EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) Western blot analysis showed that shcircFNTA can decrease FNTA expression in UMUC3 cells (upper panel) and oecircFNTA can increase FNTA expression, and that this was more profound than using oelinearFNTA in J82 cells (lower panel). (B) A RNA pull-down assay was used to test the binding of circFNTA to 11 miRNAs (predicted by online database) in UMUC3 cells. The results showed that miR-328-5p, miR-370-3p and miR-920 can be pulled-down by circFNTA. (C) Relative miRNA expressions were quantified by qRT-PCR to test miRNA overexpression efficacy. Transwell invasion assay (D) confirmed that only oemiR-370-3p can reverse oecircFNTA induced invasion in J82 cells, MTT assays (E) confirmed that oemiR-370-3p can reverse oecircFNTA induced cisplatin chemo-resistance (2 μg/ml) in J82 cells. Scale bar, 100 μm. Transducing the miR-370-3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Western blot (F) Transducing the miR-370-3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Transwell assay (G). (H) MTT assays also confirmed that transducing with a miR-370-3p inhibitor in UMUC3 cells can partly reverse cisplatin chemo-resistance (2 μg/ml) reduction by shcircFNTA. (I) The structure of the circFNTA (WT and Mut) binding sites of miR-370-3p. Mut and WT circFNTA's were constructed based on the miRNA target sites. (J) The transfection efficacy of oecircFNTA WT and oecircFNTA Mut was tested by qRT-PCR. (K) Transfection with oecircFNTA WT could increase J82 cells invasion, while oecircFNTA Mut cannot. Scale bar, 100 μm. (L) Tramsfection with oecircFNTA WT could increase J82 cells cisplatin chemo-resistance (2 μg/ml), while oecircFNTA Mut did not have this effect. (M) The structures of target sites of miR370-3p at the FNTA 3'UTR. (N) Luciferase reporter activity after transfection of WT and Mut FNTA 3'UTR reporter constructs into UMUC3 cells (left panel), comparing transduced miR-370-3p inhibitor vs Ctrl and J82 cells (right panel), and comparing oemiR-370-3p vs control PLV.
Large conformational changes of the PHD identified by FRET.Figure 3(A) Design of a functional dynamin mutant to probe PHD stalk interactions. Minimal structure of the PHD and the PHD-Stalk interface showing the location of important residues discussed in this study. The unresolved loop L1NS in the stalk hosts the IAEDANS label and is sensitive to the two tryptophans (W525 and W542) in the PHD. The distance between two introduced cysteines (S357C and native C602) used to construct a double cysteine Dyn1 is also shown. (B) Comparison of emission spectra of various IAEDANS-labeled Dyn1 RCL variants (D352C, Y354C, S357C, I365C, H367C), upon Trp excitation at 295 nm. (C) FRET transfer upon excitation is contributed by two neighboring Trps (W525 and W542); Substitution by Phe resulted in significant loss of FRET. (D) IAEDANS emission kinetics upon addition of lipid nanotubes with or without PI4,5P2 (ex=295). The arrow indicates addition of lipids.
Figure 7A) Representative pictures of WT hippocampal neurons transfected with the SBP-mCherry-GluK2 RUSH construct, followed by treating neurons with biotin and anti-SBP antibodies (mouse, green) for 20 min or 40 min to live-label the GluK2 transported to the neuronal cell surface. As a control (no biotin), neurons were incubated only with SBP antibody for 40 min. The surface/total ratio of SBP-mCherry-GluK2 constructs was estimated by calculating the ratio of SBP (green)/ mCherry (red) fluorescence signals in the same area chosen for quantification. B) Quantification results for A, indicating that incubation with biotin increases the surface/total ratio of SBP-mCherry-GluK2 over time. No Biotin n=16, 20 min n=13, 40 min n=15, all biological replicates. Graph shows mean±SEM, 1-way ANOVA p<0.0001 and Dunnett's post hoc test, No Biotin vs 20 min *p=0.0030; No Biotin vs 40 min ***p<0.0001. C-D) SEZ6 flox/flox neurons were lentivirally infected with inactivated Cre (Y324F, Ctrl) or Cre recombinase (SEZ6KO), followed by transfection with the SBP-mCherry-GluK2 construct and treated with biotin and anti-SBP antibodies (mouse, green) for 20 min. Expression of inactivated Cre or Cre recombinase were confirmed by immunostaining with an anti-Cre antibody (rabbit, blue). SEZ6KO neurons showed a significantly reduced surface/total ratio of SBP-mCherry-GluK2 (ctl n=25, SEZ6KO n=23 biological replicates. Graph shows mean ± SEM, unpaired t-test, **p=0.0068).
Figure 3Competition assays with liposarcoma cell lines transduced with RFP-labeled NTC or YAP1 shRNAs. Flow cytometric quantification of RFP-positive cells on day 17 relative to day 3 showed that YAP1 knockdown was preferentially toxic to MLS cells. Error bars represent the mean ± SD of two independent experiments. Aggregate data from competition assays shown in (A). Statistical significance was assessed using an unpaired t-test. LS, non-myxoid liposarcoma. Competition assays with MLS 1765-92 cells transduced with an RFP‑labeled NTC shRNA or an RFP-labeled shRNA against the YAP1 3' UTR following transduction with EV or the YAP1 coding sequence. Flow cytometric quantification of RFP‑positive cells on day 17 relative to day 3 showed that cell viability was rescued by expression of the shRNA‑resistant YAP1 cDNA. Error bars represent the mean ± SD of two independent experiments. Expression of total and exogenous V5‑tagged YAP1 in MLS 1765‑92 cells shown in (C). One of at least two independent experiments with similar results is shown. Cell viability and expression of FOXM1 and PLK1 in MLS cell lines following siRNA-mediated YAP1 knockdown. Error bars represent the mean ± SD of three independent experiments, unpaired t-test. The blots represent one of at least three independent experiments with similar results. Flow cytometric cell cycle analysis of MLS cell lines following shRNA‑mediated YAP1 knockdown. Error bars represent the mean ± SD of three independent experiments, two-way ANOVA; ns, not significant. Senescence‑associated β‑galactosidase (SABG) staining intensity in MLS cell lines following shRNA-mediated YAP1 knockdown. Error bars represent the mean ± SD of ten random microscopic fields, two-way ANOVA). Expression of CDKN1A, CDKN2A, total and phosphorylated RB1, and TP53 in MLS cell lines following shRNA‑mediated YAP1 knockdown. One of at least two independent experiments with similar results is shown. Expression of cleaved PARP and cleaved caspase 3/8 in MLS cell lines following shRNA-mediated YAP1 knockdown. One of at least two independent experiments with similar results is shown.
Figure 4(A) Top: Scheme showing the experimental set up for laser irradiation in DDX5-GFP transfected U2OS cells depleted of BRCA2 (siBRCA2) or control cells (siC). Middle: Live cell imaging of the recruitment of GFP-53BP1 or DDX5-GFP to DNA damage tracks at different time points as indicated. Exposure and processing were adjusted to best demonstrate stripes and anti-stripes. Scale bar indicates 10 µm. Bottom left: Western blot showing the siRNA mediated knock-down of BRCA2 from U2OS cells transfected with DDX5-GFP. Bottom right: Quantification of the percentage of transfected cells that exhibit DDX5-GFP "anti-stripe" pattern (reduced GFP signal at DNA damage tracks compared to the signal in the nucleus) at the times indicated in cells depleted of BRCA2 (siBRCA2) or treated with control siRNA (siC). The data represent the mean ± SEM from three independent experiments. (B) DDX5 ChIP-qPCR signal values at RBMXL1 and ASXL1 loci in U2OS DIvA cells transfected with the indicated siRNAs and either untreated cells (-OHT) or after tamoxifen addition (+OHT). The data represent the mean ± SEM from three independent experiments. The green line represents the background levels of DDX5 signal. The statistical significance of the difference was calculated with unpaired one-tail t-test and the p-values show the significant differences between untreated cells (-OHT) and after tamoxifen addition (+OHT). (C) Top: Representative images of immunofluorescence of U2OS DIvA cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) and either untreated cells (-OHT) or after tamoxifen addition (+OHT), as indicated. Scale bar indicates 10 µm. Bottom: Quantification of the number of γH2AX foci per nucleus (left) and DDX5 nuclear intensity (right). The data represent at least 800 cells per condition from three independent experiments. The red line in the plot indicates the median and each symbol represents the value of a single cell. The statistical significance of the difference was calculated with Mann-Whitney U-test and the p-values show the significant difference.
Defects in brain and spinal cord in Epg5−/− mice.Figure 2.(A) Nissl staining of the fifth layer of the cortex in 10-mo-old Epg5+/− and Epg5−/− mice. (B) The number of pyramidal cells in the fifth layer per millimeters squared. (C and D) H&amp;amp;E staining of the hippocampus shows reduced pyramidal cell numbers in Epg5−/− mice. Means ± SEM of three mice are shown in B and D. (E) The normally developed cerebellum in Epg5−/− mice. (F) The thickness of the molecular layer in the cerebellum in mutant and control mice. (G and H) Purkinje cells, stained by calbindin, are not reduced in number in Epg5−/− mice. Means ± SEM of five mice are shown in F and H. (I) Arrows indicate Pyknotic neurons in the anterior horn of Epg5−/−mice. (J) The number of motor neurons in the spinal cord (SC) in Epg5+/− and Epg5−/−mice. (K) The numbers of Nissl-stained interneurons in the spinal cord are similar in control and Epg5−/− mice. Means ± SEM of three mice are shown in J and K. (L) Eosinophilic spheroids (arrows) accumulate in the thoracic spinal cord of Epg5−/− mice. (M) Eosinophilic spheroids are swollen degenerating axons (arrow). (N) EM pictures showing degenerated axons (arrows) in the spinal cord of Epg5−/− mice. (O and P) GFAP staining in Epg5+/− and Epg5−/−mice. DCST, dorsal corticospinal tract.
Figure 5(A) p47 requires its association with FTCD for its localization to the Golgi at the end of mitosis. HepG2 cells were treated with FTCD siRNA and then the indicated HA-tagged FTCDs were expressed as described in Figure 4C. The mitotic cells were collected, cultured for 35 mins, and then fixed. The cells were stained with polyclonal antibodies to p47 and HA and a monoclonal antibody to GM130, and observed by confocal microscopy. Panels a-l show the distribution of p47 or/and GM130 and panels m-o show HA-FTCD expression. Scale bar = 10 μm. (B) Insets in (A). p47 (red) and the GM130 (a Golgi marker, green) were visualized. Scale bar = 5 μm. (C) The results of quantification of (A). The results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiments. Asterisks indicate a significant difference at P<0.01 compared with the mock-treated cells (Bonferroni method). (D) p97 requires its association with FTCD for its localization to the Golgi at the end of mitosis. The cells were treated in the same way as (A) and stained with polyclonal antibodies to p97 and HA and a monoclonal antibody to GM130. Panels a-l show the distribution of p97 or/and GM130 and panels m-o show HA-FTCD expression. Scale bar = 10 μm. (E) Insets in (D). p97 (red) and the GM130 (a Golgi marker, green) were visualized. Scale bar = 5 μm. (F) The results of quantification of (D). The results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiments. Asterisks indicate a significant difference at P<0.01 compared with the mock-treated cells (Bonferroni method).
Figure 7Workflow of tamoxifen (TAM)-induced Ythdf1 deletion in AOM/DSS-induced Lgr5-creERT2:Ythdf1fl/fl mice. Colons from mice in (A) treated with or without TAM on day 80 of AOM/DSS induction. Colon tumor number from mice treated with or without TAM on day 80 of AOM/DSS induction. Data are shown as mean ± SEM (7 mice for each group). ****p<0. 0001 (t-test). Immunoblot analysis of 2 pairs of colon tumors from mice treated with or without TAM. Workflow of tamoxifen (TAM)-induced Ythdf1 deletion in Apcmin/+ mice. Kaplan-Meier survival plots for Apcmin/+; Lgr5-creERT2:Ythdf1fl/fl mice (8 mice for each group) treated with or without TAM. Log-rank (Mantel Cox) test. **p<0. 01 (t-test). A proposed model of amplified β-catenin activity by m6A-YTHDF1-mediated translation.
Analysis of striatal tissue from ALN reprogrammed dCAM mice by single cell RNA-seq.Figure 5A, Scheme depicting experimental preparation of cells of 13 wpi mice striatal regions (n=2, one technical replicate). Papain dissociated cells are prepared for scRNA-seq using droplet-based separation and barcoding. Uniform Manifold Approximation and Projection (UMAP) visualization of QC-selected cells for GFP and ALN (n=3,899). Color labeling highlights nine main cell groups based on Leiden clustering and identification based on marker genes. Rectangle highlights astrocytic and neuronal cell clusters. 4,273 highly variable genes (HVG) were detected. B, Subclustering of 1,110 cells identified four groups of astrocytic and neuronal identity. Layout is based on UMAP visualization presented in a. Clustering of markers genes selected based on expression levels between clusters. Expression Z-scores are hierarchically clustered by rows. C, GFP control and ALN reprogrammed cells selected from the neuronal and astrocytic clusters are visualized based on detection of GFP (red cells), marker gene Ascl1, Myt1l and Gad1/Gad2 (Gad1/2) (blue cells), and the co-detection of both (yellow cells). Numbers of GFP positive cells (red), marker gene positive cells (blue) and double positive cells (yellow) are indicated for the astrocytic and neuronal clusters respectively.
Fig.Figure 6A. Schematic representation of the strategy to induce systemic inflammatory response syndrome (SIRS) via intravenous TNF-α injection. B. Survival graph after intravenous TNF-α injection of Casp8WT vs. Casp8ECko mice within the first 24h (n=11 WT, 11 ECko; Log-Rank test). C. Representative images of the small intestines 24h after TNF-α injection in Casp8WT and Casp8ECko mice (blue arrowheads point to Peyer's Patches). D-E. Representative pictures of Peyer's Patches (D) and quantification of lymphatic vessel density (E) in Casp8WT, Casp8ECko and Casp8ECko/MLKLko mice injected with TNF-α (n=7 WT, 5 ECko, 15 ECko/MLKLko. Peyer's Patches are pooled from 3-5 mice per genotype). Scale bars: 200µm. Data information: All data is shown as mean ± SEM. * P<0.05, *** P <0.001, **** P <0.0001; ns: not significant. F. Representative pictures of TUNEL staining together with CD31 and Lyve-1 in Peyer's Patches of Casp8WT, Casp8ECko and Casp8ECko/MLKLko mice injected with TNF-α. TUNEL+ LECs, but not BECs could already be seen in Casp8ECko, but neither in Casp8WT nor in Casp8ECko/MLKLko mice. White dotted areas outline location of the lymphatic plexus. Yellow arrowheads point to TUNEL+ECs). Scale bars: 50µm. G. Schematic representation of the Enbrel® treatment protocol to inhibit TNF-α signaling. H. Survival graph of Casp8WT and Casp8ECko mice after Enbrel® treatment (n=7 WT, 5 ECko + IgG, 8 ECko + Enbrel; Log-Rank test). Representative images of small intestines (I) of Casp8ECko mice after Enbrel® treatment Scale bars: 100µm. Representative images of H&E staining (J) with histopathological scoring (K) of Casp8WT and Casp8ECko mice after Enbrel® treatment (n=6 WT, 4 ECko + IgG; 8 ECko + Enbrel (from which 3 mice were diseased, 5 mice healthy); one-way ANOVA with tukey's multiple comparison). Scale bars: 100µm. Data information: All data is shown as mean ± SEM. * P<0.05, *** P <0.001, **** P <0.0001; ns: not significant.
Figure 9A. Deconvolved fluorescence micrographs of NLS-GFP in the indicated yeast strains at either 30°C or after 2 h at 37°C. Scale bar is 5 μm.B. Plot of the mean nuclear to cytosolic fluorescence intensity ratio of NLS-GFP from cells represented in A. Error bars are SD from the mean from 3 independent replicates of 150 cells from each strain. p values from un-paired student's T-test where ns is p > 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001. Only chm7Δ and chm7Δapq12Δ cells have N:C ratios below 1.2 (dotted red line).C. Re-plotting of data from B showing the percentage of cells with a N:C NLS-GFP fluorescence ratio <1.2. Error bars are the SD from the mean from 3 independent replicates of 150 cells/strain. p values from student's T-test where *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001. D. Model of a proposed stepwise recruitment and activation of Chm7 and other ESCRTs at defective NPCs (bottom, red), or NE holes.
Correlative light-electron microscopy analysis of GFP-LC3 puncta demonstrates they are AVs.Figure 4.Correlative light-electron microscopy analysis of GFP-LC3 puncta demonstrates they are AVs. (A) Phase, (B) fluorescent, and (C) TEM of the same field of 293/GFP-LC3 cells 48 h after siRNA depletion of α-COP. In B the image was inverted to allow better visualization of the GFP-LC3-positive structures. (D) Higher magnification of cell shown with asterisk in A and C. Top region of cell of interest is enlarged in E to show the two AVs structures indicated by arrows in B. (F and G) Higher magnification of AVs shown by arrows in E.
Figure 3Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle or with the indicated concentrations of AEA (***P<0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc). Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, the inhibitor of NSM GW, AEA or the combination of GW and AEA (***P <0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc). Western blot against NSM and GAPDH (used as loading control) and graph showing mean ± SEM NSM protein levels in extracts from cultured neurons treated with vehicle or with 50μM AEA (*P = 0.0230, n = 6 independent cultures, Student's t-test). Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, or with JNJ, PF or URB in the presence or absence of AEA (**PJNJ = 0.0013, *PPF = 0.0143, ***PURB = 0.0002, **PJNJ+AEA = 0.0069, **PPF+AEA = 0.0077, ***PURB+AEA <0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc). Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, with PF or with SR141716 + PF (*PPF = 0.0109, *PSR+PF = 0.0343, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc). Immunofluorescences against the dendritic marker MAP2 of cultured neurons from ASM-KO mice treated with vehicle, AEA, JNJ, PF or URB. Scale bar, 50 μm. Mean ± SEM number of apoptotic cells measured by TUNEL assays in cultured neurons from ASM-KO mice treated with H2O2 (as positive control), vehicle, AEA, JNJ, PF or URB (n = 3 independent cultures).
Differences in temporal regulation of the proteome and phosphoproteome between WT and CRY-deficient cells.Figure 2A) Fold-change in relative amplitude (RA) was calculated for each of the proteins found to be rhythmic in both genotypes by RAIN analysis (i.e. no RA cut-off). The mean fold change (log) in RA was an increase of 14% in CKO cells compared to WT cells (One sample t-test, ****p<0.0001). B) For each genotype, all proteins were divided into 10 deciles of equal number, ranked by abundance. The mean abundance of each decile was plotted against the proportion of the decile that was rhythmic. Linear regression lines are shown for each genotype, and the slopes were significantly non-zero (F test, WT **p=0.0016, CKO ****p<0.0001). The slopes were also significantly different to each other (F test, p=0.0001). C) Fold-change in relative amplitude (RA) was calculated for each of the phosphopeptides found to be rhythmic in both genotypes by RAIN. On average, the RA was increased in CKO cells compared to WT cells by 6% (One sample t-test, *p<0.05). D) The same analysis in B) was carried out for phosphopeptides. The slopes were significantly non-zero in CKO but not WT (F test, WT p=0.3 (ns.: non significant), CKO *p=0.04). The slopes were also significantly different to each other (F test, p<0.0001). E-H) Circular histograms showing the number of proteins at each rhythmic phase. Phase is defined and estimated by RAIN, as the time of the first predicted peak in a 24-hour period. Concentric circles represent the counts scale, with the outermost circle marking the upper end of the counts. Black arrow indicates the phase of CRY1. E-F) The distributions of rhythmic proteins in CKO cells were significantly different to WT cells (***p<0.001, Watson's two-sample test). G-H) This was also the case for the proteins rhythmic in only one genotype (***p<0.001, Watson's two-sample test). I-J) Circular histograms showing the number of phosphopeptides at each rhythmic phase. The distributions of rhythmic phosphopeptides in CKO cells was significantly different to WT cells (***p<0.001, Watson's two-sample test).
Figure 7(A) Murine Krt86 is shown schematically with exons as rectangles and the transcription start site marked by an arrow. Sites assayed in the pre-mRNA or ChIP studies of panels B-G are indicated. Graphs present pre-mRNA (B, C) assays of Krt86 using skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P6 (B, C) All values are derived from 3 independent experiments and presented as mean ± S.D. Pre-mRNA measurements were analyzed statistically using two-way ANOVA with post-hoc Sidak's multiple comparison test at a 95% confidence interval. Graphs present ChIP (D-G) assays of Krt86 using skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P7 (D-G). ChIP assays were performed with antibodies to FOXN1 or AFF4 as indicated. All values are derived from 3 independent experiments and presented as mean ± S.D. ChIP assays were analyzed statistically using one-way ANOVA with post-hoc Tukey's multiple comparison test at a 95% confidence interval. ****, p ≤ 0.0001.
Figure 5A. PGAM1 was immunoprecipitated from HepG2, HAT1 KO Clone 1 HepG2, and HAT1 KO Clone 1 HepG2 cells reconstituted with either wildtype HAT1 or mutant HAT1 (T188A). The levels of succinylation, PGAM1, HAT1, and β-actin were analyzed by Western blot analysis in the cells. B. Relative PGAM1 activity was measured by PGAM1 activity assays in the indicated tumor cells. N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01. C. The decrease of 2-PG and increase of 3-PG were measured by ELISA assays in HepG2, HAT1 KO HepG2, and HAT1 KO HepG2 cells reconstituted with either wildtype HAT1 or mutant HAT1 (T188A). N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01. D. HAT1-catalyzed PGAM1 succinylation was analyzed by mixing purified HAT1, PGAM1, and succinyl-CoA/acetyl-CoA in the in vitro succinylation assays. Western blot analysis was performed with indicated antibodies. E. Purified HAT1, PGAM1 or PGAM1 mutant, and succinyl-CoA were mixed in the in vitro succinylation assays and relative PGAM1 activity was measured by PGAM1 activity assays in vitro. N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01. F. The decrease of glucose and increase of lactate in the culture medium and the relative amounts of 2-PG and 3-PG were analyzed by ELISA assays in HepG2 cells depleted endogenous PGAM1 and reconstituted expression of PGAM1 or PGAM1 mutant (K99R). N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01.
Figure 3Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread pachytene-like (Pac.-like) and zygotene-like (Zyg.-like) spermatocytes of different genotypes. Unsynapsed axial elements of sex chromosomes (X,Y) are indicated. Arrowheads indicate nucleolar-associated round bodies. Scale bar: 10 µm.