EMBO/sd-character-level-ner huggingface dataset:
MMP was monitored by TMRE fluorescence at every 1 h. FCCP (200 µM) and antimycin A (50 µM) were used as pretreatments for 30 min. Data were normalized by basal state of WT A549 cells (B-D) Data at 1 h (C), 5 h (D are presented as mean ± SEM; n = 4 (B-D) biological replicates, ****p < 0.0001, **p < 0.01, *p < 0.05 by paired T-test (C), or one-way ANOVA followed by Dunnett's multiple comparison test (D
PARP1 and OAS1 compete for the 2'-hydroxyl of the terminal ADPr of PAR (shown in purple), which may result in "capping" of PAR chain termini with adenylates. The 2'-hydroxyl of the terminal adenylate (shown in red) can be further modified by OAS1 (OAS1 typically adds 2-3 AMPs), but presumably not by PARP1. 2'-positions modified by PARP1 are shown in blue. Besides the terminal ribose, PARP1 also attaches ADPr to the 2'-hydroxyl of the second ribose moiety of ADPr (shown in blue in parentheses), producing a branched PAR structure. We propose that 2-5A capping of PAR by OAS1 results in shorter and potentially more branched PAR chains (bottom).
nanoCAGE sequencing was applied to determine transcription start sites in shERα BM67 cells. The number of detected transcription start sites (peaks) and RefSeq transcripts when sampling increasing number of RNAseq reads are indicated to evaluate the complexity of nanoCAGE RNAseq libraries.
C-F Cell morphology of strains DWA350 and DWA307 following growth in LB at 30°C (C and D) and at 42°C in LB + 0.5% w/v xylose (E and F). Arrowheads indicate minicells. Cell membranes were stained with FM5-95. Insets show the corresponding phase contrast light microscopy images. Scale bar, 5 μm.
(A) Schema of the analysis of gene expression in islets isolated from control larvae and larvae subjected to β-cell ablation. β cells were ablated by exposing nitroreductase (NTR)-expressing transgenic larvae to metronidazole (MTZ) from 3-4 dpf. Islets were then isolated, and their RNA extracted and analyzed by microarray. Out of the 470 genes that were upregulated more than 50%, 33 genes encoded proteins that harbored a signal peptide for secretion (according to the algorithm of SignalP). Excluding genes that encode enzymes or proteins with a transmembrane (TM) domain, we selected 11 genes for overexpression studies in zebrafishlarvae (C-E).
E After transfection with the indicated siRNAs, cells from (D) were cultured for 2 weeks. The colonies formed were stained and photographed.F The area of the colonies in (E) was quantitated using an image analysis system and represented as percentage of the total area of the plate. *P = 0.024.
F-H Viability assessment of RKO (F), MiaPaCa-2 (G) and HeLa (H) cells treated with DMSO, IMT1 alone and in combination with FG4592. Data are expressed as mean values ± SD of n=6, 5, 6 independent experiments for RKO, HeLa and MiaPaCa-2, respectively. Statistical significance was calculated with one-way ANOVA. Paired IMT1 vs IMT1 + FG4592 comparisons: RKO: **p<0,0028, MiaPaCa-2: **p= 0,0011, HeLa: *p= 0,0029.
(D) Pull-Down of GST-CTC but not GST-CTD or GST-CTE (middle IB) by immobilized MBP-β2AR C-terminus (left) but not MBP alone (right), all of which were present at comparable amounts (bottom and top IBs, respectively). GST fusion proteins were detected by an anti-GST antibody and MBP fusion proteins by an anti-MBP antibody.(E) Quantification of (D) (***p<0.001, ANOVA).
A Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of freshly isolated CD8 T cells from wildtype (WT) or Peli1 KO (KO) mice (6 weeks old). Data are presented as a representative blot (left) and summary graphs of densitometric quantifications of the indicated proteins (presented as phosphorylated/total protein ratio) based on three independent experiments.
H. Frequency (in percentage) of pre-miR-181a-1 colocalization with vesicles double positive for organelle markers. Each data point corresponds to one axon. Total number of puncta, axons and independent experiments analyzed: 745 puncta, 23 axons, n=5 (CD63/pre-miR-181a-1/Rab7a), 577 puncta, 18 axons, n=3 (CD63/pre-miR-181a-1/LysoTracker). Values are median with interquartile range. Abbreviations: ns, not significant; CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1.
Volcano plots displaying the genes that are differentially expressed in immature enterocytes 2 upon SARS-CoV-2 infection of colon organoids. infected vs. mock infected cells The statistical significance (-log10 adjusted p-value) is shown as a function of the log2 fold change. MAST tests were used to generate P‐values, bonferroni multiple hypotheses correction was used to compute FDR values. Labeled dots in blue in all panels are gene names of selected differentially expressed genes between the compared two populations. Labeled dots in red in all panels are gene corresponding to interferon if detected.
A TFIISmut shows upstream splice site selection. The splicing was assessed by RT-PCR and capillary electrophoresis in 3‐week‐old soil grown plants. The chart represents the average relative contribution of the mRNA forms found in the total pool of amplified products. The error bars represent ± SD (n = 3). To the right of the charts, the structures of the examined transcripts are shown. The black boxes, white boxes and black lines represent constitutive exons, alternative regions and introns, respectively. The black arrows show the locations of the primers. Representative splicing assays are shown.B Directionality of splice site selection in TFIISmut. For each type of alternative splice event, the black and white boxes show the contributions of opposite direction splicing events. The numbers represent the percentage of splice events supporting the direction of the splice site event change (also shown on horizontal axis). The numbers on the right‐hand panel represent the number of affected splicing events versus total number of splicing events analysed. The white bars represent downstream 3′ and 5′ splice site selection (3′SS/5′SS), exon skipping (ES) and intron retention (IR), while the black bars represent upstream 3′/5′ splice site selection (3′SS/5′SS), exon inclusion (ES) and intron splicing (IR).
(D) Representative images of lungs, livers, intestines, and kidneys from mice 8 weeks after orthotopical injection of 22Rv1-M cells transfected with either Scramble-shRNA or ESM1-shRNA. Scale bar: 0.5 cm. Tumors were removed and weighed. Quantitative summary of tumor luminescence in different metastases measured in photons/second are shown.
B. Ponatinib inhibits LPS-mediated cytokine release in THP1 macrophages in vitro. Data information, data are shown as the mean of three technical replicates. Error bars denote SEM.
(B) MIA Paca-2 cells were transfected with HA tagged ArfGAP1 or RFP1 (control) for 4 days and then cell numbers were counted. Six replicated wells were counted each group. Columns represent Mean ± SEM. Student's t-test *P = 0.028.
A Theoretical calculation of the rescaled radius of paraspeckles versus the rescaled transcription rate for the case in which the A and C blocks were deleted by CRISPR/Cas9 in the steady state. The graph is derived by assuming that NEAT1_2 is produced at a constant rate. The produced NEAT1_2 RNPs diffuse in the solution and the free diffusion is hindered by the attractive interactions between NEAT1_2 RNPs with the interaction parameter χ (we used χ = 1.0). NEAT1_2 was degraded at the constant rate k0. The radius was rescaled by the length scale $\sqrt{D/k_{0}}$ and the transcription rate was rescaled by the inverse time scale k0(D/k0)3/2/v0.
(D) Treatment with 1,25D3 enhances co‐immunoprecipitation of p27KIP1 with CDK4 in U937 cells. Extracts of U937 cells were probed for p27KIP1 and CDK4, and immunoprecipitated with an anti‐CDK antibody and probed for p27KIP1 co‐immunoprecipitation.
A-D. EV-producing cells expressing or not Cx43 (HEK293Cx43+ or HEK293Cx43-) were transiently transfected with shRNAs targeting hnRNPA2B1, hnRNPQ or non-target shRNAs, for 48 h. miR-133b levels were assessed in EVCx43+ and EVCx43- (A; n=6-12 biological replicates) and EV-producing cells (B; n=4-9 biological replicates). miR-509-3p levels were assessed in EVCx43+ and EVCx43- (C; n=6-15 biological replicates) and EV-producing cells (D; n=4-9 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.
(I) RNA ISH showing the co-localization between circLMP2A and miR-3908 in SNU-4th cells. circLMP2A probes were labelled with Cy3, and miR-3908 probes were labelled with FITC. Nuclei were stained with DAPI.
(B) A standard uptake reaction was performed. After 70 min of incubation, the vacuoles were reisolated, washed, and treated with protease. The vacuoles were reisolated again, resuspended in reaction buffer without luciferase, and incubated for the indicated times at 27°C. Aliquots were withdrawn to assay the total luciferase remaining. As a control, luciferase was incubated in reaction buffer for the same periods of time and assayed for activity.
(B) Diagram showing the percentage of up- and down-regulated genes in the RNA-seq experiment comparing NTC vs. shB DVL3 (left panel) and EV vs. DVL3-WT (right panel) in MDA-MB-468 cells.
(b) Upper panel: Representative images of BOEC colonies from PBMC cultivation 10 days post-seeding. Dotted line in each image outlines a BOEC colony. Lower panel: Stabilized BOEC cultures after passaging of colonies (scale bar, 250 µm). (c) Proliferation doubling time for NAFLD and healthy BOECs based on passages 2 and 3. Sample sizes are n = 9 healthy, n = 7 NAFLD; with 3 biological replicates per donor. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, not significant (t-test).
Cell cycle analysis by flow cytometry of BT474 and BTRH cells (50,000 events) treated with EV20/MMAF (10 nM) for the indicated times.
E A working-model for the feedback regulation of synaptic activity. Prolonged neuronal excitation downregulates VSP34-mediated synthesis of endosomal PI(3) P. PI(3) P depletion causes the activation of Calpain 2 and Cdk5, resulting in repression of SV recycling, neurotransmission and kinetic impairment of SV endocytosis to limit SV re-supply and downregulate neuronal network activity.
(J) Myeloid subtype (CD11b+Gr1mid), and c-Kit positivity inside myeloid fraction in BM (n = 3-5).
MoLC were transfected with control (siCTR) or S100A9-specific (siS100A9) siRNA and checked for S100A9 expression by RT-qPCR (left) and western-blot (right). Shown is a representative experiment out of six. RT-qPCR data were represented as means of duplicates ± SD.
(A) Cartoon representation of the NHE9 homodimer shown from the extracellular-side in the endosomal lumen (left), and along the membrane plane (right). Ion translocation 6-TM core transport domains (blue), dimerization domains (orange) and linker helix TM7 (grey), are coloured as in Fig. 1E with the respective helices numbered.
A. RT-PCR products with divergent and convergent primers showing circularization of has_circ_0079480 and has_circ_0087391. cDNA, complementary DNA; gDNA, genomic DNA.
C, D Quantification of the mitochondrial number and morphology in patient 1 (C) and the Mbtps1-cKO mouse model (D). (C) Left panel: n = 13 biological replicates of normal individuals, n = 10 biological replicates of patient 1. Middle and right panel: n = 121 biological samples of normal individuals and patient 1. (D) Left panel: n = 23 biological replicates of Mbtps1-loxP mice; n = 15 biological replicates of Mbtps1-cKO mice. Middle panel and right panel: n =113 biological replicates of Mbtps1-loxP mice and Mbtps1-cKO.
G-J Time-controlled knock-down of metabolic enzymes in the dorsal compartment of ubi-AT1.03NL wing discs using the apGalts driver. Images of FRET efficiency in control (G), apGalts>PfkRNAi (H), apGalts>GapdhRNAi (I), and apGalts>Glo1RNAi (J) wing discs after RNAi induction for 48 h (G), 120 h (H, J), and 93 hours (I). A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars = 50 µm. K Line graphs showing the FRET efficiency in the ventral and dorsal compartment of individual control (n=27), apGalts>PfkRNAi (n=7), apGalts>GapdhRNAi (n=10), and apGalts>Glo1RNAi (n=6) wing discs. Loss of Pfk, Gapdh or Glo1 in the dorsal compartment of the wing disc reduces the levels of ATP. Paired t-test, ns= not significant, **p≤0.01, ***p≤0.001.
Analysis of p62/SQSTM1 levels in patient and control fibroblasts transduced with control or VPS16-expressing lentivirus under basal conditions and after 2 hours of serum-starvation.
ChIP analysis of naïve WT or Stat4-KO CD4+ T cells cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days. The cells were restimulated with IL-6 or LIF for 30 minutes, crosslinked with formaldehyde and immunoprecipitated with the anti-STAT3 antibody, followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1-p10 primer pairs. The results are presented relative to the amount of input DNA.
(h-k) Representative TEMs of wild-type and hlh-30 animals after 24 h starvation. IEC, intestinal epithelial cell; EPI, epidermis; BB, brush border; GON, gonad; BWM, body wall muscles, aj, apical junction; ld, lipid droplet. Scale bar, 2 μm.
WT and C2βD1212A/D1212A mice were subjected to tMCAO followed by 24h of reperfusion. (A) Gene expression of TNFα, IL-1β and IL-6 in the cortex and basal ganglia (B. ganglia). The mRNA levels are given as the fold change normalized to rps29 relative to the corresponding contralateral (healthy) hemisphere. Data represent mean ± SEM (n=8-10 mice per group; *p=0.05; **p<0.01, Unpaired t-test with Welch's correction). Data represent mean ± SEM (n=5-7 mice per group; **p<0.01; Mann-Whitney test).
G. The final circadian periods of Cry1-/- SCN slice double-transduced in complementary reverse orders did not differ between each other, p=0.7.
(B) Convertase labeled perimCherry/cytoGFP bacteria (Green) exposed to C5b6MAC or Conv-MAC. Conditions were similar to those in (A), however C9-Cy5 was used to detect MAC pores (Red). 100 nM of C5 and C6 or C5b6 were used in combination with 100 nM C7, 20 nM C8 and 100 nM C9-Cy5. Conv + C5b6MAC and conv-MAC images were taken in separate experiments in which laser settings were adjusted to the staining intensity of C9-Cy5 to properly visualize pore distribution. Scale bars = 3 µm.
(A) Nuclear extracts of murine intestinal crypts were immunoprecipitated with anti-SRCAP antibody or rabbit IgG followed by ADASDS-PAGE, sliver staining and mass spectrometry.
(E) Immunoblot analysis of BBS4 expression in the brain lysates of Bbs4+/+, Bbs4GT/GT, and Bbs4KO/KO mice. β-actin staining served as a loading control. A representative experiment out of two biological replicates is shown.
(H) MTT assays confirmed that both shADAR2 constructs can partly overcome shAR-dependent effects on cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.
(A) Schematic of the generation and analysis of the Cx3cr1-Cre;Rheb1-loxp GL261 model.
H Cellular glucose uptake and lactate production were measured in Dgcr8−/− ESCs expressing Pkm2 shRNAs and further infected with miR‐290 cluster.
(h) Coimmunoprecipitation of Flag-Parkin and Fbxo7 in the mitochondrial and cytosolic fractions of HEK293T cells overexpressing both proteins. CxVα and GAPDH are markers for mitochondria and cytoplasm, respectively. All western blots are representative of experiments performed at least three times. Full-length blots are presented in Supplementary Figure 9.
Hela cells were co-transfected with the RFP-based TDP-NLS reporter, GFP or GA175-GFP, as well as PSMD11 or empty vector. F) RFP-NLSTDPWT mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.
(D) Sphinganine derivatives DHS1P and S1P show comparable binding affinity to HDAC1, similar to the known HDAC inhibitor TSA.
Double immunolabeling of SYCP3 (red) and RAP1 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.
Under the electron microscope (II), macrophages infected with wild-type Salmonella exhibited multivesicular structures resembling autophagosomes (A-D) Often, electron-dense material, presumably damaged organelles, were readily observed within the multivesicular structures (B-D). In contrast, macrophages infected with type III secretion-defective sipD mutant did not exhibit the large number of multivesicular structures observed in wild-type-infected cells. Bars (A and E) 500 nm; (B, C, D and F) 250 nm.
B. FACS profiles of indicated BM subsets from WT and PKCδ KO mice 20hr after BrdU injection. Data information: All data are presented as mean±SEM, *p<0.05 and **p<0.001, with significance determined by two-tailed Student's unpaired t-test analysis.
(C-D) qRT-PCR analysis with RNA isolated from control or BECLIN1 siRNA transfected THP-1 cells expressing 3X-Flag epitope or Flag-IRGM as indicated.
B, qPCR of SOX2 expression measured in NS 6 h after IR (5 Gy, fold vs. non-irradiated cells, ctrl). *: t-test, p<0.02.
(A) HDAC6 KO mice accumulate ubiquitin‐positive protein aggregates in the brain. The hippocampus and cerebral cortex regions from 6‐month‐old wild‐type and ubiquitin KO littermates were subjected to immunostaining with a ubiquitin antibody and counterstained with hematoxylin. The red arrows indicate ubiquitin‐positive neuritic aggregates and black arrows indicate cytoplasmic aggregates. These ubiquitin‐positive structures were rarely observed in control littermates. Scale bar, 50 μm
(F) ET27 (PsigX-gfp) cells were infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are fluorescence from GFP (upper panels) and corresponding phase contrast images (lower panels), captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm.
Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with MICU1 WT-HA or MICU1 SD-HA, together with MCU-flag and EMRE constructs, following carbonate extraction at pH 7.4, pH 10.5, pH 11.5 or pH 12.5. The cells were treated with 5 μM MG132 for 4 h before fractionation
(C) Western blot analysis of Parkin expression in liver biopsies of chronic hepatitis C patients. Samples 1 and 2 represent liver biopsies of HCV-negative patients and samples 3-7 represent HCV-positive chronic hepatitis patients. (B and C) The relative intensity of Parkin and PINK1 expression normalized to β-actin analyzed by ImageJ.
F. Quantitation of resident monocytes (Ly6C Hi, ivCD45-CD64-CD11cLoSiglec-F- CD11bHiLy6CHi or Lo) and neutrophils (Neu, ivCD45-Ly6GHiCD11bHi) in IgG or high dose CD8 depleted young (left) or aged (right) mice treated with indicated Ab.
B Representative images of GFP-LC3B puncta in HeLa-GFP-LC3B cells transfected with USP19-specific siRNA or scrambled siRNA during growth in normal medium (DMEM) or EBSS medium for 3 h. Arrows denote representative autophagosomes. Scale bar, 200 μm.
(H) In vitro ubiquitylation of the indicated cyclodextrins (2 mM) by HOIL-1, visualised by Coomassie staining. Met1-linked ubiquitin tetramer was also included in these assays.
F, G The time in the open arms (F) and closed arms (G) in the EPM in susceptible mice, compared to control mice (n = 19, 33 mice. Susceptible vs control, for open arms, P < 0.0001; For closed time, P < 0.0001, Student's t test).
(G) Asynchronous cultures of the indicated cells were used to generate cell extracts, which were then incubated with agarose beads coupled to anti-GFP nanobodies.
(a) HeLa cells were transfected with 0.2 μg GFP-LC3 and 0, 0.2 or 0.6 μg of Bif-1SH3-Myc expression plasmids for 24 h. The total amount of plasmid DNA was adjusted at 0.8 μg per transfection with the pcDNA3 vector. The cells were then cultured in EBSS or complete medium for 3 h and the number of GFP-LC3 dots per GFP-positive cell was determined using a fluorescence microscope (mean ± s.d.; n = 40).
(D) Left hand group of four images show cells grown in glutamine-replete (2.00 mM) and glutamine-depleted (0.15 mM) conditions for 24 h, then incubated with an anti-CD63 antibody (yellow) for 30 min at 4oC, washed with PBS, chased at 37oC for 30 min, then fixed, immunostained and imaged. Right hand group of four images show cells grown in glutamine-replete and glutamine-depleted conditions for 24 h, incubated with Tfn-Alexa Fluor® 488 (yellow) for 30 min at 4oC, shifted to 37oC for 30 min, then washed, immediately fixed and imaged.
B A plasmid encoding the truncated mutant composed of Smad6 amino acids 422-441 (Myc-Smad6(422-441)) or wild-type Smad6 MH2 domain (Myc-Smad6-MH2) was co-transfected with the HA-tagged full-length Pellino-1 plasmid into HEK293 cells. Cell lysates were immunoprecipitated (IP) with anti-Myc or anti-HA antibody, and immunoblotted (IB) with anti-HA or anti-Myc antibody, respectively. The vector, CS3MTBXA-6xMyc, was transfected as a negative control. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates. Data are representative of at least three independent experiments.
(E) Native BrdU staining of PC3 cells transfected either with mock or siCHD1 (SmartPool) were grown for 48 h and then treated with NCS for 2 h prior to staining with anti-BrdU and γH2AX antibodies. Scale bar 5 µm.
Quantitative mRNA expression analysis of the indicated genes in LPLs from colitis mice (n=3 per group).
(b) To quantify changes in protein dynamics, each of the strains was labelled in lactate-based minimal media containing either normal lysine (Lys0), lysine-D4 (Lys4) or lysine-13C6 15N2 (Lys8). Samples (10 OD600 units) were taken at day 1 (Lys0), day 2 (Lys4), day 3 (Lys8), day 4 (Lys4) and day 5 (Lys8; see Methods). The mixed lysates were then separated by one-dimensional SDS-polyacrylamide gel electrophoresis and the proteins were digested with LysC. The resulting peptides were analysed by liquid chromatography-tandem mass spectrometry and the raw data were processed by MaxQuant. We performed two biological replicates of the complete time course experiments for each mutant, and three replicates for the WT, and observed good reproducibility for all the individual experiments (Supplementary Fig. S2).
A Schematic of the cyp-34A4 gene. Large boxes indicate coding sequences. Small boxes indicate UTRs. The location and nature of individual mutations is shown. Sequence deleted in od107 is shown with a horizontal black bar.
Distinct conformational states of the RHD of FAM134 proteins were obtained from conformational sampling (total n=10.000) using coarse-grained molecular dynamic simulations (10 μs) and subsequent clustering. Representative conformations corresponding to the top 3 populated clusters are shown for each system. Conformation states in light colours represent the most populated states in the reported simulation.
Flow cytometry analysis of MitoSOX staining in Gata6-WT and Gata6-KOmye pMɸ (gated on F4/80hi/+ Tim4+) unstimulated (-) or after LPS (100 ng.ml-1), IL-10 (10 ng.ml-1) and αIL-10R (5 µg.ml-1) stimulation for 16 h in vitro. Data shown are pooled from 2 independent experiments and normalized to WT unstimulated.
(B, a-h) Series of pictures displaying the neglected appearance of pups delivered by Shank2-/- dams