtx calculated by densitometric quantification in six such immunoblots. AV, autophagic vacuoles. Mean + s.d. *P 0.05 compared to wild-type values. (b) Filter retardation analysis of the same fractions as in a, blotted for htt. Negative signal indicated absence of aggregates retained in the filter. (c) Immunoblot for ubiquitin in homogenates (Homog), cytosol and the autophagic fractions described in a, using 6% (top) and 16% (bottom) gels. Dotted line separates stacking and running parts of gel. (d) Immunoblot for htt, LC3 and p62, of brain homogenates (Homog) and fractions enriched in APHs from the same mouse groups as in a. (e) Immunoblot for htt and p62 of membranes from autophagic vacuoles shown in d subjected to co-immunoprecipitation for htt under low stringency. Input for 111Q-htt autophagic vacuoles was one-third of that for 18Q-htt to avoid antibody saturation. Levels of htt (top) and p62 (bottom) in the input, immunoprecipitate (IP) and flow through (FT) are shown. Full-length blots in Supplementary Figure 21.
Figure 6A) A model of HP1-mediated protein stability regulation of Jmjd1a/b. In wild-type cells, HP1 tethers Jmjd1a/b to chromatin through HP1-H3K9me2/3 interaction. We speculated that HP1 might be released from chromatin in Suv39h/Setdb1/G9a-deficient cells since H3K9me2/3 might be absent in these cells. In this situation, HP1 might no longer ensure protein stability of Jmjd1a/b. B) Strategy to establish Suv39h/Setdb1/G9a-deficient mESCs. By disrupting G9a alleles in the #33-6-SuvDKO line by CRISPR/Cas, we established an mESC line, 3S1G, which carries the conditional knockout alleles for Setdb1 and conventionally knocked out alleles for Suv39h and G9a. C) Impact of Suv39h/Setdb1/G9a depletion on HP1-mediated protein stability regulation of Jmjd1a/b. Jmjd1a/b were destabilized in mESCs lacking Suv39h/Setdb1/G9a, even though HP1 was present. D) Functional evaluation of H3K9me2/3 for confining HP1 to chromatin. Subcellular localizations of HP1α, β and γ in TT2 and mESCs lacking Suv39h/Setdb1/G9a were compared. α-Tubulin, Ezh2, and histone H3 were used as representative markers for each fraction.
Figure 2(A-D) The serial dilutions of post-second immunization serum samples were analyzed by ELISA using plates coated with purified RSV rA2-Line19F-FFL virions. The serum samples were serially diluted, and the detection of antibodies were measured by optical density (OD) at 490 nm. End-point titers of the serum samples were determined as the reciprocal of the highest dilution providing an absorbance twice that of the negative control (PBS immunized animal). (E) Virus neutralization titers following second vaccination. End-point serum dilution titer that resulted in 50% inhibition of RSV infection (ID50) in RSV-LINT assay is shown. The mean value for each group is indicated.
Differential RNA expression in SAM stem cells during development.Figure 2(a) Expression of CLV3, mCherry, TEL2, PAN, and the meristem marker genes STM and KNAT1. Asterisks indicate timepoints of significantly different expression (Wald test, Benjamini & Hochberg corrected, q<0.05) between stem and non-stem cells for each time point (N = 2). + = stem cell nuclei; - = non-stem cell nuclei, E = nuclei from embryos, D7/14/35 = nuclei from 7/14/35 day-old plants, S14 = nuclei from 14 d-old above-ground seedlings. (b) Global gene expression correlations at different developmental stages of stem (+) and non-stem cell (-) nuclei. (c) Number of DEGs between stem and non-stem cells at each timepoint. The banded portion of the bars indicates the number of transcription factor genes (also in parenthesis). (d) Overlap of genes with higher expression in stem cells (excluding mCherry) and significant GO-terms for shared DEGs (increased expression during at least 3 timepoints). See also Appendix Fig. S2 for p-values of overlapping gene sets. p-values were calculated with Fisher's exact test and Bonferroni-corrected (see also Appendix Table EV3).
Figure 6(A) 293T cells were co-transfected with HA-GRWD1 wild-type (WT) (0.5 ug) or HA-GRWD1 Δ1-136 (3.5 ug) and RPL11-FLAG (1.5 ug) as indicated for 42h, and then immunoprecipitated with an anti-FLAG antibody. Immunoprecipitates (IPs) and 1.6% of inputs were immunoblotted with the indicated antibodies. (B) 293T cells were co-transfected with His-Xpress-MDM2 (0.6 ug), RPL11-FLAG (0.9 ug), and HA-GRWD1 WT (0.5 ug) or HA-GRWD1 Δ1-136 (4.5 ug) as indicated for 42h and subjected to immunoprecipitation with an anti-FLAG antibody. Immunoprecipitates (IPs) and 1.6% of inputs were immunoblotted with the indicated antibodies. (C) H1299 cells were co-transfected with the indicated expression vectors (p53, 7.5 ng; HA-Ub, 0.5 ug; His-Xpress-MDM2, 2 ug; RPL11-FLAG, 1 ug; HA-GRWD1-FLAG WT, 1.5 ug; HA-GRWD1-FLAG Δ1-136, 1.5 ug; GFP, 0.04 ug) for 48 h, and then analyzed by immunoblotting with the indicated antibodies. GFP serves as a control protein to show equal transfection efficiencies. (D) HFF2/T cells stably expressing HA-GRWD1 wild-type (WT) or Δ1-136, E7, and KRAS G12V were established using retroviral vectors and analyzed by immunoblotting with the indicated antibodies. *, endogenous RAS proteins. (E and F) Anchorage-independent growth of HFF2/T cells overexpressing E7 and activated KRAS G12V with HA-GRWD1 WT or Δ1-136 (or without HA-GRWD1). At 9 days after seeding, colonies were analyzed as above. Representative images are shown in (E). Scale bars, 500 um. Average numbers of colonies (>200 um in diameter) per photographed area with SDs are shown in (F). Seventy randomly selected areas were counted.
Figure 6Body weight gain and cumulative food intake of male OXGR1 adrenal-specific reexpression mice (OXGR1REAG). Male OXGR1KO mice (8 weeks) were adrenal-specifically injected with control HBAAV2/9-GFP (OXGR1KO control) or HBAAV2/9-OXGR1 (OXGR1REAG). Two weeks after injections, mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 12 weeks (n = 8 per group). Representative image of body composition and fat and lean mass index of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group). Weight index of gWAT and iWAT in male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group). Immunoblots and quantification of p-HSL and ATGL protein in gWAT of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group). Oxygen consumption of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group). RER of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).
In vitro uptake depends on GTP hydrolysis and on the V-ATPase.Figure 7In vitro uptake depends on GTP hydrolysis and on the V-ATPase. (A) Standard uptake reactions were performed in the presence of different concentrations of GTPγS. Luciferase uptake was assayed as in the legend to Fig. 3 A. Four independent experiments were averaged, using the sample without inhibitor as the 100% reference. Error bars indicate SD. GTPγS did not influence luciferase activity by itself (not shown). (B) Standard uptake reactions were performed in the presence of FCCP (CMA, 20 μM) or concanamycin A (20 μM), or in the absence of ATP.
Figure 1A. Colony formation assay (CFA) with imatinib with representative images of colonies formed by each cell line within the panel. Error bars represent mean ±S.E.M., n=9, *P < 0.001 by two-tailed unpaired Student's t-test (H1915 P= 0.0004, H2110 P=0.0001). B. Western blot analysis after 1 hour treatment with imatinib. C. MTT assay for NSCLC cell lines and Beas-2B cells with dasatinib or DMSO. Error bars represent mean ±S.E.M., n=9, *P<0.001 by two-tailed unpaired Student's t-test (H1915 P=1.09×10-5, H2110 P=0.009 and A549 P=6×10-6). D. Western blot analysis after 1 h treatment with dasatinib.
Model of PPM1H specificity for Rab GTPases.Figure 7Although PPM1H phosphatase is likely to be dimeric in cells, a 1:1 complex is shown for simplicity. The switch 2 helix is red and the phosphate site is yellow. The flap domain (305-414) encodes the primary specificity for phosphorylated Rabs while the anchor (33-79) contributes to folding of the enzyme. The β-motif (green, residues 480-496) is also indicated. The conserved loop motif (386-396) with a central Leu392 residue may couple substrate binding to catalysis.
CRMP4 protein levels are altered via a dynein-dependent activity (A) Representative images of healthy or C9orf72 iPSC-derived MNs treated with Sema3A or untreated in the distal compartment, 6 hours post treatment.Figure 3(A) Representative images of healthy or C9orf72 iPSC-derived MNs treated with Sema3A or untreated in the distal compartment, 6 hours post treatment. Red: denotes Tubulin, Green: denotes CRMP4. Scale bar: 5 μm. (B) Quantification of CRMP4 intensity levels in healthy or C9orf72 iPSC-derived MNs with Sema3A treatment or untreated. 14 untreated healthy axons, 12 healthy axons with Sema3A treatment, 49 untreated C9orf72 axons and 31 C9orf72 axons with Sema3A treatment were monitored from 3 different chambers. One-way ANOVA, Tukey's multiple comparisons test, n = 3, Data presented as mean ±SE, *p = 0.0338; ****p<0.0001. (C) Illustration of the experimental procedure for MNs in an MFC treated with the fluorescently tagged retrograde tracer CTX in the distal compartment. Neuronal cell bodies in the primary neuron whose axons have traversed into the distal compartment were also labeled by the retrograde tracer. (D) Representative images of healthy or C9orf72 human-derived MN cell somata with Sema3A treatment, Sema3A + dynein inhibitor treatment, or untreated. Gray: denotes CTX, Green: denotes CRMP4. Scale bar: 5μm. (E) Quantification of CRMP4 intensity (normalized to GAPDH+mCherry/area) levels at the somata of healthy or C9orf72 human-derived MN after Sema3A treatment, Sema3A + dynein inhibitor treatment, or untreated. Analysis performed in 3 independent chambers per condition. 19 healthy untreated cell somata, 26 healthy cell somata with Sema3A treatment, 20 healthy cell somata with Sema3A + dynein inhibitor treatment and 14 C9orf72 cell somata from each condition were monitored. One-way ANOVA, Newman-Keuls multiple comparisons test, n=3, Data presented as mean ±SE, *p=0.0207; ***p=0.0004. (F) Representative images of healthy or C9orf72 human-derived MN proximal axons with Sema3A treatment, Sema3A + dynein inhibitor treatment or untreated control. Green: denotes CRMP4, Red: denotes GAPDH. Scale bar: 5μm. (G) Quantification of CRMP4 intensity levels (normalized to GAPDH+mCherry/area) at the proximal axons in healthy or C9orf72 human-derived MN after Sema3A treatment, Sema3A + dynein inhibitor treatment, or untreated control. Analysis performed from 3 independent chambers in each condition. 21 healthy untreated proximal axons, 24 healthy proximal axons with Sema3A treatment, 16 healthy proximal axons with Sema3A + dynein inhibitor treatment, 12 C9orf72 untreated proximal axons, 8 C9orf72 proximal axons with Sema3A treatment and 13 C9orf72 proximal axons with Sema3A + dynein inhibitor treatment were monitored. One-way ANOVA, Newman-Keuls multiple comparisons test, n = 3, Data presented as mean ±SE, *p =0.0334, ****p<0.0001.
RNA depletion results in reduction of B-type compartmental interactions Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for aXL control and RNase A treated cells.Figure 2Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for aXL control and RNase A treated cells. The arrows represent similar patterning of long-range interactions in samples treated with RNase after crosslinking.   Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for bXL control and RNase A treated cells. Black arrow indicates the reduction in long-range interactions in the bXL RNase A dataset. The arrows represent perturbed patterning of long-range interactions in samples treated with RNase before crosslinking.  C-D) Pearson correlation heatmaps showing the genomic interactions at 500kb resolution for chromosome 4, with the 1st principal components (1st PC) below for aXL (C) and bXL (D) samples. The bXL RNase A sample shows reduced Pearson correlations across the genome. E) Violin plot showing the first eigenvalues (calculated by the trans-chromosomal data) in control and RNase treated cells before and after crosslinking. The bXL RNase A sample show reduction of the negative eigenvalues, indicative of perturbation of B-type compartments. p-value: Wilcoxon rank-sum test. In the violin plots, the horizontal bands represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates. F) Saddle plots showing the compartmental interactions for both cis- and trans-contacts across the conditions. The bXL RNase A samples display reduced B-B type interactions.
FXR1 works together with the dynein-BICD2 complex to inhibit cytoplasmic Nup granules formation.Figure 6A HeLa cells stably expressing GFP alone or GFP-FXR1 were immunoprecipitated using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (mean, *P < 0.05; N = 3). B, C HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Arrowheads indicate blebbed regions of nuclei. The percentage of interphasic cells with Nup granules was quantified (C), 900 cells were analysed (mean ±SD, ***P < 0.001; N = 3). D Lysates of HeLa cells stably expressing GFP alone or GFP-FXR1 were subjected to immunoprecipitation using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (mean, *P < 0.05; **P < 0.01; N = 3). E, F HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block and released for 12 hours and analysed by immunofluorescence microscopy for FXR1 and mAb414. The magnified framed regions are shown in the corresponding numbered panels in (E). The percentage of cells with cytoplasmic Nup granules was quantified in (F), 2700 cells were analysed (mean ±SD, ***P < 0.001; N = 3).