EMBO/sd-character-level-ner huggingface dataset:
B. Miro1-EF is less susceptible to ubiquitination by Parkin. HEK cells overexpressing HA-ubiquitin, myc-Miro1 or myc-Miro1-EF and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. HA-ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin (red), Miro1 (green) and Parkin (green; representative images are from the same experimental material on separate SDS-PAGE-WB membranes). The high-contrast grayscale shows the green channel of this blot, and the bar chart shows quantification of co-immunoprecipitated Miro from 3 different blots (**P < 0.01 and ***P < 0.001, repeated measures ANOVA). Note that EYFP-Parkin co-immunoprecipitated with ubiquitin only after A&O treatment
J Quantification of the relative number of BrdU+ cancer cells found in DMSO DEBIO-0932 treated BrM-PDOC respect to the corresponding PDOC treated with DMSO. Values are shown in box-and-whisker plots where every dot represents a patient (mean value obtained from all PDOC from the same condition and patient) and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=19 patients with DMSO treated PDOC, n=14 DEBIO-0932 10 µM and n=15 DEBIO-0932 1 µM, each patient is an independent experiment). P value was calculated using two-tailed t-test. Dots are colored according to the primary source of the metastasis.
D Analysis of pSTAT3 levels in MDSCs after co-culture with GFP-expressing or TRF2-overexpressing cancer cells treated with or without sodium chlorate (NaClO3). Data information: In (D), data represents mean of n = 3 independent experiments +/- SEM (***p < 0.001; Student's t-test).
(B) Representative merged and single channel pictures of cardiac CD31, DAPI and WGA immunostaining in mouse hearts as indicated. The quantification of the myocardial capillary density as capillaries/cardiomyocyte (CM) ratio, 7 days (d) after cryoinjury or sham surgery is shown as bar graph (B´). Scale bar: 20µm. **p=0.0039.
D Normalized luciferase activity by transfecting 300 ng of each PTEN−luciferase reporter. Open bar: vector transfection control; filled bar: TFEB transfected. Student's t−test, n = 4; *P = 0.01; **P = 0.005
Collagen abundance in ECM produced by NIH-3T3 cells expressing empty vector or Slc25a1 cDNA grown in the presence or absence of TGFβ, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated empty vector expressing cells.
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.
Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), or control (A-I) combined or not with a treatment with NU-7441 (E,F) followed by western blot analysis.
(a) Electron micrographs of the IM (black arrowheads) associated with the ER (black arrows) from Atg4BC74A-expressing NIH 3T3 cells. The IM appears to expand between the two ER cisternae and forms an autophagosome. The white arrowhead indicates vesicular clusters visible in the vicinity of the IM. The white arrow indicates the autophagosome.
(C) Blood and brain tissue levels of CNP520 in the rat after a 30 µmol/kg (15.4 mg/kg) oral dose. Shown are unbound fraction (main graph) and total levels (insert) of CNP520 in the blood and brain, using values for rat plasma protein binding of 97.8% and unspecific binding to brain homogenate of 99%. Values are mean ± SD, with n = 5 per time point.
D Cell growth of the indicated stable cell lines treated with trastuzumab (Trz) or vehicle (veh) as measured by SRB assay. Trastuzumab-sensitive HER2+ breast cancer cell lines (S) and trastuzumab-insensitive HER2+ breast cancer cell lines (partial responsive, PR; resistant, R) were treated with 1 µg/mL or 100 µg/mL trastuzumab, respectively. Data represent the mean ± s.d. P-values were calculated using RM ANOVA with a post-hoc LSD test. cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive.
A Representative kymographs of LysoTracker-labeled lysosomes in proximal dendrites of hippocampal neurons infected with scrambled shRNA (shSc), LAMTOR2 shRNA (shLAMTOR2), or Raptor shRNA (shRaptor). Scale bar, 5 µm. B Quantitative analysis of lysosomal movement from kymographs (N = 44, 24, and 19 neurons for shSc, shLAMTOR2, and shRaptor, respectively, from 3-10 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Sidak's post-test ***P < 0.001 as compared to shSc; n.s., not significant.
C) Kaplan Meier survival curve for neuroblastoma patients with low (red) or high (blue) expression of H2AFZ (H2A.Z.1) obtained from R2 genomics.
B. Illustration of saltatory tangential migration performed by an interneuron. Nucleus is marked with a red asterisk. All images taken on a spinning disc confocal microscope at 10x magnification.
(f) The physical interaction between Aurora B and survivin was analysed reciprocally and reduced in cells expressing high levels of nuclear ATG5. Neither ATG5 nor HSP90 were detectable in the Aurora B-containing immune precipitate, but ATG5 molecularly interacted with survivin. Results representative of three independent experiments.
Representative images of wild-type (WT) and Ctns−/− mouse fibroblasts transfected with the ptfLC3 vector under resting conditions (fed), or after serum starvation (Serum Starv.) in the presence or absence of the alkalinizing drug chloroquine (CQ). GFP and RFP staining was analyzed by confocal microscopy. Examples of red-only puncta (mature autophagosomes) are indicated with arrows. Scale bar: 5 μm.The percentage of mature autophagosomes (red-only vesicles) was calculated based on the ratio between the number of red-only puncta and the total number of autophagosomes (number of green and red + red-only puncta). The graph is representative of three different experiments with similar results. Results are mean ± SEM (n = 7 cells per condition). In addition to the experimental conditions shown in (A), the Aa/serum starvation condition is included.
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. (
Samples were TCA precipitated, treated with NEM, and LMF1 was purified via its C-terminal His tag. Samples were resolved by reducing or nonreducing SDS-PAGE. The lanes marked -LMF1 are HEK293∆LMF1 cells included as a negative control. Samples were probed with an antibody for TRX.
(A) Identification of critical amino acids for HA-LC3 conversion. 293 cells were transfected with the indicated CD16:7-263-281 mutants and HA-LC3A, subjected to anti‐CD16 aggregation and lysed for western blotting against the indicated molecules. Asterisks mark mutations with reduced activity. Shown is one representative experiment of six repetitions.
B, Schematic depicting the endogenous protein, and the artificial mini-nesprin-2G and MUT-mini-nesprin-2G constructs. Both constructs contain the first two and last two spectrin repeats of the endogenous protein; the mutant construct presents two mutations on the actin-binding domain that prevents it from binding to actin.
D In vitro ubiquitination was performed with recombinant GST-Mdm2 (left) or GST-Mdm2 C464A mutant (right) and Flag-NAT10 or Flag-NAT10 GE in the presence of E1, E2 and Ub in vitro as described in Experimental Procedures. The reaction products were resolved by SDS-PAGE, followed by western blot for evaluation of the indicated proteins.
(A) Genes up- (red) and downregulated (blue) in the tumor cells from lymph nodes (Vav1∆C LN) and spleen (Vav1∆C SP) of recipient mice transplanted with Vav1∆C-expressing CD4+ T cells. As comparative control, we used CD4+ T cells from spleen (C SP) and lymph nodes (C LN) of control recipient mice. Rows represent individual genes. Columns represent experimental replicas. Total number of genes is indicated on the left. Relative changes in abundance are shown in color gradients according to the color scale shown on the right.
A PRK5 domain structure: SP signal peptide (aa 1-39), LRR leucine‐rich repeat, RPT internal repeat, LC region of low complexity, TM transmembrane domain (aa 282-304), EM Y‐based sorting/endocytosis motif (YSSM; aa 670-673).
G. The luc activity driven by the indicated A-capped monocistronic mRNA in the presence of Endouc or EndoucK242A in HEK 293T cells. The m7G-capped Rluc mRNA was used as an internal control to normalize the activities of A-cap Fluc transcripts. The egfp mRNA was used as a negative control, and the luc activity of cells transfected with egfp mRNA and A-cap Fluc mRNA containing full-length huORFchop-1-105-nt was set to 1. The data were averaged from three independent experiments and presented as mean ± S.D. (n = 3). Student's t test was used to determine significant differences between each group (**: P<0.005, ***: P <0.001).
Quantification of the Filipin III intensity at primary cilia from (A). Rapamycin-induced peroxisome targeting to the ciliary pocket restored the insufficiency of ciliary cholesterol in the Rab10−/−and KIFC3−/−hTERT-RPE1 cells, but not in ORP3−/−and EHD3−/− hTERT-RPE1 cells (*p<0.05, **p<0.01, ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 35-40 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.
(O-R) ZK1053.4::GFP is weakly expressed in wild-type embryos (O-P) and sqst-1 mutants (R), but forms a large number of aggregates in epg-7 mutants (Q). (O) DIC image of the embryo shown in P.
D) Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in pid-4 mutants. Colour scheme represents the odds ratio of overlaps representing the strength of association.
(C) Forty mice were inoculated with MM cells and treated or not with BoxA. Tumor growth was detected via BLI. Lines that do not reach day 34 correspond to mice that were sacrificed for ethical reasons.
E. MCF-7 cells were transfected with control siRNA, TRIM46 siRNA, or a siRNA-resistant TRIM46 construct together with TRIM46 siRNA. Western blotting (left) and RT-qPCR (right) were performed as in A.
(c,d) Histological analyses (haematoxylin and eosin staining) of lungtumours in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 18 weeks after AdCre inhalation. (c) Nuclear and cellular polymorphy, more dense and sometimes irregular chromatin as well as more nucleoli in tumour cells of KRas;Atg5fl/+ mice as compared with KRas;Atg5fl/fl tumours. Sections were analysed 18 weeks after AdCre inhalation. Scale bars, 50 μm. (d) KRas;Atg5fl/fl tumours exhibited markedly increased apoptotic figures and necrotic areas in the centre of larger tumours (asterisk).
(E) Viability, as measured by Alamar Blue reduction, of promastigotes. All data are means ± SD from three independent experiments. *, Alamar blue reduction was significantly different (p<0.05).
Apoptotic mitochondria were decorated with antibodies against Bax (green) and Tom22 (red). The Bax signal was recorded in the STED mode. Shown are the individual color channels as well as an overlay (from top to bottom) of several apoptotic mitochondria. We used Lagrange interpolation to double the number of pixels. Note that throughout this manuscript except for contrast stretching no image manipulation was applied. Scale bar: 0.5 µm.
C. iPOND experiment using HUWE1-knockout 293T cells, corrected with wild-type or PIP-mutant HUWE1. The PIP-mutant shows reduced crosslinking to nascent DNA, indicated defective recruitment to replication forks.
(A) Schematic representation of TREM2 processing by ADAM10/17. Cleavage occurs C-terminal of residue His157. The entire ectodomain (residues 19-171) was used for immunization of rats to generate TREM2 antibodies. sTREM2 (soluble TREM2), CTF (C-terminal fragment).
Control or CEP41-depleted HUVECs were transfected with expression vectors encoding nothing (MOCK) or HIF1α and treated with MG132 or not, and then the cells were cultivated under hypoxia. Whole cell lysates were used for immunoblot assays for HIF1α, phospho-AURKA, and AURKA, and the protein levels were normalized against β-ACTIN in the same blots. NOR, normoxia; HYP, hypoxia.
D, E Aβ40 and Aβ42 concentrations in CSF of male and female APP24 mice (homozygous; 2 (n = 13), 3-4 (n = 16), 7-8 (n = 15), 18-19 (n = 14), 24 (n = 16), and 30 (n = 18) months of age). CSF Aβ40 followed a significant quadratic trend (F(1, 86) = 6.678, P = 0.011) and CSF Aβ42 best fitted a cubic trend (F(1, 86) = 30.599, P < 0.001).
(K) qPCR levels of LOX-L2 in PSCs, normalized to RPLP0 (60S acidic ribosomal protein P0) and relative to control
(D) Immunoblot showing the levels of pulled-down PhotoClick Cholesterol (streptavidin-HRP) in ER and MAM fractions. Note how the levels of pulled-down C99 in the ER are negligible when compared to those from MAM. Isolated MAM and ER fractions were assessed by western blot (shown in AppendixFig S1E).
(C) Confocal images of ATG9A colocalization with p62/SQSTM1 in HCT-116 ATG9A-HA KI- ATG13 WT, ATG13 KO or ATG13 KO cells reconstituted with ATG13 WT, ATG13 Δ2AA and ATG13 ΔHORMA. Cells were grown in full DMEM media, fixed, labelled with antibodies for HA and p62/SQSTM1 and imaged (Scale bar=10 µm). Images are from the same single independent experiment (D) Quantification of average surface area of p62/SQSTM1 puncta in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates. A break was inserted in the Y axis to accommodate the wide range of p62/SQSTM1 puncta sizes. (E) Quantification of ATG9A colocalization with p62/SQSTM1 in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates.
(B) LN229 cells stably transduced with 6x-Histidine-tagged Ubiquitin (His-Ubi) were then stably transduced with indicated shRNAs against AMOTL1 or a scrambled shRNA control (sh-Co.). These cells were detached, lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.
A. Schematic representation of the RNF43 protein. SP; signal peptide, PA; protease-associated domain, TM; transmembrane domain, RING; E3 ligase catalytic domain, DIR; Dishevelled-interaction region, SRR; Serine-rich region, HRR; Histidine-rich region, PRR; Proline-rich region. The region in which oncogenic truncations occur (aa 504-563) is indicated.
The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated with LPS (20 ng/mL, 20 min) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.
(E-F) Immunofluorescence was conducted to detect the expression levels of cGAS, NF-κB, and pyroptosis associated molecules (GSDMD, caspase-1, and IL-1β) in BV2 cells. n = 3 in each group. Scale bars, 50 μm.
F Representative images of spheroid invasion assay from sg-CTRL and sg-HMGCL (#2, #3 and #4 clones) PANC-1 cells after 10 days of culture in presence of Matrigel. Photos are representative of n=3 independent experiments.
Effect of VISP1-Flag and the autophagy inhibitor 3-MA on accumulation of SGS3-Flag and SGS3ΔCC-Flag in N. benthamiana leaves. The co-infiltrated leaves were treated with 3-MA (10 mM) or buffer at 48 hpi and were harvested for western blotting 12 h later. Accumulation of AtSGS3 transcript was analyzed by RT-PCR.
Oxygen consumption (F-G) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day.
F Relative mRNA levels of ERRα, Mfn2, and Nrf1 were evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT. P-values versus WT: ERRα ***P < 0.0001, Mfn2 **P = 0.002 and Nrf1 **P = 0.009 (n = 6-7 and 7-8 for WT and SOD1G86R, respectively, at 65 days; n = 5 and 6 for WT and SOD1G86R, respectively, at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).
(D-F) Quantification of the Rango-3 E gradients (top panels) and average cellular Rango-3 E (bottom) in involving untreated oocytes (controls) or oocytes injected with RanT24N mRNA, importin β(71-876) protein or treated with 20 µM IPZ. Data for each panel are from at least 2 separate experiments. Means ± SDs, t-test, oocyte numbers indicated in brackets.
A. Schematic diagram of the social interaction device indicating the social and the empty chambers.B, C. Mice were simultaneously exposed to an empty container and a caged unfamiliar juvenile mouse (social enclosure, stranger 1). Lrig1-mutant mice exhibit social interaction defects as determined by the time spent interacting (sniffing) with the stranger enclosure (B) and the percentage of total interaction time with stranger in the three-chamber social interaction test (C). Dashed line in (C) represents chance-level performance (i.e. 50%) when mice equally explore the social enclosure and the empty container. Data represent means ± SEM of independent determinations performed in n=8-9 mice of each genotype, and the statistical significance between wt and knockout mice are *p<0.05 by Students t test.
(A) In vivo fundusimage of an AAV-injected rd1mouse expressing ReaChR-mCitrine driven by the hSyn promoter (AAV2-hSyn:ReaChR-mCitrine); transduced RGCs are visible as green puncta.
B,C EDTA-SMP were solubilized at the indicated pH values, extracted with 1% (w/v) digitonin and subjected to BN-PAGE in order to separate dimers (Vd) and monomers (Vm), which were identified by Coomassie blue (panel B) or in-gel activity staining (panel C) and analyzed by densitometry, which is reported in the bottom part of each panel. Values report the dimer/monomer ratio, where the ratio at pH 7.4 was taken as 100%. Data are an average ± s.e. of 3 independent experiments. The p-values calculated with the Student t test are shown * p≤0.05 *** p≤0.001.
A. Effects of GOLPH3 KD or OE on glycosphingolipid synthetic flux as assessed by [3H]-sphingosine pulse (2 hr) and chase (indicated times), lipid extraction and HPTLC separation and radioactive counting (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]).
insulin tolerance test (ITT) from WT and Parkin-KO mice fed with chow (CD) or high fat diet (HFD). (n=8; WT) (n=6; Parkin-KO).
HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). J Cell survival by CellTiter-Glo after 24 hours (mean ± SEM, n=4 biological replicates, **P<0.01, ***P<0.001, ****P<0.0001, ANOVA).
Immunoblotting of BM67 cell lines extract and its quantitation following incubation with 100 µg/mL cycloheximide as indicated. Graphs represent mean ± standard deviation (n = 5).
Graph showing the body weight of WT, KO and KO+ AAV-mWwox at different days after birth (n= 4 per group).
E. MEFs cells were transiently transfected with GFP-REV3L761-1029 and fixed with 4% formaldehyde. The distribution of GFP- REV3L761-1029 was detected by autofluorescence (green), chromocenters were visualized by H3K9me3 immunostaining (red, top panel) or HP1α (red, bottom panel) and DNA was counterstained with DAPI. Line scans represent the colocalization of proteins within each image (right panels). Scale bar= 5 μm
(e) Time-lapse microscopy. HeLa H2B-mCherry-α-tubulin-EGFP cells were transduced by lentiviral gene transfer as indicated. Only ATG5-overexpressing cells demonstrated evidence for mitotic catastrophe (see also Supplementary Movies). As with the GFP control, the Beclin 1 construct also included GFP, hence the intensity in green was very high. Scale bar, 10 μm.
C. Distribution of bone marrow cell types in vehicle- and tamoxifen-treated mice. The pie charts are colored accordingly to panel B. Only cell types with more than ten cells in any of the conditions are included.
Anti-HA co-IP showing that GAPDH fused to the HIV-1 MHR (GAPDH-MHRhiv), but not HA-tagged GAPDH (GAPDH) binds to Flag-CLIP170 (C) in 293T cells. Results are representative of three experimental replicates.
(A) Predicted protein domains for Mcp3. Mcp3 is predicted to contain a canonical N-terminal mitochondrial targeting signal (black bar) that contains a hydrophobic stretch (hatched part). The predicted MPP and the Imp1/2 cleavage sites are indicated by a black or grey arrow head, respectively. The two predicted transmembrane domains are highlighted as grey bars.
U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 2 h starvation (EBSS) or 4 h BafilomycinA1 (BafA1, 200nM), lysed in total cell lysis buffer and subjected to western blot analysis. Blots were probed for the presence of HA-tag (ATG2A), p62/SQSTM1, LC3B, pan-GABARAP (GABARAP, GABARAP-L1 and GABARAP-L2) and vinculin (loading control).
PIP strip lipid binding assay with recombinant PTPIP51_ΔTM, PTPIP51_TPR, and PTPIP51_ΔTPR.
(C) Mitochondria were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG and control cells. The samples were analyzed by SDS-PAGE and Western blot. Mitos, mitochondria.
Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stimulated with plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) antibodies for 72 h and the proliferative activity was measured. Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, *p < 0.05). The mean value for the activity of Ifih1-/- thymocytes in an unstimulated condition was set as one. For comparative analysis, the same data shown in Appendix Fig S1B was used for Cd4cre A1fl/fl mice.
Immunofluorescence microscopy quantitative analysis for colocalization of M.tb with p47phox (K and L) as described above in WT BMMs. Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.
(G) Cell cycle analysis in Ntras-silenced H5V cells (n = 3 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (G) two-way ANOVA,
(J) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs transfected with DNase I (15 μg, 6hr).
A Schematic representation of PINK1 variants phosphorylating Ub at Ser65. hPINK1, human PINK1; TcPINK1, Tribolium castaneum PINK1; PhPINK1, Pediculus humanus corporis PINK1.