EMBO/sd-character-level-ner huggingface dataset:
Mitochondrial fission and dysfunction contribute to muscle atrophy through AMPK.Figure 7(A) Left panel: mitochondrial fission activates AMPK. HEK293 cells were transfected with a mock vector (control), Bnip3, DRP1, Fis1 or co‐transfected with DRP1 and Fis1. Cells were collected 24 h later and lysates were analysed by immunoblotting. Right panel: immunoblotting analysis shows AMPK phosphorylation in EDL and soleus muscles during muscle atrophy. P‐AMPK/AMPK ratio analysed by densitometric analysis is indicated below the panels. (B-E) Inhibition of AMPK blocks Bnip3‐ and DRP1/Fis1‐mediated atrophy. (B) Adult TA muscles were transfected with Bnip3 together with d.n.AMPK or mock vector and cross‐sectional area was measured (*P0.001) n=480. (C) Adult TA muscles were transfected with DRP1 and Fis1 together with d.n.AMPK or mock vector. Twelve days later muscles were collected and cross‐sectional area was quantified (*P0.001) n=400. (D, E) RNAi‐mediated knockdown of AMPK α 1 and α 2 inhibits Bnip3‐ and DRP1/Fis1‐dependent atrophy. Mouse TA muscles were co‐transfected with Bnip3 or DRP1 and Fis1 and pSuper vectors expressing shRNAs against lacZ (control) or AMPK. Twelve days later, TA muscles were collected and the cross‐sectional area was quantified (*P0.001) n=450.
BET inhibitor effects on FGF2 secretion by LX-2 cells and FGFR expression in UM cells.Figure 7
Figure 7(A) SGC7901 and MGC803 cells were transfected with ATF4 shRNA or shcontrol. Western blottings and qRT-PCR were used to analyze the expression of UHMK1. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Data information: Data represent mean ± SD. **P< 0.01, ***P< 0.001. (B) Overexpression of ATF4 enhanced the expression of UHMK1 in BGC823 and HGC27 cells. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Data information: Data represent mean ± SD. **P< 0.01, ***P< 0.001. (C) The nucleotide sequences showing the -1348/-948 region in the human UHMK1 gene. The red text indicates the candidate ATF4 binding sites. (D) The effect of ATF4 on the activity of the WT-UHMK1 promoter and its mutations, as evaluated by using luciferase reporter assays. Data information: Data represent mean ± SD. **P< 0.01, ***P< 0.001. (E) ChIP assays were used to examine the binding of ATF4 to the UHMK1 promoter in SGC7901 and MGC803 cells. Input DNA and nonspecific IgG were included as controls. (F) ATF4 deficiency and overexpression regulated the activity of the UHMK1 promoter in GC cells. Data information: Data represent mean ± SD. **P< 0.01, ***P< 0.001.
H3K122succ is enriched at active TSS.Figure 2A IGV browser snapshots showing the distribution of reads around the TSS of the actively transcribed PPM1D gene (chr17:60,587,313-60,643,064; top panel) and of the inactive CD40 gene (chr20:46,114,180-46,129,721; bottom panel) for the indicated histone modifications, input and RNAseq. B Venn diagrams showing the overlap between genes with H3K122succ peaks detected at their TSS regions and genes with CpC island associated to their promoters (CGI) (upper panel), as well as housekeeping genes (HKG) (bottom panel). P-values were calculated through hypergeometric tests. C Venn diagram showing the number of H3K122succ peaks detected overlapping with H3K4me3 peaks and H3K9ac peaks. D Correlation matrix displaying the degree of overlap between H3K122succ, H3K4me3, H3K9ac and H3K27ac peaks from ChIP experiments at TSS (peaks within ± 500 bp of TSS). Total number and fraction of overlapping peaks are displayed. E Correlation matrix displaying the overlap between our H3K122succ ChIP-seq peaks and DNase-seq and FAIRE-seq enriched regions at TSS (peaks within ± 500 bp of TSS). Total number and percentage of overlapping peak regions are displayed. F H3K122succ occupancy profile over a meta gene as a function of expression level (based on RNA-seq quartiles: Q1 - least expressed, Q4 - most expressed). All genes were normalized to the same length. G Boxplots visualizing expression values of genes (reads per kb; RPK) with peaks for indicated different combinations of histone PTMs at their TSS. Histone modifications are (from left to right): none (no PTM, black); only H3K122succ (green); H3K122succ plus one tail modification (H3K4me3, or H3K9ac, or H3K27ac, green and light orange); H3K122succ plus two tail modifications (green, yellow and light orange); H3K4me3 and H3K9ac and H3K27ac without H3K122succ (yellow, light orange and dark orange); H3K122succ and all three tail modifications (green, yellow, light orange, and dark orange). The number of genes within the groups is indicated below each boxplot. Boxes indicate the range between the first and third quartile, the central line depicts the median, and the whiskers span the range of the data while extending no further than 1.5 times the interquartile range. Each distribution was compared to the "no PTM" distribution.
Figure 2LTP was induced at hippocampal CA3-CA1 synapses after 20 min baseline recordings (arrowhead, TBS). cDKO mice expressing Venus (red) exhibited significant lower induction and maintenance of LTP (128.12 ± 3.41%) compared to Venus injected LM controls (white, 156.69 ± 4.75%, ###p<0.001). AAV mediated expression of APPsα (green) restored potentiation after start of baseline recording and resulted in an LTP curve comparable to that of LM controls. The LTP induction rate is shown as percentage % of mean baseline slope. Data points were averaged over 6 time points. The deficit of PPF in Venus injected cDKO mice at the 10 ms (#p<0.05) and 20 ms (#p<0.05) ISI was restored by expression of APPsα. n=number of slices. N=number of animals. Representative images of basal and midapical dendritic segments of CA1 neurons. The spine density deficit of Venus injected cDKOs mice (11% in basal and 18% in midapical dendrites) is rescued by APPsα to LM control levels. Images are maximum projections of deconvolved z-stacks. Scale bar: 5 µm. Spine density was normalized to LM control levels. n=number of neurons (from 5 animals per condition).
Figure 5Representative images of mitophagy showing co-localization between the mitophagy dye (red) and the lysosome dye (green) in DC and control fibroblasts treated with vehicle or 3 mM NR. White arrows: mitophagy. B The mean value of fluorescence intensity/cells in each image was scored. At least 15 images (~ 200 cells) were counted per group. The relative values in each group was normalized to Con1. C Representative immunoblots of the expression of PINK1 and PARKIN in DC cells treated with vehicle or 3 mM NR. Quantification of immunoblots from the indicated proteins is from three replicates.
Figure 2Luciferase-based imaging of short therm drug response in Vegfr3Luc;Tyr::CreERT2; BrafV600E; Ptenflox/flox mice. Panels labeled as "basal" and "induced" correspond to the bioluminescence of animals prior and 5 weeks after administration of 4OH-tamoxifen (5 mM, topical administration, 3 consecutive days) for the induction of melanomas. (Right panels) images of mice were treated the day before with one dose of the indicated compounds: αPD-L1 antibody (clone 10F.9G2) or the corresponding control IgG (200ug/dose); vemurafenib (Vem, 50 mg/Kg) or BO-110 (BO, 0.8 mg/kg). Scale: p/s/cm2/sr (x106). Response of xenografts of mCherry-labeled SK-Mel-147 in Vegfr3Luc nu/nu lymphoreporter mice treated with one dose (24 h) or 4 dosis of BO-110 (BO, 0.8 mg/kg). Left panels correspond to Vegfr3-Luciferase (neolymphangiogenesis) and right panels to mCherry fluorescence emission (tumor content). Scale, Vegfr3Luc: p/s/cm2/sr (x106) and mCherry: p/s/cm2/sr (x109). qRT-PCR analysis of relative mRNA levels of MDA5 16 h after treatment of HLEC with 0.5 ug/ml BO-110 (BO), 10 µM vemurafenib (Vem), or vehicle control (V). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by t-test.   qRT-PCR analysis of relative mRNA levels of VEGFR3 16 h after treatment of HLEC with 0.5 or 1 ug/ml BO-110 (VO), 10 µM vemurafenib (Vem), or the corresponding vehicle control (V). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by ANOVA.   Luciferase signal driven by FLT4 (VEGFR3)-promoter transduced into HLEC treated with vehicle (v) or BO-110 (BO) at the indicated doses (ug/ml) as indicated in methods. Results were normalized to vehicle control. N=4 biological replicates. Error bars correspond to mean ± SD. Statistical significance was determined by ANOVA. Tubulogenic activity of HLECs in the presence of BO110. Images correspond to cells plated in matrigel and imaged at the indicated time points after treatment with 0.5 µg/ml BO-110. Analysis of apoptotic cells at the indicated time points. HLEC cells were treated with vehicle (V) or 0.5 µg/ml BO-110 (BO) for the indicated time points. Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test. Relative mRNA levels of VEGFC and VEGFD in the indicated melanoma cell lines 8 h after treatment with vehicle (V) or 0.5 µg/ml BO-110 (BO), as determined by qRT-PCR. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.
Figure 3(A) The expression pattern of atgl-1 viewed by Patgl-1::GFP transcriptional fusion reporter. Patgl-1::GFP expression is high in intestine. Relatively low fluorescent signals are found in nerve ring, motor neuron commissure, ventral nerve cord and tail. Scale bar: 20 μm. (B) atgl-1 is expressed in neurons. There is no fluorescent signal when Patgl-1::GFP1-10 and Prab-3::GFP11 are expressed separately. When Patgl-1::GFP1-10 and Prab-3::GFP11 are co-expressed, the GFP signals are found in the head region, tail and ventral nerve cord, where most neuronal cell bodies are located. The white lines mark the outline of worms. Scale bar: 50 μm. (C) atgl-1 is expressed widely in neurons. The left panels depict the whole worm. Scale bar: 50 μm. The insets in the right panels are enlarged views of the ventral nerve cord in the white dotted boxes of left panels. The signals of mCherry and GFP merge very well. Scale bar: 20 μm. (D) Visualizing LDs in head neurons with the xdIs109 reporter in different genetic backgrounds. The accumulation of LDs in head neurons of atgl-1(xd314) mutants can be rescued by expressing wild-type atgl-1 in neurons (Punc-119::atgl-1), but not in intestine (Pvha-6::atgl-1), glia (Phlh-17::atgl-1) or hypodermis (Pajm-1::atgl-1). Scale bar: 5 μm. (E) Quantification of the number of LDs in head neurons in different genetic backgrounds. Each dot represents one worm. The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to the control xdIs109. ns: not statistically significant. ****P < 0.0001. Data show mean ± SEM. n ≥ 7. (F) Neuron-specific knockout of atgl-1 causes LD accumulation in neurons, similar to atgl-1(xd314) mutants. Scale bar: 5 μm. (G) Quantification of the number of LDs in head neurons in different genetic backgrounds. Each dot represents one worm. The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to the control xdIs109. t-test was used between the groups under the crossbars. ns: not statistically significant. ****P < 0.0001. Data show mean ± SEM. n ≥ 9
SD-anti SD interaction in bacteria and SD-anti SD like interaction of HCV IRES at the eukaryotic mRNA exit channel.Figure 6(A, B) Comparative solvent side views of the small subunit with the bacterial SD-chamber on the 30S from the 70S POST state (PDB 2HGR) (Yusupova et al, 2006) and the corresponding region of the eukaryotic 40S in the classical 80S*HCV IRES complex. Analogous proteins and rRNA helices are highlighted in the same colours, respectively. (A) Crystal structure of the 30S subunit with mRNA. The SD-helix is established between the mRNA (orange) and the 3' end of the 16S rRNA (blue).(B) Atomic model of the 40S subunit with the HCV IRES model (pink). The mRNA-part and S2 of the IRES are shown in orange and cyan, the 3'-end of the 18S rRNA is shown in blue. (C) Overview and zoomed view of Classical 80S*HCVIRES. The cryo-EM map of the Classical 80S*HCVIRES was filtered according to resolution.(D and E) mRNA exit channel from in Classical 80S*HCVIRES and POST state (PDB 5AJ0) (Behrmann et al, 2015). The two structures are aligned at the 40S body. We note eS28 as part of the 40S head is differently located because of the head tilt. The surface representation was created using the pdb models. Shown are the 18S rRNA (yellow), ribosomal proteins (gray), eS26 (cyan) eS28 (purple), HCVIRES (pink).
FAK is selectively recruited to LAMP-1-positive SCVs.Figure 1(A) FAK immunoblotting was performed on macrophages isolated from the peritoneum of WT and FAKΔmyeloid mice. (B) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 0-5 hours before immunoblotting with the indicated antibodies. (C, E and G) WT PEMs expressing GFP-FAK were incubated with either ΔinvG Salmonella (C), LPS-coated beads (E) or E. coli (G) for a total of 5 hours before analysis by immunofluorescence. Cells were co-stained with antibodies recognizing LAMP1 (red). Dapi was used to visualize nuclei and bacteria (blue). Bars represent 10 µm. Arrowheads indicate bacteria or beads in enlarged panel where bars represent 2 µm. (D, F and H) The percent of LAMP1-positive (gray bars) and LAMP1+GFP+ (black bars) ΔinvG Salmonella (D), LPS-coated beads (F) and E. coli (H) was quantified. At least 100 bacteria were counted per condition; N = 3.
Sensitized CRISPR screens identify TMEM41B as a novel regulator of autophagy.Figure 1 A Schematic representation of pooled CRISPR screening workflow. H4 cells stably expressing Cas9 were transduced with a lentiviral sgRNA mini-pool library, selected for stable integration, expanded and treated for 24 h with the mTOR inhibitor AZD8055 (activated autophagy) or DMSO as vehicle control (basal autophagy). Endogenous p62 or NDP52 were visualized by immunostaining and cells separated by FACS into populations with high or low signal. Abundance of sgRNAs was quantified in cell populations by next generation sequencing  B, C Relative abundance of sgRNAs in cell populations with high versus low signal were visualized as log2 fold chances for basal as well as activated autophagy conditions for (B) p62 or (C) NDP52. Entire data is reported in Dataset EV1  D-F Validation of TMEM41B. H4 Cas9 cells were infected with sgRNAs targeting TMEM41B or a non-targeting (NT) control, treated with 500 nM AZD8055, 50nM Bafilomycin A1 (BafA1) or DMSO vehicle control for 24 h, and analyzed by immunoblotting. (E, F) p62 and NDP52 band intensities are depicted as total levels relative to vehicle control in H4 Cas9 NT cells (E), or as flux by calculating the ratio in BafA1-treated cells versus vehicle control (F). Data are presented as mean ± SD (n=6 independent experiments) with paired t-test values  G LAMP1 and p62 were co-stained and imaged using an LSM700 confocal microscope. Arrowheads point at p62-filled and empty LAMP1-positive organelles. Scale bar: 5 µm  H LAMP1-positive organelles were manually quantified for containing a p62-filled or empty lumen. A total of 50-60 LAMP1-positive organelles were analyzed in each experiment. Data are presented as mean ± SD (n=3 independent experiments) with paired t-test values  I HeLa cells were transduced with Cas9 and TMEM41B or NT sgRNAs followed by infection with luciferase-expressing S. typhimurium. Luciferase readings were taken up to 8.5 h post-infection. Results from one representative experiment are presented as mean ± SD (n=8 technical replicates)
Fig.Figure 1The refined models of four crystal structures of the ribosome bound to aminoacyl- or peptidyl-tRNA analogs are displayed in their respective unbiased electron density Fo-Fc maps (contoured at 2.5σ). The maps were calculated using phases produced by rigid body refinement of the ligand-free test structure put into the Fo data set.A, B. A-site bound substrates ACC-Puromycin (A) and ACCA-Pro (B). The refined models of four crystal structures of the ribosome bound to aminoacyl- or peptidyl-tRNA analogs are displayed in their respective unbiased electron density Fo-Fc maps (contoured at 2.5σ). The maps were calculated using phases produced by rigid body refinement of the ligand-free test structure put into the Fo data set.C, D. P-site bound substrates ACCA-Leu-Phe (C) and ACCA-Pro-Pro (D).
a–c, Mass spectra of purified ARF1-strep in untreated (a), IpaJ-treated (b) or IpaJ C64A-treated (c) cells, with the observed monoisotopic mass reported in daltons.f3a-c, Mass spectra of purified ARF1-strep in untreated (a), IpaJ-treated (b) or IpaJ C64A-treated (c) cells, with the observed monoisotopic mass reported in daltons. *Observed 1 Da shift in molecular mass is accounted for in MS/MS data (Supplementary Fig. 7). d, Structure of N-myristoylated ARF1 N terminus in untreated (left) and IpaJ-treated (right) samples. R group following Asn 3 denotes ARF1-strep protein residues 4-240. e, Reconstitution of ARF1-HisN-myristoylation (N-myr) by NMTp in bacterial cells and IpaJ cleavage reaction in vitro (see Supplementary Information and Methods). f, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-His isolated from bacteria either not expressing (lane 1) or expressing (lane 2) NMTp. Bacterial cell lysates treated with recombinant IpaJ or IpaJ C64A as indicated. The expression levels of ARF1-His were determined by Coomassie blue stain. g, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-strep purified from HeLa cell lysates left untreated or incubated with IpaJ or its catalytic mutant as indicated. The expressed amounts of ARF1-strep are shown (bottom panel). h, In-gel fluorescence assay visualizing protein extracts isolated from HeLa cells incubated with azide myristic acid, azide palmitic acid or geranylgeranyl alcohol azide and subsequently labelled with Alexa Fluor 647 alkyne by click chemistry. Arrows indicate proteins that are proteolytically demyristoylated by IpaJ.
Figure 2A) Schematic of the germline-specific pie-1::gfp::h2b::pie-1 sensor (top) and schematic of the experiment (bottom). B) Representative fluorescence images of transgene expression in wild-type, rpb-9 (mj261), hrde-1 (tm1200) and prg1 (n4357); prg-2 (n4358) animals in the parental (P0) and inheriting (G1-G6) generations. Scale bar = 50 µm.
Mito-C counteracts dengue virus replicationA, Huh7 cells were treated with Mito-C at the indicated concentrations and simultaneously infected with dengue virus (MOI = 5).Figure 4A, Huh7 cells were treated with Mito-C at the indicated concentrations and simultaneously infected with dengue virus (MOI = 5). Infectious titers are presented as % of DMSO in Mito-C concentration of 0.625, 2.5 and 10µM conditions, upon 48h of viral infection. Errors bars show the standard error of the mean (SEM) (n=3). B, Huh7 cells were treated with or without (DMSO) Mito-C, infected with dengue virus and fixed after 48h of infection. Cells were then immunostained for endogenous TOM20 (green channel), viral non-structural protein NS5 (red) and DAPI (Blue). Cropped areas illustrate the mitochondrial morphology in described conditions. C, Quantification of the mitochondrial phenotypes observed in b. Errors bars show the standard error of the mean (SEM). (15-20 images each with an average of 20-25 cells from triplicate independent experiments were analyzed).
Macrophages recruited after irradiation are polarised towards an immunosuppressive, pro-tumourigenic phenotype.Figure 2A, Quantification of iNOS expression on gated macrophages (CD11b+ F480+) from MC38 and KPC tumours, with representative histograms. Data are presented as mean ± SEM and analysed by Mann-Witney (n=6 mice/group). B, Quantification of CD206 expression on macrophages (CD11b+ F480+) from MC38 and KPC tumours, with representative histograms. Data are presented as mean ± SEM and analysed by Mann-Witney (n=6 mice/group). C, Quantification of the percentage of TAMs (CD11b+ F480+) in MC38 control tumours compared with irradiated tumours (n=6 mice/group). D-E Quantification of iNOShi (D) macrophages as a percentage of total cells in MC38 tumours (n=6). D-E, Quantification of CD206hi (E) macrophages as a percentage of total cells in MC38 tumours (n=6). F, The total number of iNOShi TAMs were divided by CD206hi TAMs to derive a M1/M2 ratio in MC38 and KPC tumours receiving mock or 10Gy irradiation. Data are presented as mean ± SEM and analysed by Mann-Witney (n=6 mice/group). G-H, TAMs (CD11b+F480+) were isolated by FACS. Expression of selected immune stimulatory and immunosuppressive genes in TAMs was determined by RT-qPCR (n=3). Data are presented as mean fold change ± SEM compared to TAMs from mock-irradiated tumours (n=3). Statistical significance was determined by Mann-Witney. I-J, Assessment of TAM suppression of T cells was assayed by evaluation of inhibition of T cell proliferation. TAMs were isolated by magnetic bead separation using F480 microbeads and co-cultured at a 1:1 ratio with CFSE labelled CD8+ T cells. CFSE dilution was analysed by flow cytometry to measure proliferation. Percentages of CFSElo T cells were analysed by Kruskal-Wallis with Dunn's multiple comparisons test. Representative histograms are shown (right panel). Experiments were repeated twice for each tumour cell line (n=3 mice/group) *P <0.05 **P <0.01 ***P <0.001.
Figure 2(A) Time-lapse observation of the establishment of bipolarity in acentrosomal cells. HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed with a 63× objective. Magenta and green represent mCherry-NuMA and EGFP-centrin1, respectively. Arrowheads indicate the assembling NuMA after NEBD. Two-way arrows indicate the bipolarity. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (B) Time-lapse observation of the dynamics of NuMA and chromosomes. Centrinone-treated HeLa cells expressing mCover-NuMA were observed with a 63× objective. Magenta and green represent SiR-DNA and mClover-NuMA, respectively. Arrowheads and two-way arrows indicate the assembling NuMA after NEBD and bipolarity, respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (C) The timing of bipolarity establishment in the acentrosomal pole, chromosome alignment, and anaphase onset Each plot shows the cumulative percentage of each event at individual time points (N=49 cells from two independent experiments). (D) The distribution of kinetochores during acentrosomal spindle pole separation was confirmed in fixed cells. Two-way arrow indicates the bipolarity of elongated NuMA structures. Red, gray, green, and blue represent NuMA, CENP-C, GT335, and DNA, respectively. Scale bar, 5 μm. (E, F) Time-lapse observation of the structure of NuMA and microtubules upon centrosome removal. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were observed with a 60× objective. Red and green represent NuMA and SiR-tubulin, respectively. Z-projections of 17 sections, every 1 μm. Scale bar, 5 μm. Time zero corresponds to NEBD. (F) An example of NuMA formed several asters (arrowheads) at the time of NEBD.