EMBO/sd-character-level-ner huggingface dataset:
Uniform global response to presence of an extra chromosome.Figure 4(A) Two‐dimensional annotation enrichment analysis. Pathways altered in the cell line with chromosome 3 trisomy in comparison with the cell line with chromosome 5 trisomy are plotted (Benjamini‐Hochberg FDR threshold 0.02). Each dot represents one category as defined in the KEGG and GO database, the colors mark groups of related pathways as described in the inset. Axis-position scores of the pathways; negative values indicate downregulation, positive values indicate upregulation. See Supplementary Information for further details. (B) Pathway alterations identified in HCT116 and RPE‐1 cells show similar trends. (C,D) Box‐plots of all proteins within representative categories identified as significantly altered by the two‐dimensional annotation enrichment analysis. Examples of upregulated (lysosome, carbohydrate catabolic process) and downregulated (spliceosome, chromatin remodeling) pathways show uniform response among all cell lines with extra chromosomes. All differences between DNA and protein levels are statistically significant (unpaired T‐test with Welch's correction, P<0.05). For more information, see also Supplementary Figure S4 and Supplementary Table S2.
Fig.Figure 6The morphology of MOLM-13 after FST knockout by CRISPR/Cas9 in vitro. Scale bar = 10 μm. The clonogenicity of MOLM-13 after FST knockout by CRISPR/Cas9 in vitro. Scale bar = 10 μm. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). The engraftment of MOLM-13 after FST knockout was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation. The engraftment of MOLM-13 after FST knockout was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). E The effect of FST knockout on the survival of NSG mice-engrafted with MOLM-13 cells. Cas 9: Cas 9 only (10 mice); sgRNA#3: Cas 9 + sgRNA#3 (10 mice); sgRNA#4: Cas 9 + sgRNA#4 (8 mice). Data information: survival curves were analyzed by Log-rank test. *P<0.05. F The knockdown efficiency of different FST-specific antisense oligoes (ASOs) in MOLM-13 cells was detected by RT-qPCR after three days treatment in vitro. The knockdown and RT-qPCR experiments were performed in triplicates. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). G MOLM-13 cell growth was measured after 3 days treatment of FST-ASO in vitro. The ASO treatment experiments were performed in triplicates. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). H Intraperitoneal injection of FST-ASO (10mg/kg weekly, 6 mice) significantly prolonged the survival of MOLM-13-engrafted NSG mice. The random sequence was used for negative control (Neg-ASO, 6 mice). Data information: survival curves were analyzed by Log-rank test. *P<0.05.
Figure 2A The schematics of WT NEAT1_2 and mutants with deletions in the 5′ terminal regions are shown in Figure 2A. The positions of NEAT1 probes used in SRM (blue) and EM (orange) are shown. B SRM images of the paraspeckles in ∆0-0.8kb and ∆0-1.9kb (∆5′) cells treated with MG132 (5 μM for 6 h) detected by NEAT1_5′, 1k, and 2k (green) and NEAT1_3′ (magenta) FISH probes. Scale bar, 500 nm. C Graph showing the proportion of paraspeckles with localization of the NEAT1 5′ ends to the core and shell or the shell in WT, ∆0-0.8 kb, and ∆5′ cells treated with MG132 (5 μM for 6 h). (WT: n = 159, ∆0-0.8 kb: n = 54, ∆5′: n = 48) D (left) EM observation of the paraspeckles in ∆5′ cells treated with MG132 (5 μM for 17 h) using NEAT1_5′ probes. Scale bar, 100 nm. (middle) Graph showing the proportion of localization of NEAT1_5′ probes (347 gold particles) within the paraspeckles in ∆5′ cells. (right) Graph showing the proportion of the localization of NEAT1_5′ probes in each paraspeckle in ∆5′ cells (n = 20). The box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top). E Schematics of the NEAT1_2 configuration in the WT and ∆5′ mutant.
Figure 5Control or CEP41 siRNA-transfected HUVECs cultivated under either static or shear stress states and examined in a wound healing assay in (A). Scale bars, 600 μm. Quantification of the extent of wound closure in (A) displayed graphically by measuring the distance between the lines at the indicated time points in (B). Data are presented as mean ± SD of three independent experiments (n ≥ 3 scratches per experimental condition). Statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test (***P < 0.01, ns: non-significant). in vitro angiogenesis assay was performed in static or shear stress-exposed HUVECs transfected with the indicated siRNAs for 18 h in (C). in vitro angiogenesis assay was performed in static or shear stress-exposed HUVECs transfected with the indicated siRNAs for 18 h in (C). Scale bars, 600 μm. Quantification of tube node number in (D) and tube length in (E) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition. Control or CEP41-deficient HUVECs cultivated under laminar shear stress and immunostained with ARL13b-specific antibodies to measure deciliation. Representative images are indicated by rectangles with numbers and shown in magnified images in the right panels. The resulting data were compared to those of cells under static conditions in (F). Scale bars, 20 μm. Quantification of ARL13b-positive ciliary cells (G) and of relative intensity of GT335/ARL13b signals in the cilia (H) in images (F) is the results of three independent experiments with ≥ 200 cells per condition (mean ± SD (G), median (H)). * Control, CEP41-, or CCP5-depleted hTERT-RPE1 cells cultured under serum starvation for 48 h and fixed at 18 h after serum retrieval for immunostaining with both ARL13b- and GT335-specific antibodies in (I). Images of the results 18 h after serum addition. Representative cells are indicated by rectangles. Scale bars, 20 μm. Quantification of ARL13b-positive ciliated cells (J) and relative intensity of GT335/ARL13b signals in the cilia (K) under either serum starvation or serum retrieval (18 h) conditions are the results of three independent experiments with ≥ 200 cells per condition (mean ± SD (J), median (K)).
Figure 8The mRNA expression of SHH effector genes (Gli1/2 and Glis1-3) on laser microdissection captured samples were evaluated by real-time RT-PCR analysis. The results are in arbitrary values after normalization for GapDH. Each sample was isolated from distinct mice (n = 3). Representative IF staining of Gli1 (left panel, red) and Glis2 (right panel, red) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7. Samples were counterstained with DAPI (blue). Dotted lines, basement membrane. 3D reconstruction of Glis2 staining (see B, red) and its association with DAPI-labeled nuclei (blue). Nuclear transparency was set at 0 or 70% as indicated. Representative IF double staining of Glis2 (red, bottom panel) and AcTub (green, top and bottom panels) along stem cell-transit amplifying cell axis in lower incisor CLE at P7. Samples were counterstained with DAPI (blue). Note the association of Glis2 with primary cilia particularly in the transition zone between stem cell and transit amplifying cell regions. Volume of Sox2-positive region on the lower incisor CLE at P7 was evaluated in WT vs. Glis2 KO mice upon IF staining. Number of animals analyzed is indicated (n). Representative IF staining of Prom1 using specific antibodies targeting either its extracellular loop (top panels, green) or cytoplasmic C-terminal end (bottom panels, red) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7 WT and Glis2 KO mice. Samples are counterstained with DAPI (blue). Dotted lines, basement membrane. Arrowheads marks approximate boundary of stem cell regions. Representative IF double staining of Glis2 (red) and AcTub (green) as a marker of primary cilia in stem cell region of WT vs. Prom1 KO mice. Note the reduction of Glis2 expression in Prom1-deficient animals.
Figure 6A. Experimental design and representative traces for analysis of synaptic transmission in retinas from wild-type (black) and RIBEYE KO mice (red). Presynaptic bipolar neurons were depolarized in voltage-clamp mode from -70 mV to -10 mV for 0.5 s as indicated on top to open Ca2+-channels and trigger release. Presynaptic Ca2+-currents and postsynaptic EPSCs were measured simultaneously as indicated in the traces; dotted box displays an expansion of the initial Ca2+-currents and EPSCs.B. RIBEYE KO does not alter presynaptic Ca2+-currents. Summary graphs show average peak Ca2+-currents and Ca2+-current.C. RIBEYE KO severely impairs EPSCs triggered by presynaptic depolarization. Plot shows integrated EPSC charge as a function of time; both initial and sustained neurotransmitter release are impaired.D. RIBEYE KO strongly reduces initial synchronous release induced by presynaptic depolarization as indicated by the summary graph of the peak EPSC amplitude.E. RIBEYE KO suppresses both the initial synchronous and the sustained phase of release induced by presynaptic depolarization. Summary graphs display integrated EPSC charges for the initial phase (0-50 ms) and the sustained phase (50-500 ms).F. RIBEYE KO does not alter the kinetics of EPSCs. Summary graphs depict the mean synaptic delays (left), rise times (center) and decay time constants (right) of EPSCs.
TGFβ selectively promotes translation in serum-stimulated fibroblasts.Figure 1Growth curve of NIH-3T3 cells treated with TGFβ (2 ng/mL for all experiments) or vehicle alone (mock) for the indicated time. The cell number at the indicated days relative to the number at the start of the treatment (d0) is shown. The dotted line represents the cell number at d0. NIH-3T3 cells were treated with TGFβ or mock for 48h and incubated with O-propargyl-puromycin (OPP) for the last 60 min. The translation rate was determined by flow cytometry for OPP incorporation into proteins. A representative plot is shown on the left, and the quantification is shown on the right. Values are relative to mock-treated cells. Western blot of NIH-3T3 cells treated with TGFβ or mock for 48h. Schematic of extracellular matrix (ECM) production in vitro. Western blot of ECM from NIH-3T3 cells grown in the presence of absence of TGFβ and of cell lysates from NIH-3T3 cells grown on a parallel plate. Note the absence of tubulin and the electromobility shift of collagens in ECM extracts. ECM was produced from NIH-3T3 cells according to (d), and collagen abundance was measured by picrosirius red staining, normalized to the packed cell volume before ECM extraction, and expressed relative to mock-treated cells. NIH-3T3 cells were treated with TGFβ or mock for 48h. The medium was replaced in the last 12h of the treatment (with the same treatments as before), and glucose consumption from the fresh media were measured using the YSI bioanalyzer. ATP production rate from glycolysis (JATPgly) and mitochondrial oxidative phosphorylation (JATPox) from NIH-3T3 cells treated with TGFβ or mock for 48h, calculated using Seahorse data shown in (k) and EV Figure 1f. Pearson correlation of mitochondrial ATP production rate (JATPox) and OPP incorporation of NIH-3T3 cells treated with TGFβ or mock for 2h, 4h, 8h, 24h and 48h, relative to mock-treated cells. Also see EV Figure 1k and l. Note that the datapoints of the 2h and 4h measurements overlap. NIH-3T3 cells were treated with TGFβ or mock for 48h. The medium was replaced in the last 12h of the treatment (with the same treatments as before) Glutamine consumption measured as in (g). NIH-3T3 cells were treated with TGFβ or mock for 48h, and the oxygen consumption rate (OCR) before and after treatment with mitochondrial inhibitors was measured using the Seahorse bioanalyzer. Oligo, Oligomycin; Rot/Anti-A, Rotenone/Antimycin-A; PCV, packed cell volume. NIH-3T3 cells were treated with TGFβ or mock for 48h and reduced (GSH) and oxidized (GSSG) glutathione were measured by LC-MS/MS. Shown is the GSH/GSSG ratio. NIH-3T3 cells were treated with TGFβ for 48h or mock and incubated with CM-H2DCFDA for the last 30 min. ROS were measured by flow cytometry for CM-H2DCFDA. Values are relative to mock-treated cells.
Peroxisomes directly contribute to the cholesterol supply into ciliary membranes (A) Quiescent G0-phase wild-type, Rab10−/−, KIFC3−/−, ORP3−/−, and EHD3−/− hTERT-RPE1 cells transfected with PEX3-GFP-FRB (red) and tdTomato-BicD2-FKBP (white) were treated with or without 100 μM rapamycin for 30 min, and then immunostained with anti-acetylated-tubulin (blue) antibody.Figure 8Quiescent G0-phase wild-type, Rab10−/−, KIFC3−/−, ORP3−/−, and EHD3−/− hTERT-RPE1 cells transfected with PEX3-GFP-FRB (red) and tdTomato-BicD2-FKBP (white) were treated with or without 100 μM rapamycin for 30 min, and then immunostained with anti-acetylated-tubulin (blue) antibody. Cholesterol was stained with Filipin III (Green). Arrows indicate the enrichment of ciliary cholesterol. The scale bars represent 5 μm. Quantification of the Filipin III intensity at primary cilia from (A). Rapamycin-induced peroxisome targeting to the ciliary pocket restored the insufficiency of ciliary cholesterol in the Rab10−/−and KIFC3−/−hTERT-RPE1 cells, but not in ORP3−/−and EHD3−/− hTERT-RPE1 cells (*p<0.05, **p<0.01, ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 35-40 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively. Model for the molecular machinery for the supply of cholesterol into the ciliary membrane. The Rabin8-Rab10-KIFC3 complex associates peroxisomes with cholesterol to load them on microtubule arrays. KIFC3-mediated microtubule minus-end-directed movement guides peroxisomes to the ciliary pocket for the supply of cholesterol to the ciliary membrane. The ORP3-EHD1-EHD3 complex is involved in cholesterol trafficking at the ciliary pocket. In the pathological context of ZS, insufficient ciliary cholesterol might impair the cilium-related Shh signal competence, causing ciliopathy-related symptoms.
Figure 6A. Scheme to assess the influence of the highly connected nodes on germ layer acquisition. B. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zfp354c-/- mESCs in the 3KI background to ectoderm. C. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zfp354c-/- mESCs in the 3KI background to mesendoderm. D. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zff354c-/- mESCs in the 3KI background to definitive endoderm. E. Scheme to assess the influence of the highly connected nodes on the spontaneous fate acquisition between mesendoderm and ectoderm. F. Differentiation of wild type (WT), Sp1+/-, Nr5a2-/- and Fos-/-Jun-/- mESCs in the 3KI background to definitive endoderm.
a, Western blotting analysis of OFD1 protein levels in control or OFD1 knockdown MCF7 (C-19) cells, quantified OFD1 level was normalized with &bgr-tubulin.f4a, Western blotting analysis of OFD1 protein levels in control or OFD1 knockdown MCF7 (C-19) cells, quantified OFD1 level was normalized with alpha-tubulin. b, Quantification of percentage of cells with primary cilia in MCF7 or C19 cells subjected to 72 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia for 500 cells per well in triplicate samples. ***P 0.001, two-tailed unpaired student's t-test. c, Quantification of length of primary cilia in C19 cells subject to 48 h or 72 h serum starvation. d, e, Representative confocal images of primary cilia with variable length formed in MCF7 or C19 cells subject to 72 h serum starvation. Data shown represent 100 cells per well in triplicate samples. d, e, Representative confocal images of primary cilia with variable length formed in C19 cells subject to 72 hserum starvation. Data shown represent 100 cells per well in triplicate samples. f, Scanning electron microscope analysis of primary cilia (marked by arrows) formed in C-19 cells subject to 72 h serum starvation and cycling Atg5+/+ MEFs in normal medium for 24 h. Similar results were observed in three independent experiments.
Figure 4a, Alternative models for inheritance of molecular factors governing the durations of cell cycle phases. b, Simulation of the "strength of coupling" as a function of the number of unique phase-coupling molecule types under the many-for-all model. Each simulation generated 200 cells for which an R2 value was calculated. R2 values were averaged across 200 simulations. The shaded area represents the standard deviation of R2 across the simulations. , Simulation of coupling strength as a function of the number of unique phase-coupling and phase-specific factors. Phase-coupling factors have shared control over a pair of cell cycle phases, whereas phase-specific factors affect only one cell cycle phase. Strength of coupling is represented by mean R2 value simulating the effect of perturbing a single phase-coupling factor by significantly increasing its abundance or activity. Perturbation was simulated by increasing the abundance of a phase-coupling factor by 10 fold. simulating the effect of increasing a phase-coupling factor by 10 fold f, Schematic of prolonging all phases by adding CDK2 inhibitor. RPE cells were treated with 2 μM CVT-313 and the durations of each phase were quantified for a full cell cycle. g, Shifts in phase durations for RPE cells treated with 2 μM CVT-313. A boxplot representing the distributions of durations in untreated cells is underlaid for comparison. *, P < 1 × 10-5, 2-sided Kolmogorov-Smirnov test. (n = 117 cells). h, Pairwise correlation between cell cycle phase durations upon treatment with CVT-313. i, Schematic of shortening phases by overexpression of cyclin D. Cyclin D was overexpressed in RPE cells and the durations of each phase were quantified for a full cell cycle. j, Shifts in phase durations for RPE cells overexpressing cyclin D. A boxplot representing the distributions of durations in untreated cells is underlaid for comparison. *, P < 1 × 10-5, 2-sided Kolmogorov-Smirnov test. (n = 113 cells). k, Pairwise correlation between cell cycle phase durations upon overexpression of cyclin D. P indicates P-value.
Expression of four GA-biosynthesis and -degradation enzymes in wild-type plants upon mannitol treatment and in GOF lines showing an altered growth phenotype in both independent lines.Figure 5A The expression level of two GA-degradation and two GA-biosynthesis enzymes in expanding leaf tissue (third leaf at 15 DAS) of wild-type plants 20 min, 40 min, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h after mannitol treatment. The fold changes (FC) were calculated relative to control conditions.Data information: In A, data are presented as mean ± SEM, n = 4 independent experiments, *= FDR<0.05 (unpaired two-sided Student's t-test) B The expression of GA2-OX6 and GA20-OX1 in expanding leaf tissue (third leaf - 15 DAS), 8 h after transfer to DEX-containing medium to induce overexpression. The FC was calculated relative to control conditions.Data information: In B, data are presented as mean ± SEM, n = 3 independent experiments, * = FDR < 0.1 (mixed model analysis, user-defined Wald tests).  C The effect of ERF6, ERF9, ERF98 and WRKY15 on the GA2-OX6 promoter determined with transient expression assays. The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Data information: In C, data are presented as mean ± SEM, n = 3 independent experiments.
Figure 3Lysates from COS-7 cells transfected with siRNA as indicated were processed for Western blot with antibodies recognizing the indicated proteins.Band intensities of blots as in A are presented as % control siRNA. Two-tailed Student t-test, Mann-Whiteney post hoc test, ***p < 0.001, N = 3. NP-40 soluble lysates were prepared from COS-7 cells transfected with siRNA as indicated and equal protein aliquots (1.5 mg) were incubated with GST-PAK-CRIB domain. Specifically bound proteins were detected by Western blot with antibody recognizing Rac1. An aliquot of the starting material (SM) equal to 5% of that added to GST-PAK-CRIB was analyzed in parallel. Band intensities of blots as in A were presented as % control siRNA. Bars represent mean sem. One-way ANOVA, Bonferroni's post hoc test, ***p < 0.001, N=3. COS-7 cells transfected with siRNA as indicated were fixed and processed for immunofluorescence using Phalloidin-647 and antibody recognizing CLCs. Scale bar = 10 m. Perimeter and area of cells as in A were measured and a perimeter:area ratio was plotted as a % of the control siRNA treated cells. One-way ANOVA, Bonferroni's post hoc test, ***p < 0.001, N=3. Lysates from 21 day in vitro cultured hippocampal neurons transduced with lentivirus (at 7 days in vitro) driving expression of control shRNAmiR or shRNAmiRs specific for CLCa, CLCb, or both viruses in combination. The lysates were processed for Western blot with antibody that recognizes all forms of CLCs. Hippocampal neurons transduced as in G were fixed at 21 days in vitro and processed for immunofluorescence with polyclonal antibody specific for the pre-synaptic protein synapsin and monoclonal antibody specific for the post-synaptic protein PSD95. The lentivirus drives expression of GFP. The areas indicated by white boxes in the low power images (squares) are shown enlarged immediately below. The scale bars = 10 µm for the low mag and 2.5 µm for the high mag.
Fig.Figure 1A-D The morphology of uninjected, FLT3/WT mRNA and FLT3/ITD mRNA injected (150 ng per embryo) embryos on day 2 post fertilization (dpf). Data information: ov: otic vesicles Scale bar = 500 μm. E-H Whole mount in situ hybridization (WISH) of notochord-specific marker col9a2 in uninjected, FLT3/WT mRNA and FLT3/ITD mRNA injected embryos on 2 dpf. Data information: ov: otic vesicles; cm: cephalic mesoderm; nc: notochord. Scale bar = 500 μm. FLT3 signaling was detected by Western blotting in 293FT cells-transfected with FLT3/ITD mRNA (I) FLT3 signaling was detected by Western blotting in zebrafish embryos-injected with FLT3/ITD mRNA (J). K The effect of FLT3 inhibitor quizartinib (Qui) on the dorsalization and axis-duplication phenotype-induced by FLT3/ITD mRNA injection in zebrafish. Data information: the experiments were performed in triplicates and the data are presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). NS, not significant. Quantification of fst expression by RT-qPCR (L) after FLT3/ITD overexpression in zebrafish embryos at 6 hpf. Data information: the experiments were performed in triplicates and the data are presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). NS, not significant. Quantification of fst expression by Western Blotting (M), after FLT3/ITD overexpression in zebrafish embryos at 6 hpf. Quantification of fst expression by WISH (N) after FLT3/ITD overexpression in zebrafish embryos at 6 hpf.