EMBO/sd-character-level-ner huggingface dataset:
Figure 5A Absorbance of each RNA substrate at 260nm monitored between 25°C and 95°C, recorded at 1°C intervals; n=3 UV melting curves per RNA substrate (technical replicates), mean ± SD. B The m-fold predicted folding free energy (ΔG) of hairpin structures of pure CAG and CGG interrupted repeat tracts with different configurations. Filled symbols show sequences also used for UV melting analyses. Grey dotted line indicates ΔG of pure (CAG)8. C The folding free energy (ΔG) of interrupted hairpin structures of repeat expansions found in SCA8 patients (Figure 1I) and pure repeat tracts of the same length, as predicted by m-fold. Patient-derived alleles are as follows: Sporadic 5 - 48 repeats in length - (CAG)7(CGGCAG)18(CAG)5; Family 7 A-1 - 53 repeats in length - (CAG)8(CGGCAG)14(CAG)2CGG(CAG)5CGG(CAG)8; and Family 7 A-2 - 52 repeats in length - (CAG)7(CGGCAG)16(CAG)4CGG(CAG)8. Each symbol represents a single predicted hairpin structure; multiple hairpin structures, including branched hairpins, are predicted for each SCA8 patient allele and for pure (CAG)53 Black dotted line shows ΔG=0.
Involvement of homologous recombinations in repairing DNA DSBs.Figure 4RAD51 foci in XRCC4wt and XRCC4m/m fibroblasts irradiated with 0.5 Gy of γ-rays. Values are the mean ± SD of three independent experiments. ***P < 0.001; **P < 0.01 (XRCC4m/m versus XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.Immunofluorescence micrographs of RAD51 foci in XRCC4wt and XRCC4m/mfibroblasts. Arrowheads indicate the CENP-F-positive cells.
mATG9 and ATG16L1 Meet in Recycling Endosomes.Figure 3(A and B) (A) HeLa cells were incubated for 4 hr at 37°C or 18°C and processed for LC3II western blot. Some cells were incubated overnight with 20 mM NH4Cl in full medium (to block lysosomal degradation) at 37°C or 18°C (B). (C) HeLa cells were transfected with pEGFP-LC3 for 20 hr and incubated for 4 hr at 37°C or 18°C. (D and E) (D) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr and incubated for 4 hr at 37°C or 18°C. The size and the number (E) of vesicles were scored (a minimum of 20 cells were examined for each condition). Error bar, SEM. ∗∗∗p < 0.001 and ∗∗p < 0.01. (D and E) (D) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr and incubated for 4 hr at 37°C or 18°C. The size and the number (E) of vesicles were scored (a minimum of 20 cells were examined for each condition). Error bar, SEM. ∗∗∗p < 0.001 and ∗∗p < 0.01. (F and G) (F) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr, incubated for 4 hr at 37°C or 18°C, and labeled with EEA1 or RAB11 to visualize early endosomes or recycling endosomes. Histogram in (G) shows the amount of colocalization of ATG16L1 in EEA1 or in RAB11 compartment (Manders' coefficient). Error bar, SEM. NS, not significant; ∗∗p < 0.01. (H) HeLa cells were transfected with pmStrawberry-ATG16L1 for 20 hr, fixed, and labeled for mATG9. The histograms show quantification of colocalization and the size of the vesicles labeled with ATG16L1 and mATG9 at 37°C or 18°C. Error bar, SEM. ∗∗∗p < 0.001 and ∗∗p < 0.01. (I) HeLa cells were transfected with pEGFP-RAB11 or pEGFP-Myosin Vb tail (MVb) and incubated during the last 16 hr with Bafilomycin A1 (Baf) or DMSO and processed for LC3-II western blot. Control cells were transfected with pEGFP empty vector. (J) HeLa cells were transfected with ATG16L1-mStrawberry and RAB11-EGFP or MVb tail-EGFP. The size of ATG16L1 vesicles was scored in RAB11 and MVb tail overexpression conditions. The inserts show just the ATG16L1 vesicles as quantified in histogram. Error bar, SEM. ∗∗∗p<0.001. (K) HeLa cells were transfected as in (C) and loaded with transferrin Alexa-647 for 30 min. Histogram and the inserts show the correlation between ATG16L1 and the loaded transferrin (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001. (L) HeLa cells were transfected as in (C) and labeled for mATG9. Histogram and the inserts show the correlation between ATG16L1 and mATG9 in RAB11 and MVb tail overexpression conditions (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001. (M) HeLa cells were transfected with mStrawberry-ATG16L1 for 20 hr, starved in HBSS for 4 hr, or maintained in full medium, loaded with transferrin Alexa-488 (Tf). Pictures and histogram show the correlation between ATG16L1 and transferrin in basal and starving condition (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001. (N) HeLa cells were transfected with mStrawberry-ATG16L1 for 20 hr, starved in HBSS for 4 hr, or maintained in full medium and then fixed and labeled with anti-mATG9 antibody. Pictures and histogram show the correlation between ATG16L1 and mATG9 in basal and starved cells (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.
a) CCI-779 reduces aggregate load in mice expressing mutant huntingtin.figf8(a) CCI-779 reduces aggregate load in mice expressing mutant huntingtin. Peroxidase immunohistochemistry of brains of mice expressing mutant huntingtin at 16 weeks of age. Mice treated with CCI-779 had fewer aggregates per unit area in the striatum than mice treated with a placebo. Overall cell staining in the tissues of mice treated with CCI-779 was weaker than in those treated with a placebo and in nontransgenic untreated littermates (data not shown). Aggregates, when visible, were smaller in mice treated with CCI-779 than in those treated with a placebo, as apparent in the higher magnification images of single cells with aggregate (bottom panels). Six coronal sections per mouse were analyzed; these corresponded to the bregma ± 1 mm. We counted aggregates in five different high-power fields (60× objective) per slide on each side of the brain, sampling fields from the dorsal to the ventral part of the striatum just underneath the external capsule. The striata of mice treated with CCI-779 had 70% fewer aggregates than the striata of mice treated with a placebo (P = 0.0001, two-tailed t-test). (b) CCI-779 blocks mTOR signaling in vivo. Immunohistochemistry of mice with Huntington disease at 16 weeks of age with antibody to phosphorylated S6, in the cortical area and striatum. Levels of phosphorylated S6 were lower in mice treated with CCI-779 than in those treated with a placebo.
Figure 6A, B) m6A-IP specifically enriched sams mRNA isoforms that retain the AG dinucleotide at the distal/productive 3'SS. (A) Representative gel-like images (n=3) of semi-quantitative RT-PCR analysis of sams-3, sams-4 and sams-5 in input and immunoprecipitated (IP) RNAs from the smg-2 (yb979) mutant. The splicing patterns were analyzed and presented as in Figure 2B. (B) Relative enrichment of total and each of mRNA isoforms compared to eef-1A.1 mRNA by RT-qPCR. Error bars indicate standard error of mean (n=3). ( C) Examples of normalized Nanopore currents for unmodified and m6A-modified in-vitro transcribed sams-3/sams-4 RNAs at nucleotide positions -4 through +4 relevant to the m6A site. One hundred reads are lotted for each. Color codes are indicated. (D) Distribution of mean (top) and standard deviation (bottom) of normalized Nanopore currents at nucleotide positions -4 through +4 relevant to the m6A site for 5,000 of the unmodified and 2,680 of the m6A-modified sams-3/sams-4 RNAs and 85 of an endogenous sams-3 mRNA isoform E2/E3L. Color codes are indicated. Central bands represent the median, boxes represent the 25th and 75th percentiles, and whiskers represent the lowest and highest values. A red line at position 0 in the top panel indicates a cut-off line (mean current = 1.7557) that discriminates between the unmodified and m6A-modified sams-3/sams-4 RNAs with accuracies of 64.58% for both. ( E) Machine learning-based classification of sams RNA reads in Nanopore direct RNA sequencing data. The statistics in (D) as well as duration time of the Nanopore current at nucleotide positions -50 through +50 of up to 80% of mapped reads from the unmodified and methylated in-vitro transcribed sams-3/sams-4 and sams-5 RNAs were pooled and used for training with a machine learning algorithm Gradient Boosting and accuracy of the classifier was tested with 20% of the mapped reads. The classifier was also applied to reads identified as unproductive sams-3, -4 or -5 mRNA isoforms in the analysis of endogenous mRNAs shown in Figure 1. Numbers of reads used in the tests are indicated at the top; percentages of reads classified as "m6A-modified" are shown with color code. ( F) Relative importance of the statistics at each position for Gradient Boosting.
Increased macroautophagy in PS1/APP mice and human brains.Figure 1.(A-D) EM images of cortical neuropil show an absence of AVs and normal neurite profile in 9-mo-old NTg mouse brains (A, arrowheads outline normal neurites) and a marked accumulation of AVs within enlarged or dystrophic neurites in PS1/APP mice (B, arrowheads outline dystrophic neurite profiles in C) and biopsied brain material from an AD patient (B, inset). At higher magnification (C), AVs include autophagosomes (arrows) and multilamellar bodies (arrowhead). In normal dendrites of PS1/APP mice, multiple AVs are frequently seen (D, arrows). (E and F) LC3 quantification analyzed from immunoblots of LC3-I and LC3-II (top) in prefrontal cortical homogenates from cases of nonaffected (Cont), early stage (preclinical) AD (AD-ES), and moderate AD (AD-MS; E), and from brains of 18-22-mo-old PS1/APP (PA) mice (n = 3; F) compared with nontransgenic (NTg) controls (n = 3; *, P < 0.01). Error bars represent SEM. (G-L) LC3 immunofluorescence in 9-mo-old PS1/APP mice can be seen mainly as puncta in dystrophic dendrites of the cortex (G, arrows) and along adjacent dendrites. LC3 (H, arrows) is strong in dystrophic neurites in the periphery (asterisks) of a thioflavin S-labeled plaque core (H, inset) but is less so in neurites closest (H, arrowheads) to the Aβ deposit. LC3 is diffuse and uniform in neurons of NTg mice (I and J) but is predominantly vesicular and distributed more to the dendrites (arrows) than the cell soma (arrowheads) in 9-mo-old PS1/APP cortex (K and L).
Genes with different APA patterns across tissues.Figure 5Positive correlation between a gene's median APA Shannon index across tissues and its APA switch score. Spearman correlations between differences in major PAS usage and in gene expression level for each tissue pair comparison (P value < 4 x 10-10 in all comparisons). Spearman correlation between the expression level of a gene and its major PAS usage for genes with different major PAS location. P values were obtained from binomial tests to examine whether the number of genes with positive correlations is larger than the random expectation of 50%: ***P < 0.001. Upstream: upstream of last stop codon, 3' UTR(S): the only PAS in the 3' UTR, 3' UTR(F): the most proximal PAS in the 3' UTR, 3' UTR(M): the middle PAS in the 3' UTR, 3' UTR(L): the most distal PAS in the 3' UTR. The boxplot shows the Q1 to Q3 quartile values (the box limits), the median (the horizontal green line) and values within the 1.5 * IQR (the whiskers). Spearman correlation between the expression level of a gene and its first, middle and last PAS usage for genes with exactly 3 PASs in 3' UTR and major PAS in 3' UTR(M). P values are from binomial tests, **P < 0.01; ***P < 0.001. The boxplot shows the Q1 to Q3 quartile values (the box limits), the median (the horizontal green line) and values within the 1.5 * IQR (the whiskers). Spearman correlation between tissue APA differences and APA variability between replicates in each tissue pair.
HOPS, p53 and mitochondria.Figure 6A HOPS localization at mitochondria. RKO cells were stained by MitoTracker™ (red), immediately fixed and stained to visualize HOPS (green). Nuclei were DAPI stained (blue). Samples were analysed with fluorescent microscope and representative images were shown. Scale bars, 10µm. B RKO cells were lysed and processed for mitochondria isolation. WCL, cytoplasm and mitochondria were analysed using anti-HOPS antibody. GAPDH, Lamin B1 and Tom20 are purity controls of mitochondrial fraction. C HOPS and p53 translocation to mitochondria. RKO cells were treated with etoposide as indicated and processed for mitochondrial extraction and subsequent immunoblotting for p53, HOPS and the reported extraction purity controls. D HOPS/p53 binding at mitochondria. RKO cells were treated with etoposide and subjected to mitochondrial purification, and subsequent immunoprecipitation using anti-HOPS antibody. Samples were immunoblotted to detect p53, HOPS and the reported controls. E HOPS and p53 mitochondrial translocation in vivo. Hops+/+ and Hops−/− mice were treated with etoposide and thymus, spleen and testis were harvested. Organs were homogenized and mitochondrial fraction were isolated and analysed by immunoblotting versus p53. GAPDH and Tom20 are purity controls. p53 mitochondrial protein levels were semi-quantified using Tom20 as loading control and p53 levels at time 0 were assumed as 1. Data information: assays involving animals were performed three times in three different mice per condition. In E data are presented as mean ±SD. * P < 0.05; *** P < 0.001, by two tailed Student's t-test. F p53-dependent mitochondrial membrane potential (Δψm) induction in MEFs. Hops+/+ and Hops−/− MEFs were treated with etoposide and then incubated with JC-1 vital dye. The images were collected by fluorescent microscopy and representative images are shown (left panel). Scale bars, 10µm. Fluorescence was analysed by TECAN plate reader and plotted in the graph (right panel). Data information: assays involving animals were performed three times in three different mice per condition. Experiments on mitochondria from MEFs were performed five times. In F data are presented as mean ±SD. * P < 0.05; *** P < 0.001, by two tailed Student's t-test. G p53 MOMP induction in HOPS deficiency. Mitochondria from Hops+/+ and Hops−/− MEFs, were purified and incubated with p53 recombinant protein. Cytochrome C release from mitochondria as metabolic marker was assayed by immunoblotting. Samples were tested to detect p53 as control and Tom20 as mitochondria structural marker. Data information: assays involving animals were performed three times in three different mice per condition. Experiments on mitochondria from MEFs were performed five times.
Figure 5A) Enrichment analysis of RPB-9-bound proteins in rpb‑9::ollas (mj604) versus wild type by IP/MS. [p<=0.05, log2(fold change)>1 or <-1, Welch t-test]. n=4 technical replicates (pooled). B) Transgene transcript length is not affected in rpb-9 (mj261) mutants. Scheme of the gfp unspliced transcript showing the positions of RT-qPCR primers (top) and pairwise ratios (3′ to 5′) of unspliced gfp amounts along the transcript as indicated by the primer pairs (bottom). (n=3, error bars represent SD). ns= not significant (two-tailed t-test).  E) TE binning according to increasing 22G siRNA density in wild-type animals (grey, n=3). Mean normalized rpb-9 (mj261) mRNA reads (red, n=3) are overlaid with mean normalized wild-type mRNA reads (grey). (PolyA-selected RNA-seq libraries). The CEMUDR1 and Chapaev-2 transcripts are indicated with red (rpb-9 (mj261)) and grey (wild type) box plots. Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers. F) Distribution of Class I, Class II and Class II genes (as defined in Figure 4A-C) across bins as defined in (C)).  H) Distribution of normalized reads mapping over the CEMUDR1 and Chapaev-2 DNA transposons.
Figure 3A Immunoblotting of caspase-1 activation in Ubrs-knockdown BMDMs derived from the 129/Sv mice. Cells were transfected with the indicated siRNA for 60 h, and then treated with WT LT (+) or its E687C mutant (-) for 3 h. Immunoblotting of caspase-1 activation in E2s-knockdown BMDMs derived from the 129/Sv mice. Additional Ube2o siRNAs were used to confirm the inhibitory effect of Ube2o knockdown on LT-induced caspase-1 activation (C). Ube2o knockdown efficiency was measured by qPCR and are shown as mean values ± SD from three replicates. RFP-ASC specks formation assay of the effect of Ube2o knockdown (siRNA Ube2o-01) on LT-induced inflammasome activation in RAWRA cells. The percentages of cells showing RFP-ASC specks (mean values ± SD from three replicates) are in (D). F Co-immunoprecipitation interaction of UBE2O and UBR2. Myc-UBE2O and Flag-UBR2 were co-expressed in 293T cells and cell lysates were subjected to anti-Flag immunoprecipitation followed by immunoblotting analyses as shown.
Figure 4(A-C) SHP099 (A, 75mg/kg; B, C: 100mg/kg) reduces B16F10 tumor growth in syngeneic mice. Panels depict tumor growth from initiation of treatment to endpoint (left) and tumor weight (right) at endpoint; no. mice/group: 6-11 control; 7-9 SHP099. (D) Extravasation of FITC-dextran (green, pointed by arrowheads) in tumors treated with SHP099. Vessels identified by CD31 (red); DAPI (blue) identifies cell nuclei. Confocal images (scale bar: 200μm). Representative images from 3 tumors. (E) "Vascular sleeves" (Collagen IV+ CD31-) identified in a representative SHP099-treated tumor. Representative confocal images from 6 tumors (scale bar: 50μm). (F) Reduced CD31+ vascular area in SHP099-treated tumors compared to controls (n=6/group); quantification by ImageJ. (G) The ratio of Collagen IV+/CD31+ area is increased in SHP099-treated tumors compared to controls (n=6/group); quantification by ImageJ; Coll: collagen. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001. (H) Cleaved caspase-3 detection in a CD31+ vessel (representative of 6 SHP099-treated tumors); arrowheads point to cleaved caspase-3+CD31+ cells. Cleaved caspase-3+ tumor cell is marked by *. Confocal images (scale bar: 10μm). (I) Increased cleaved caspase-3 fluorescence intensity in CD31+ cells from SHP099-treated tumors compared to controls (n=3/group); at least 100 cells counted/sample. (J) Ki67+ proliferating cells in representative of 3 SHP099-treated and 3 control tumors; CD31 identifies the vessels; DAPI identifies cell nuclei. Confocal images (scale bar: 50μm). (K) Quantification of proliferating cells within SHP099 and control (n=4/group) tumors by ImageJ. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001. (L) Hypoxic tumor identified Hypoxyprobe (green) representative of 3 SHP099-treated tumors; confocal images (scale bar: 100μm), left; quantification of hypoxic areas in control (n=3) and SHP099-treated (n=3) tumors by ImageJ. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001. (M) Representative control (of 5) and SHP099-treated (of 5) B16F10 tumor sections through the maximum diameter; H&E staining (scale bar: 10mm). (N) Cleaved caspase-3+ fluorescence intensity in SHP099 and control tumors (n=3/group). Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.
Figure 5A. Crystal structure of V1 in the autoinhibited state 2 conformation (PDB 5D80) (Oot et al., 2016). HCT of wild type H stabilizes an open catalytic site (arrow) with inhibitory MgADP bound to an adjacent site. B. Model of V1 in complex with subunit C and Oxr1p. In this complex, V1 is in state 1, with C bound between EG2 and EG3. Oxr1 (red) is wedged between the N-terminal domain of EG2, the subunit AB pair next to the open catalytic site, and Cfoot. C. Structural changes in EG2, EG3 and subunit C upon Oxr1 binding. D. Oxr1 causes disassembly of holo V-ATPase. In this model, Oxr1 binding destabilizes the interaction between EG2, Cfoot and the distal domain of aNT, which leads to release of V1 from the membrane.
Ubiquitin accumulates in dense cytosolic aggregates and forms K63-linked chains.Figure 2A) Immunoelectron microscopy of HeLa cells infected with S. Typhimurium strains for 12 h (ubiquitin - 10 nm gold particles; scale bars, 0.5 µm). Arrows indicate ubiquitin accumulations near bacteria within vacuoles. (B) Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue) for 10 h and immunolabelled for ubiquitin (all chain types - Ub, red) and lysine-48-linked (K48-Ub, green) or lysine-63-linked (K63-Ub, green) ubiquitin chains (false coloured, scale bars, 5 µm). Arrows indicate SCV-associated ubiquitin and K63-Ub labelling.
a) Schematic representation of the GFP–ActA-Q79C series of constructs.figf4(a) Schematic representation of the GFP-ActA-Q79C series of constructs. FL, N and C comprise ActA residues 30-612, 30-389 and 390-612, respectively. (b-e) The series of pEGFP-ActA-Q79C constructs was transfected into COS-7 cells. Cells were fixed with 4% Paraformaldehyde after 18 h and stained with an anti-Arp2 antibody (b), an anti-VASP antibody (c), an anti-ubiquitin antibody (d) or an anti-p62 antibody (e; red). Scale bars, 10 μm. Arrowheads indicate Arp2-, VASP-, ubiquitin- or p62-positive Q79C. Ub, ubiquitin.
Figure 5A. Immunoblot of phospho-S622 and pan-Drp1 in wild-type CNs treated with or without 10 or 50 μM pimozide and with or without 100 U/mL rIFN β. * denotes a potential Drp1 cleavage product of approximatively 50 kDa. B. Immunoblot of phospho-S622 and pan-Drp1 in N2A cells depleted of STAT5 A and B by CRISPR/Cas9 (DKO) or with nontargeting control (NTC). Vinculin was used as a loading control. Immunoblot of phospho-S622 and pan-Drp1 in N2A cells depleted of STAT5 A and B by CRISPR/Cas9 (DKO) or with nontargeting control (NTC). Vinculin was used as a loading control. C. Quantification of B. D. Projections of 3D images from immunostaining for phospho-S622 Drp1 (yellow), Hsp60 (mitochondria, green), and calnexin (ER, magenta) in N2A cells depleted of STAT5 A and B (δStat5) or IFN-β (δIfnb) or with nontargeting control (NTC). Cells with or without rIFN-β treatment for 6 h. Scale bar equals 5 microns. Arrows indicate triple-positive foci. E. Quantification of D. F. Pgam5 promotor contains STAT5 binding motifs. Schematic of the upstream region of pgam5, encompassing the pgam5 promotor and pxmp2 with its terminator. The TTCT/CnA/GGAA motif is the optimal STAT5A and B binding motif and is specific to STAT5. OR indicates the origin of replication, as predicted by GPminer. Distances are in bp. G. Kinetics of the expression of pgam5 by qPCR in CNs on rIFN-β treatment. H. PGAM5 levels by rtPCR in NTC, δIfnb, and δStat5 N2A cells with and without rIFN-β for 6 h. Error bars are SD from 1 representative graph of 3 independent experiments. I. Immunoblotting against PGAM5 in Ifnb+/+ ­and Ifnb-/- CNs with or without rIFN-β treatment for 2 h and quantification. J. Quantification of the impact of rIFN-β on Drp1 phosphorylation after silencing of genes encoding the kinases CDK5, Camk2a, and ERK2 and the phosphorylase PGAM5. K. Kinetics of the impact of rIFN-β on S622 and S643 phosphorylation. Quantified in J. L. Immunostaining of PGAM5 and Hsp60 in NTC and δSTAT5 N2As transfected with PGAM5 or empty vector control and quantification of mitochondrial section area. Scale bars equal 5 microns. Error bars mean SEM from 10 individual cells. M. Optineurin immunostaining in Ifnb+/+ and Ifnb-/- MEFs overexpressing PGAM5 and quantification. Empty vector was used as control. Additional controls are shown in Fig EV5L. Error bars are SD from N=30 cells. N. Oxidative stress in δIfnb N2A cells decreases on overexpression of PGAM5 and phosphomimetic Drp1 but not wild-type Drp1. Immunoblots of PGAM5. Vinculin was used as a loading control. O. Loss of mitochondrial membrane potential in δIfnb N2A cells. Quantification of TMRE by automated Incucyte-based imaging and analysis. Bars are mean + SEM.