EMBO/sd-character-level-ner huggingface dataset:
a) Fluorescence images showing colocalization patterns of the indicated GFP-p38 variants and Sph1 (green) with endogenous p62 (red) in SY5Y cells.f5(a) Fluorescence images showing colocalization patterns of the indicated GFP-p38 variants and Sph1 (green) with endogenous p62 (red) in SY5Y cells. Arrows indicate colocalization. (b) Quantitative FRAP analysis of mCherry-p62 expressed alone (control) or co-expressed with the indicated GFP-Sph1 and -p38 variants. Top: time course of changes in relative fluorescence intensity (RFI); bottom: percentage of p62 in the diffusing fraction and diffusion time (t1/2). Values are mean+s.e. of ten cells analysed over three different experiments. Representative images are shown in Supplementary Fig. S8. (c-d) Top: IBs for p62 and actin in control and p62 knockdown (p62 KD #325 or #788) SY5Y cells (c); and in control and p62 KO MEFs (d). Bottom: autophagic clearance of GFP-ANK1-p38 Agm in the same cells in response to serum removal. (e) Fluorescence images of GFP-p38, -ANK1-p38 and -NA Sph1 Agm in SY5Y cells immunostained for endogenous NBR1. Arrows indicate colocalization. (f,g) Top: IBs for NBR1, p62 and actin in control and NBR1 knockdown (NBR1 KD #161) SY5Y cells (f), and in control and p62/NBR1 double knockdown (Double KD #A or #B) SY5Y cells (g). Bottom: autophagic clearance of GFP-ANK1-p38 Agm in the same cells. For all densitometric analyses, values are mean+s.e. (n=3). *Significant versus serum+ condition. See also Supplementary Fig. S8-S11.
Figure 3Vector copy number (VCN) cartography shows integrated LV genome (assessed by qPCR) along the rostro-caudal axis (slices 1-10) in LV.hARSA-injected S1.1 and S2.2 NHP in a side-by-side comparison with ARSA mRNA expression (assessed by qPCR analyses using probe and primers annealing to sequences conserved in both the human and Macaca fascicularis and expressed as fold increase to region-matched blocks of the contralateral hemisphere). Grading of colors for VCN ranged from white (VCN<0.001; corresponding to CT>37) to dark orange (VCN=1-3). The highest VCN is found in close correspondence to the injection sites, as confirmed by comparison with post-surgery MR images (yellow and orange circles indicate viral suspension close to the injection sites). Grading of colors for ARSA expression ranges from white (<1.5 fold the physiological level) to dark purple (100-fold the physiological level).
EGFR mediates Akt activation induced by T.Figure 5A, mHEVc cells were transfected with control siRNA or EGFR siRNA. Expression of EGFR and actin were assessed by immunoblot 48 h post-transfection. mHEVc cells expressing either control siRNA or EGFR siRNA were challenged with T. gondii. Cell lysates were used to examine the expression total Akt or phospho-Ser473 Akt by immunoblot. B, Mouse microglia were treated with or without AG1478 (1 µM) 1 h prior to challenge with T. gondii. Cell lysates were used to examine the expression total Akt or phospho-Ser473 Akt by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control siRNA and EGFR siRNA or control and AG1478-treated cells to indicate that band densities from infected control cells or infected cells subjected to EGFR blockade were compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.
a–c) Two canonical TRAF6-binding sites in AMBRA1 are essential for its interaction with TRAF6.figf5(a-c) Two canonical TRAF6-binding sites in AMBRA1 are essential for its interaction with TRAF6. HEK293 cells were co-transfected with vectors encoding FLAG-TRAF6 together with AMBRA1WT and mutant constructs. Protein extracts were immunoprecipitated using an anti-FLAG antibody. AMBRA1 and FLAG-TRAF6 were analysed by western blotting. (a-c) Two canonical TRAF6-binding sites in AMBRA1 are essential for its interaction with TRAF6. HEK293 cells were co-transfected with vectors encoding FLAG-TRAF6 together with AMBRA1WT and mutant constructs. Protein extracts were immunoprecipitated using an anti-FLAG antibody. AMBRA1 and FLAG-TRAF6 were analysed by western blotting. (d) HeLa cells were transfected with AMBRA1 RNAi oligonucleotide (siRNA AMBRA1). Then, cells were co-transfected with vectors encoding ubiquitin-HA and AMBRA1WT or AMBRA1AA, respectively. Protein extracts were immunoprecipitated using anti-ULK1 antibody; ubiquitin-HA, ULK1 and AMBRA1 were analysed by western blotting. (e) HeLa cells stably interfered for AMBRA1 were transduced with lentiviral vectors encoding β-galactosidase (negative control) AMBRA1WT or AMBRA1AA respectively; protein extracts were analysed by western blotting using anti-ULK1, anti-AMBRA1 anti-ATG13, anti-pATG13 and anti-actin antibodies. (f) HeLa cells were transfected as in e and some of them were treated with chloroquine for 1 h. LC3, p62, AMBRA1 and actin were analysed by western blotting. Quantification of LC3II and p62 are shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.03 for LC3 and P = 0.045 for p62. (g) GFP-LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with the indicated lentiviral vectors and quantification of LC3 puncta per cells is shown on the right. Bars represent mean±s.e.m. of triplicate samples with 50 cells analysed per sample; n = 3 independent experiments. Significance is P = 0.001 and P = 0.003 (n = 3). Scale bar, 10 μm.
Figure 2A, Scheme of the transgenic construct (top), and sagittal brain section of a Fluc-EGFP mouse from line 1214 at 3 months of age immunostained for EGFP (bottom). B, Representative images of the indicated brain regions of a Fluc-EGFP mouse, stained for EGFP (green) and the neuronal marker NeuN (magenta). Insets show higher magnification of the areas indicated by the boxes. C, Left, Western blot of acute brain slice lysates from Fluc-EGFP mice and non-transgenic littermates. The slices were treated with 1 mM 4-PBA or vehicle control, and subjected to heat shock at 43°C for 15 min, as indicated above the blot. Middle, quantification of specific luciferase activity of Fluc-EGFP upon indicated treatments, normalized to vehicle-treated slices kept at 37°C. Right, quantification of Fluc-EGFP protein levels in the indicated conditions. N=3 mice. Colored asterisks indicate comparisons to the corresponding vehicle-treated 37°C control group (one-sample t-test), black asterisk indicates comparison between 4-PBA and vehicle-treated heat shock groups (two-tailed t-test). D, Representative Western blot of hippocampal lysates from Fluc-EGFP mice at the indicated ages. Short bracket with an arrow indicates the high molecular weight species observed in older mice. Long bracket indicates the part of the lane that was used for Fluc protein quantification (see Materials and Methods). Several lanes on the blot between 6-month-old and 12-month-old samples were digitally removed. E, Fluc-EGFP specific luciferase activity measured in the indicated brain regions of Fluc-EGFP mice at the indicated ages. Values are normalized to the 3-month-old group. N=5 Fluc-EGFP mice for each age group. One-way ANOVA with Bonferroni's multiple comparisons test. ANOVA: Hippocampus, *p = 0.0205; Cortex, **p = 0.0077; Cerebellum, n.s. Significant pairwise comparisons to the 3-month-old group are indicated on the graphs. F, Fluc-EGFP protein quantity measured in the indicated brain regions of Fluc-EGFP mice at the indicated ages. Values are normalized to the 3-month-old group. N=5 Fluc-EGFP mice for each age group. One-way ANOVA with Bonferroni's multiple comparisons test. ANOVA: Hippocampus, *p = 0.0278; Cortex, n.s; Cerebellum, **p = 0.0058. Significant pairwise comparisons to the 3-month-old group are indicated on the graphs. Protein quantity normalized to total protein instead of tubulin is shown in Appendix Fig. S2C.
a) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig.figf6 (a) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages exposed to DMSO (negative control), mild heat shock, IL-1β or IFN-γ.  (b) Dansylcadaverin staining of untreated macrophages (Basal) or macrophages exposed to mild heat shock, IFN-γ or IL-1β and infected for 8 h with wild-type HSV-1, normalized to the signal obtained in basal conditions and presented in arbitrary units.  (c-f) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages incubated at 37 °C (c) or exposed to mild heat shock (d), IL-1β (e) or IFN-γ (f) and infected for 8 h with wild-type HSV-1 with the addition of DMSO (negative control), 3-methyladenine, bafilomycin, brefeldin A or MG-132 at 2 h after infection. Results in a,c-f are normalized to results obtained for CD8+ T cells stimulated with macrophages incubated with DMSO in each condition and are presented in arbitrary units. Data are from three independent experiments (mean and s.e.m. of triplicate samples).
Figure 1Human DCs were incubated with a non-cytolytic dose (0.2 μg/ml) of purified PLY for 45 min. Immunofluorescence staining shows that PLY (green) co-localize with MRC-1 in DCs and EEA-1 (purple, early endosomes). All scale bars, 5 μm. Images are representative of three independent experiments. Human DCs were incubated with a non-cytolytic dose (0.2 μg/ml) of purified LLO for 45 min. Immunofluorescence staining shows that LLO (green) co-localize with MRC-1 (red) in DCs and EEA-1 (purple, early endosomes). All scale bars, 5 μm. Images are representative of three independent experiments. Human DCs were incubated with a non-cytolytic dose (0.2 μg/ml) of purified SLO for 45 min. Immunofluorescence staining shows that SLO (green) co-localize with MRC-1 (red) in DCs and EEA-1 (purple, early endosomes). All scale bars, 5 μm. Images are representative of three independent experiments.
FIS1 is required for the formation of MAPAs.Figure 7Time-lapse analysis of live HeLa cells transfected with OTC-Δ-GFP and Mito-MTX-DsRed and treated with MG132. White arrowheads show a OTC-Δ-GFP-positive Mito-MTX-DsRed-negative structure that eventually segregated from the mitochondrial network. Immunoblot analysis of the MG132-induced deposition of FUNDC1, TIM23 and p62 in HeLa cells transfected with Ctrl or DLP1 shRNA. C, D. Immunoblot analysis of the MG132-induced deposition of FUNDC1 in cells transfected with ctrl shRNA, FIS1 shRNA or FIS1 shRNA together with shRNA-resistant Flag-FIS1. Quantification is shown in (D). E, F. HeLa cells were stably transfected with Ctrl or FIS1 shRNA, treated with MG132 and immunostained with indicated antibodies. Scale bar=10 µm. Quantification is shown in (F). G, H. Fractionation analysis of HeLa cells stably transfected with Ctrl or FIS1 shRNA, then transiently co-transfected with Ub-R-GFP and Flag or Flag-FIS1 and treated with DMSO or MG132. Quantification is shown in (H).
EGFR and T.Figure 9A, hmCD40 mHEVc expressing LC3-EGFP were treated with or without CD154 followed by challenge with T. gondii-RFP in the presence or absence of EGF (50 ng/ml) or AG1478 (1 µM). LC3 accumulation around T. gondii was assessed by immunofluorescence. B, mHEVc-LC3-EGFP cells were infected with T. gondii-T. gondii. Cells were treated with vehicle or rapamycin (1 µM) 2 h after challenge with T. gondii in the presence or absent of EGF or AG1478. Recruitment of LC3 around the parasite was examined at 5 h after challenge with T. gondii. . C, hmCD40 mHEVc-LC3-EGFP cells were treated with or without CD154 followed by challenged with WT, MIC1 ko, MIC1 ko+MIC1, MIC3 ko, MIC3 ko+MIC3, MIC1-3 ko, MIC1-3 ko+MIC1-3 tachyzoites. LC3 accumulation around the parasite was examined by fluorescence microscopy 5 h post-challenge. D, hmCD40 mHEVc-LC3-EGFP treated with or without CD154 were challenged with WT, MIC1 ko, MIC1 ko+MIC1, MIC3 ko, MIC3 ko+MIC3, MIC1-3 ko or MIC1-3 ko+MIC1-3 tachyzoites. Parasite load was examined 24 h post-challenge. E, HBMEC cells were challenged with WT, MIC1 ko, MIC3 ko, MIC1-3 ko tachyzoites followed by treatment with vehicle or rapamycin. Parasite load was examined 24 h post-challenge. F, HBMEC treated with or without CD154 or IFN-γ/TNF-α were challenged with WT, MIC1 ko, MIC3 ko, MIC1-3 ko tachyzoites. Monolayers were examined microscopically at 24 h post-challenge. G, hmCD40 mHEVc cells treated with or without CD154 were infected with either WT or MIC1-3 ko tachyzoites in the presence of complete medium (CM) alone or medium plus MIC3, MIC4, M2AP or MIC6 (all 10 nM). Parasite load was determined microscopically 24 h post challenge. Results shown are representative of 3-4 independent experiments.
Fig.Figure 3(A) Flow chart for the RNA-Seq and proteomic identification of differential mRNA and proteins. (B) Volcano plot of differential proteins between WT and HRD1 LKO livers. (C) Venn blot of the overlaps of the differential genes in mRNA and protein levels. (D) GO functional analysis of the differential proteins from proteomic screening. (E) Overlap of the increased genes identified between RNA-Seq and proteomics. (F) Protein levels of IRE1α, NRF1, RXRβ, NOTCH1, SIRT3 and GAPDH in the liver of WT and HRD1 LKO mice under the refed condition. (n=6 for each group).
Comparison of chimeric and full-length clade 2 and 3 spikesa, Full length spike sequences from As6526 (clade 2) and BM48-31 (clade 3) were codon optimized, FLAG tagged and synthesized.Figure 5:Full length spike sequences from As6526 (clade 2) and BM48-31 (clade 3) were codon optimized, FLAG tagged and synthesized. Silent mutations flanking the RBD facilitated replacing the native RBD with the clade 1 consensus RBD. Westernblot of producer cell lysates and concentrated pseudotypes particles. Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect Huh-7.5 cells. Shown are data for 3 replicates. Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect BHK cells transfected with human ACE2. Shown are data for 3 replicates.
Figure 3(A-I) Shown are ChIP-seq big wig files in the IGV browser depicting the binding of Oct4 (Red), Sox2 (Blue), Klf4 (Green) and c-Myc (Black) to putative regulatory regions in the nine TRs genes in MEFs undergoing reprogramming (18 hrs, D3 and D5) and in control MEFs, mESCs and miPSCs. All peaks have been normalized against input DNA. The relative sizes of the represented genomic loci and the corresponding TSSs are also indicated. The side color bar depicts the expression levels of each TR at the indicated time points.
In basal conditions, AMBRA1 is maintained in an inactive state by mTOR phosphorylation and by its anchoring to the dynein motor complex.figf8In basal conditions, AMBRA1 is maintained in an inactive state by mTOR phosphorylation and by its anchoring to the dynein motor complex. After autophagy induction, AMBRA1 and ULK1 are active; AMBRA1 enhances ULK1 kinase activity and stability by promoting its Lys-63-linked ubiquitylation and self-association through TRAF6. ULK1 primes AMBRA1 detachment from the dynein complex through a positive phosphorylation. Coupling these regulatory events may represent a positive feedback to sustain autophagy in a continuous loop. The exocyst may act as a scaffold platform to keep the complexes involved grouped together.
Figure 7Undetectable T22-GFP-H6-FdU emitted fluorescence in normal tissues, except for a transient accumulation 5h after a 100μg dose in the liver, which disappears at 24 h. Liver emitted fluorescence is transient and significantly lower than the one registered in tumor tissue. Tumor/Liver ratio= 7.5 (see tumor intensity in Fig 2B, which was registered in the same experiment) (N=5 mice/group). Scale bar, 1 cm. Representative images depicting the level of DNA double strand break (DSB) induction in histologically normal bone marrow 5h after treatment, as measured by anti-ɣ-H2AX, which is higher in free oligo-FdU-treated mice than in T22-GFP-H6-FdU (P=0.047). Low level of cells containing DSBs in histologically normal kidney after T22-GFP-H6-FdU or free oligo-FdU treatment, a finding occurring in all normal tissues analysed. (N=50, 5 mice/group; 10 fields/mouse). Representative images showing lack of histopathological alterations in H&E stained tissue or apoptotic induction in H&E stained samples of CXCR4+ (bone marrow) and CXCR4- (brain, kidney, liver, lung and heart) normal tissues 24h after the administration of a 100 μg dose of T22-GFP-H6-FdU or an equimolecular dose of free oligo-FdU (N=5/group). Note that the transient nanoconjugate distribution to liver or the DNA damage induced in bone marrow do not lead to cytoxicity on these no-tumor tissues. (N=50, 5 mice/group; 10 fields/mouse) Lack of differences in body weight among groups registered along time in the SW1417-derived CCR model and the regression of metastases protocol (N=10 mice/group). Lack of differences in body mouse weight among groups registered along time in the SW1417 cell line-derived model and the prevention of metastasis protocol (Buffer (N=11; Free oligo-FdU (N=12), T22-GFP-H6-FdU (N=12)). Lack of differences in body mouse weight among groups registered along time in the M5 patient-derived model and the prevention of metastasis protocol (Buffer (N=6); Free oligo-FdU (N=17); T22-GFP-H6-FdU (N=8). Scale bar, 100 μm
Figure 4Heatmap results for HAND2-AS1 or INO80 deficiency in HCC cells. Based on GSEA results, significantly changed genes due to depletion of HAND2-AS1 and INO80 were attributed to the BMP signaling in HCC primary cells. NES, normalized enrichment score; FWER, familywise error rate. ChIRP-seq analysis of HAND2-AS1 genomic binding at target sites in liver CSCs, using LacZ probes as negative control. A 10-kb interval centered on the called HAND2-AS1 peak is shown. ChIRP-seq analysis of HAND2-AS1 bound regions in the genome of liver CSCs. Representative ChIRP-seq of HAND2-AS1 binding of BMPR1A. Gel analysis of HAND2-AS1 RNA binding BMPR1A promoter in liver CSCs. LacZ and PVT1 served as negative controls. HAND2-AS1 deletion in HCC cells decreases BMPR1A expression and inactivates BMP signaling. ChIRP-immunoblotting analysis of BMPR1A interaction with the INO80 complex in control and HAND2-AS1-knockout liver CSCs. EMSA analysis of the interaction of HAND2-AS1, INO80 and RUVBL2 with the BMPR1A promoter region. BMPR1A promoter binding region to HAND2-AS1/INO80 was deleted (BMPR1A-p-KO) in liver CSCs using lenti-Cas9, followed by HAND2-AS1 and INO80 overexpression (oeHAND2-AS1, oeINO80) for 36 h. BMPR1A mRNA levels were examined by realtime PCR. Data are shown as means ± SD. n=5. ** P < 0.01 by two-tailed Student's t test. BMP signaling was examined in INO80-silenced liver CSCs by Western blot.
Figure 1Mode of regulation of transcription by Alu RNAs through suppression of RNA Polymerase II (left panel) and protein-accelerated self-cleaving processing properties of Alu RNAs (right panel) as described in previous studies (Hernandez et al., 2020; Mariner et al., 2008; Ponicsan et al., 2010; Yakovchuk et al., 2009). Plotting of the position of the 5'end of Alu RNA fragments across the Alu metagene to depict potential processing areas of Alu RNAs in all post-mortem hippocampal tissues (PM hippo) from patients from the CBB. Each row in the heatmap depicts the distribution of counts of the 5'ends of reads mapped across the Alu metagene for each patient. The x-axis represents a metagene combining all unique Alu RNA sequences (ALUome) aligned at the start site of their consensus sequence with numbers representing the distance from the Transcriptional Start Site (TSS) area. Heatmap density corresponds to normalized counts of the 5'end of the reads with red corresponding to higher density of these 5'ends at a specific position. XL1, XR1 and XR2 denote the Alu processing areas defined by the high-density areas in the heatmap at specific positions of the Alu metagene, with the middle letter corresponding to the arm of the Alu RNA (see Figure 1C, L=Left, R=Right), in which the area is located. Processing areas of Alu RNAs on the secondary Alu RNA structure. Secondary structure of Alu RNA adapted from Hadjiargyrou and colleagues (Hadjiargyrou & Delihas, 2013). As in our previous studies (Cheng et al., 2020; Zovoilis et al., 2016), we depict the SINE RNA processing areas (highlighted in pink) based on short RNA-seq data and mapping of the 5'ends of Alu RNA fragments. X mark the cleavage sites of Alu RNA that correspond to enriched processing areas (the high densities of 5'end fragments distribution) at the heatmap of Figure 1B. The rectangle depicts the critical region that binds and suppresses RNA Pol II based on (Mariner et al., 2008) that may be affected due to processing points.
Figure 5, Suppression of OTUB1 expression in NSCLC cell lines by specific shRNAs against OTUB1 was confirmed by immunoblotting using antibody against OTUB1. B, OTUB1 suppression decreases serum-induced ERK1/2 phosphorylation. Serum-starved A549 cells expressing shRNAs against OTUB1 or GFP were stimulated with 10% serum for the indicated time periods. Levels of phosphorylated ERK1/2 and total ERK1/2 were analyzed by Meso Scale assay. The results are expressed as a mean of pERK1/2 levels relative to total ERK1/2 ± s.e.m; p-values were determined by two-sided t-Test, n=2. C, D, OTUB1 suppression impairs the anchorage-independent growth of Western blot cancer cell lines. Representative images of soft-agar colonies formed by Western blot cells expressing shRNAs against OTUB1 or GFP. The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3. C, D, OTUB1 suppression impairs the anchorage-independent growth of lung cancer cell lines. Representative images of soft-agar colonies formed by lung cells expressing shRNAs against OTUB1 or GFP. The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3. E, Xenograft tumor growth of A549 cells expressing shRNAs against GFP or OTUB1 subcutaneously injected into nude mice. p-value was determined by 2-way ANOVA.
Fig.Figure 2a and b. Maximum intensity Z-stack projection images of 5 DAG old roots expressing the R2D2 auxin signaling reporter 12 HAT to ammonium (a) or nitrate (b) supplemented media. White arrows mark the position of the 10th and 20th cells from QC; "e" and "c" mark epidermis and cortex, respectively. Scale bar=50 µm. Graphs denote normalized relative auxin levels at the respective positions. Lines represent polynomial regression fit with 95% confidence band. Data are derived from 5 roots per condition from 3 biological replicates.
Figure 1A, Schematic representation of feeding time course. B, C, In primary endothelial cells isolated from human IGF-1 receptor endothelial overexpressing mice (hIGFREO) and wildtype littermates (WT), quantitative polymerase chain reaction (qPCR) shows that hIGFREO have increased expression of human IGF-1R but similar levels of murine insulin receptor (IR) gene expression as WT (n=3-5 mice per group). D, E, In primary endothelial cells isolated from WT and hIGFREO, immunoblotting shows that hIGFREO have increased expression of IGF-1R but similar levels of IR protein expression (n=3-4 mice per group). F, Chow fed hIGFREO had similar body mass to WT, however, hIGFREO did not gain as much weight as WT after 8 weeks of HFD (n=6-10 mice per group). G, Representative images of difference in fat and water distribution shown by magnetic resonance (MR) imaging in hIGFREO and WT. Scale bar = 1cm. I, hIGFREO had reduced white epididymal adipose depot weight compared to WT, there was no difference in heart, spleen or liver weight (n=7-11 mice per group). J, hIGFREO had similar whole-body and femur length as WT (n=7-9 mice per group).