EMBO/sd-character-level-ner huggingface dataset:
miR-27a and miR-24 directly target critical pluripotency associated factors and are restrained by c-Myc in ESCs.Figure 4Computer prediction of the binding of miR-27a and miR-24 on the 3′ UTR or CDS of these pluripotency-associated genes. Western blot analysis of the expression of target genes in miR-27a or miR-24 over-expressed V6.5 ESCs. Candidate target genes whose expression was decreased were framed. The relative luciferase activity of the reporter constructs co-transfected with scramble or miR-27a and miR-24 mimics. Error bars indicate s.d. (n=3); * indicates P-value < 0.05. Candidate target genes whose luciferase activity was decreased were framed. PC group was positive reporter control which harbored reverse complementary sequence of miR-27a or miR-24. The relative luciferase activity of the wild-type 3′ UTR or mutated 3′ UTR reporter co-transfected with scramble or miR-27a and miR-24 mimics. Data are shown as mean + s.d. (n=3); * indicates P-value < 0.05. The schema of Ago2 immunoprecipitation. F,G RT-PCR and qRT-PCR analysis of in vivo binding of the miRNAs with their targets. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001. H. Western blot analysis of c-Myc expression in siRNA treated V6.5 ESCs. I. Q-PCR analysis of primary-23a~27a~24-2, miR-23a, miR-27a and miR-24 expression in c-Myc knocked down ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001. J. The five putative c-Myc binding sites scattered within the promoter region of mouse miR-23a~27a~24-2 cluster. K. ChIP-PCR and ChIP-q-PCR analysis of c-Myc binding to the putative sites in the promoter. Error bars indicate s.d. (n=3); *** indicates P-value < 0.001. L. Relative luciferase activity of the wild-type and -15 binding site mutated promoter in NIH3T3 cells co-transfected with c-Myc or the blank vector (NC). Error bars indicate s.d. (n=3); * indicates P-value < 0.05. M. Relative luciferase activity of wild-type and -15 binding site mutated promoter in V6.5 ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05.
Figure 2A. Contrast-enhanced magnetic resonance imaging (MRI) of the liver from representative Tie2-GFP mice (red frame) or Tie2-IFNα (blue frame) that were intrasplenically injected with 5x103 CT26. Red arrows identify CRC liver metastases of representative z-sections. Tumors were characterized as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences, respectively. Each panel refers to a single mouse analyzed at different time points; n.a.=not assessed, refers to a mouse euthanized before the specified time point; scale bars=5mm.B. Percentage of mice bearing at least one CRC liver metastasis estimated by MRI analysis. Mice were treated as described in A. Mock/Tie2-GFP n=11, Tie2-IFNα n=9; the oblique black line pattern within the red columns depicts the percentage of mice euthanized or that died before the indicated time point; p-values were calculated by Fisher's exact test.C. Tumor volume quantification measured by MRI analysis of the same mice described in B. Each symbol corresponds to the same mouse analyzed at different time points; horizontal bars=mean values; note that Mock/Tie2-GFP mice were euthanized or died before day 54; p-values were calculated by Mann-Whitney test. D, E. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible genes Oas1 (D) and Irf7 (E) within brain (n=4, n=2), kidney (n=4, n=3) and metastatic liver (n=4, n=7) of Tie2-GFP or Tie2-IFNα mice euthanized 25 days post-intrasplenic injection of 5x103 CT26. The basal expression of Oas1 and Irf7 estimated in brain of control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values depicted. Mean values are shown; error bars=S.E.M.; p-value was calculated by Mann-Whitney test. F. Eighty-four or 54 days post-NaCl or 5x103 CT26 intrasplenic injection, Sham/CTRL (n=5) or Tie2-IFNα mice (n=7) were infected with LCMV (Armstrong strain, 200 pfu intraperitoneally injected). Eight days post-infection white blood cells were isolated and analyzed for LCMV specific CD8+ T cell response. Left panel: percentage of total CD8+ T cells; middle panel: percentage of CD8+/NP118+ T cells (NP118=recombinant dimeric H-2d/Ig fusion protein complexed with the immune-dominant H-2d-restricted LCMV NP118-126 peptide); right panel: percentage of CD8+/IFNγ+ T cells after in vitro stimulation with virus-specific H-2d-restricted peptide (NP118-126); data pooled from two independent experiments; mean values are shown; error bars=S.E.M.; differences were not statistically significant (by unpaired Student's t-Test). G. Kaplan-Meier survival curves of the indicated groups of mice described in A. Sham (n=3), Mock(wt)/Tie2-GFP (n=11); Tie2-IFNα (n=9); data pooled from three independent experiments; p=0.001 by log-rank/Mantel-Cox test. The inset images show representative macroscopic photographs of the metastatic progression in the liver of a control animal (red frame: Mock/Tie2-GFP, day 35 post-CT26 injection) opposed to the lack of lesions in Tie2-IFNα treated mice at later time points (blue frame: day 450 post-CT26 injection).
Figure 5(A) Treatment scheme and adjunctive therapy in the survival experiment. All animals received meropenem (25 mg kg-1 body weight, s.c.) twice daily and metamizole (2.5 mg per animal, p.o, every 6 h).
Figure 2JNK signaling defines epigenome and transcriptome to specify mesenchymal fate. (A) Volcano plots showing significantly differentially expressed genes upon treatment with the JNK inhibitor SP600125 (JNKi) as compared to DMSO at day 1, 2, 3, 4 and 7 during TGF-β-induced EMT in NMuMG cells. X-axis indicates fold change in log2 scale while y-axis denotes -log 10 of the P-value. Differentially expressed genes are depicted with red circles. Blue horizontal line represents FDR adjusted Pvalue of 0.1. (B) Venn diagram showing the overlap of upregulated genes in JNKi versus DMSO treated cells. (C) Same as in (B) for genes downregulated upon JNKi. (D) A principal component analysis (PCA) using RNA-Seq data was performed comparing two replicates (r1, r2) each of DMSO and JNKi treated samples at different time points during TGF-β-induced EMT (d1, d2, d3, d4, d7). The first two principal components are presented to display the variation between samples. The distances on the x- and y-axis explain 47% and 19% of variation in the data respectively. Biological duplicates (r1 and r2) are depicted in the same color. . (E) ChIP assay using H3K27me3-specific antibody in epithelial cells (day 0)) or NMuMG cells treated for seven days with TGF-β (mesenchymal) and DMSO or JNKi. qRT-PCRs were performed for indicated gene promoters and enrichments are plotted on the right y-axis as ratio of precipitated DNA (bound) to total input DNA. On the left y-axis RNA levels of analyzed genes derived from RNA-seq data (fold change to untreated normalized tag counts ) are plotted. Mean and SEM is plotted from two independent biological replicates. (F) FAIRE assay was performed in cells treated as in (D) and qRT-PCRs were performed for the same gene promoters and plotted as in (E).
Figure 3A, B. Control or OMA1 KO HCT116 cells expressing control (empty vector) or OMA1-Flag were maintained in normoxia or hypoxia (1% O2) for 24 hours. The glucose uptake (A) and lactate production (B) were measured by using microplate reader, and the values were normalized to the protein concentration. Error bars indicate the mean ± SD of three independent experiments, statistical significance was assessed by a two-way ANOVA, *P < 0.05. Cells were lysed and assessed by western blot with antibodies against OMA1 and GAPDH (A). The asterisk indicates a nonspecific band. C. Control and OMA1 KO HCT116 cells were treated with DMSO or 1mM DMOG in fresh medium for 12 h. Subsequent injections of glucose, oligomycin (Oligo), and 2-deoxy-glucose (2-DG) were performed as indicated. The extracellular acidification rates (ECAR) were measured with a Seahorse Extracellular Flux Analyzer XF96. The data are presented as mean ± SD (n = 3 independent experiments). D. Quantitative analysis of basal and maximal glycolytic capacity. Error bars are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a two-way ANOVA. N.S., not significant, ***p < 0.001. E, F. Lactate production (E) and ATP production (F) were measured in control or OPA1 KO HCT116 cells upon DMSO or 2-DG treatment. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). N.S., not significant, **p < 0.01, ***p < 0.001. G, H. Control or OPA1 KO HCT116 cells were maintained in normoxia or hypoxia (1% O2) for 24 hours. The glucose uptake (G) and lactate production (H) were measured, and the values were normalized to the protein concentration. Statistical significance was assessed by a two-way ANOVA; error bars are presented as mean ± SD of three independent experiments; N.S., not significant, *p < 0.05, **p < 0.01.
a, LUMIER binding assay: normalized ratio between luciferase activity bound to beads and present in lysates.f2a, LUMIER binding assay: normalized ratio between luciferase activity bound to beads and present in lysates. Lysates of 293ET cells expressing NDP52, p62 or optineurin each fused to luciferase, and the indicated Flag-tagged galectins were incubated with anti-Flag beads. Flag-tagged proteins are further characterized in Supplementary Fig. 4a b, Lysates of 293ET cells, expressing Flag-tagged proteins as indicated, were immunoprecipitated with anti-Flag beads. Lysates and immunoprecipitates (IP) were blotted for the presence of Flag-tagged proteins and endogenous NDP52. c, Confocal images of HeLa cells infected with S. Typhimurium for 1 h and stained with antisera against NDP52 and galectin 8. Arrowheads, bacteria shown in insets. d, Co-localization of NDP52 with galectin-positive bacteria in HeLa cells stably expressing YFP fused to the indicated galectins, infected with S. Typhimurium and stained with NDP52 antiserum 1 h after infection. Mean and s.d. of duplicate HeLa cultures, n > 100 bacteria per coverslip, representative of two independent experiments. Scale bar, 10 µm.
Figure 6(A) HepG2 cells were transfected with control or ING5, or treated with siRNAs against JFK. Soluble chromatin was prepared and qChIP analysis was performed on the selected promoters using the indicated antibodies. Histone H3 was detected as an internal control. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test). (B) HepG2 cells were treated with control siRNAs or siRNAs against JFK or/and ING5, or transfected with vector or JFK or/and ING5. Total RNAs were extracted and qPCR analysis was performed. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test). (C) HepG2 cells were treated with control siRNAs or siRNAs against JFK or/and ING5, or transfected with vector, JFK, or/and ING5. Cellular extracts were prepared for western blotting analysis with the indicated antibodies. (D) HepG2 cells were transfected with vector or JFK, AMPKα1, or/and ING5, or treated with control siRNAs or siRNAs against JFK, AMPKα1, or/and ING5, Cellular extracts were prepared for AMPK activity assays by ELISA. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test). (E) HepG2 cells were transfected with vector, JFK, or/and ING5, or treated with control siRNAs or siRNAs against JFK, AMPKα1, or/and ING5. Mitochondria were isolated and the rate of fatty acid β-oxidation was measured by spectrophotometry of ferricyanide-trapped reduced products. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test). (F) HepG2 cells were transfected with vector, JFK, or/and ING5, or treated with control siRNAs or siRNAs against JFK, AMPKα1, or/and ING5. Culture media was collected and the concentration of 3-hydroxybutyric acid and acetoacetic acid was measured by ketone body assay kit. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test).
Figure 2qRT-PCR analysis of CYP46A1 mRNA levels in LN229 and GBM#P3 cells transfected with lentivirus expressing CYP46A1 (lenti-CYP46A1) or control sequence (lenti-Ctrl). GAPDH was used as an internal control. Shown are means and SEM (n = 3). LN229: ***P < 0.0001; GBM#P3: ***P < 0.0001. Statistical significance was determined by two-sided Student's t‐test. Western blot analysis to confirm CYP46A1 overexpression in GBM cells. Growth curves for GBM cells in vitro infected with lenti-Ctrl or lenti-CYP46A1 derived from trypan blue staining. Shown are means and SEM (n = 3). LN229: **P = 0.003; GBM#P3: **P = 0.002. Statistical significance was determined by two-sided Student's t‐test. Colony forming assay for GBM cells infected with lenti-Ctrl or lenti-CYP46A1. Shown are means and SEM (n = 3). LN229: *P = 0.013; GBM#P3: *P = 0.012. Statistical significance was determined by two-sided Student's t‐test. Representative H&E staining of orthotopic tumours derived from GBM cells infected with lenti-Ctrl or lenti-CYP46A1. Scale bar = 2 mm. Kaplan-Meier survival curve of tumour bearing mice injected with GBM cells infected with lenti-Ctrl or lenti-CYP46A1 (n = 5 per group). A log-rank test was used to assess statistical significance. IHC for CYP46A1, PCNA and cleaved caspase-3 protein levels in the intracranial tumours. Scale bar = 100 μm.
Figure 4A-D Normalized Synaptophysin-pHluorin responses from neurons treated with VPS34IN1 (10 µM for 1h prior to imaging) and stimulated with four consecutive trains of 50 APs (10 Hz, 5 s) in A or 200 APs (40 Hz, 5 s) in C. PI(3) P depletion in VPS34IN1-treated neurons causes the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface in neurons stimulated with consecutive trains of 50 APs in B or 200 APs in D. Surface retention of Synaptophysin-pHluorin 90 s post-stimulus is plotted for each of the four successive stimulation trains. N ≥ 4 independent experiments; Two-way ANOVA; Sidak's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 E Cultured hippocampal neurons pre-treated with DMSO (0.1%) or VPS34IN1 (10 µM, 1 h) were either field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-), fixed and stained for surface-stranded Synaptotagmin-1, total Synaptotagmin-1, and vGlut1. Scale bar, 10 µm. F Fraction of endogenous Synaptotagmin-1 stranded on the surface of presynaptic nerve terminals from the images shown in E. N = 3 independent experiments (≥ 40 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 G Surface retention of Synaptophysin-pHluorin 90 s post-stimulus is plotted for each of the four successive 200 AP stimulation trains in neurons treated with SAR405 (20 µM). Data are similar to those shown in C and D. N = 3 independent experiments (≥ 11 images per condition); Two-way ANOVA; Sidak's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 H Exogenous supply of membrane-permeant PI(3) P acutely rescues defective Synaptophysin-pHluorin endocytosis induced by pharmacological inhibition of VPS34-mediated PI(3) P synthesis in hippocampal neurons. Data are similar to those shown in C and D; where indicated exogenous PI(3) P-AM (20 µM) was supplied in parallel to VPS34IN1 (10 µM) for 1 h prior to imaging. N = 3 independent experiments (15 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 I Kinetic block of Synaptophysin-pHluorin endocytosis at hippocampal synapses depleted of VPS34. Hippocampal neurons depleted of endogenous VPS34 by specific shRNA (shVPS34) or treated with non-targeting control shRNA (shCtrl) and stimulated as in C and analyzed as in D. N =3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Sidak's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001;
Figure 4A. Dot plot showing the expression levels of the top variable genes between myoblasts across 7 clusters of the reference cell atlas dataset. Confocal single plane images of third instar larval wing discs stained with anti-Ct (red) and (B) E(spl) m6-BFM-gapGFP (green), (C) E(spl) m7-HLH[VK00037]-GFP (green) Confocal single plane images of third instar larval wing discs stained with (D) E(spl) m3-HLH[GMR10E12]>GFP (green). E-G. ﻿Slingshot used for inferring cell lineages and pseudotime among the IFM myoblasts. (E) Two lineages for IFM myoblast clusters visualized in subset of 3,492 cells in UMAP plot of the reference single cell atlas dataset. (F) Pseudotime inference in Lineage 1 is from IFM_1 → IFM_2 →IFM_3→IFM_4. (G) Pseudotime inference in Lineage 2 is from IFM_1 → IFM_2 →IFM_3→IFM_5. H-I. Confocal single plane images of third instar larval wing discs stained with anti-Ct (cyan) showing the lineage of GAL4 (green) and the active GAL4 (red) in (H) E(spl) m3-HLH[GMR10E12]>gTRACE and (I) E(spl) m6-BFM-GAL4>gTRACE.
Figure 6The expression levels of the indicated proteins were detected by immunoblotting analysis in CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective controls of TNBC cells. PP2A activity was measured using a protein phosphatase assay kit. PP2A activity assay was performed to evaluate PP2A activity in CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective control TNBC cells. Immunohistochemistry (IHC) staining of p-AKT from TNBC primary tumor (n = 20) with high or low micropeptide CIP2A-BP expression. Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT. MDA-MB-231 cells were transfected with anti-CIP2A siRNAs. Whole-cell lysates of these cells were subjected to immunoblot analysis with CIP2A, CIP2A-BP, PP2Ac, p-AKT, AKT and β-actin antibodies. CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective control MDA-MB-231 cells were transfected with anti-CIP2A siRNAs. PP2A activity assay was performed to evaluate PP2A activity in the indicated cells. The indicated cells were pretreated with specific antagonist against AKT (MK-2206，3μM) for 1h. Then whole lysates of these cells were subjected to immunoblot analysis.
Figure 2A. Percentage of tumor-infiltrating T cells, Treg and NK cells were identified by flow cytometry from CT26 tumors as indicated. Each spot represents one mouse. * P < 0.05; * * P < 0.01 by Student's t-tests. B. Representative images of CD8 by IHC staining. Tissue sections from BALB/c mice bearing the indicated knockout of genes with treatment of PD1 antibody. Scale bars, 50 μm. C. Percentage of Granzyme B-expressing CD8+ T cells from control and Mettl3 or Mettl14 deficient CT26 tumors. Each spot represents one mouse. * P < 0.05; * * P < 0.01 by Student's t-tests. Mice bearing control and Mettl3 or Mettl14 null tumors were treated with CD8-depleting antibody and PD-1 antibody or PD-1 as indicated. Tumor volume was measured over time points. n, the numbers of mice. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests. Mice bearing control and Mettl3 or Mettl14 null tumors were treated with CD8-depleting antibody and PD-1 antibody or PD-1/GVAX as indicated. Tumor volume was measured over time points. n, the numbers of mice. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests. IFNγ production in serum (F) from BALB/c mice by ELISA. The results are representatives of at least three independent experiments. n, the numbers of mice. Data are mean ± s.e.m. * P < 0.05 by Student's t-tests. IFNγ production in Intratumor (G) from BALB/c mice by ELISA. The results are representatives of at least three independent experiments. n, the numbers of mice. Data are mean ± s.e.m. * P < 0.05 by Student's t-tests.
Figure 6The graphs represent the z-scores changes of each CSF biomarker as a function of CSF Aβ42/40 ratio (as proxy of disease progression) using a robust local weighted regression method. The z-scores were calculated using the mean and the SD of each CSF biomarker in the A-T- group as a reference. The solid lines depict the trajectory of each CSF biomarker. The dashed lines depict the trajectories of the plasma biomarkers. The vertical black dashed line indicates the CSF Aβ42/40 cutoff for A+. Note that the CSF p-tau biomarkers reach the 2 z-scores (depicted with an horizontal dashed line) with the following sequence: Mid-p-tau231, N-p-tau181, N-p-tau217, Mid-p-tau181 and t-tau. Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; NfL, Neurofilament light; N, N-terminal; p-tau, phosphorylated tau; t-tau, total tau.
Figure 1Isodensity meshes illustrate the K+ distribution (contoured at 3.5 σ) around EAAT1 and GltPh monomers in the OFC in side view. Middle insets show close-ups of the conserved K1-3 sites in GltPh and the K4 site in EAAT1 (left, coordinating EAAT1 residues; right, GltPh homologues). Lower right inset, comparison of the K1-K3 sites (isodensity meshes) with the Na+-binding sites Na1-Na3 (spheres) (Boudker et al., 2007; Guskov et al., 2016). See also Appendix Figure S1-S3.
a, RAW cells expressing GFP–LC3 were transfected by electroporation with control or Atg5 siRNA oligonucleotides.figf3a, RAW cells expressing GFP-LC3 were transfected by electroporation with control or Atg5 siRNA oligonucleotides. At 24 h after transfection, cells were fed with zymosan (yellow arrows) and the internalization process was followed by time-lapse video microscopy. Images obtained at 5 and 30 min after zymosan engulfment are shown. b, The percentage of GFP-LC3-associated phagosomes (n ≥ 100 per /group) was obtained from three independent time-lapse videos (2 h each) of macrophages transfected with control or Atg5 siRNA (means and ranges are shown). e, The percentage of phagosomes (n ≥ 50 per group) with GFP-LC3 in wild-type and Tlr2-/- macrophages. Means and ranges are shown (4 mice per group were used). e, Ly294002 (Ly29, 10 µM) was added 10 min before zymosan, and wortmannin was added after engulfment but before GFP-LC3 translocation in RAW-GFP-LC3 cells. Representative images are shown. f, Percentage of GFP-LC3-associated phagosomes (n ≥ 50 per group) with P(I) 3K inhibitors assessed by time-lapse video (means and ranges are shown; three independent 3 h videos). g, RAW cells expressing GFP-LC3 and the PX domain of p40(phox) fused to mCherry (red) were fed with zymosan (yellow arrows) or with beads (Supplementary Fig. 6). Internalization was followed at 1-min intervals (Supplementary Movie 5), representative images are shown. Single cells were followed for at least 2 h (n ≥ 20 per group) (Supplementary Movies 6, 7 and Supplementary Fig. 6). Mean ± s.d. duration of zymosan-PX translocation to the zymosan for beads (B) and zymosan (Z) are shown. h, Internalization of zymosan (yellow arrows) was followed at 1.5-min intervals for at least 2 h in RAW cells transiently transfected with GFP-BECN1. Representative images are shown. Translocation of GFP-BECN1 to the phagosome for beads (B) and zymosan (Z) was quantified (mean ± s.d.) using time-lapse movies (Supplementary Movie 8 and 9; n ≥ 20 cells per group). For g and h, time (in minutes) is indicated at the top left of each panel.