EMBO/sd-character-level-ner huggingface dataset:
HEK293 cells stably expressing either GFP-ATG13 (B) were transiently transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR), together with CFP-LC3B plasmid (visualized in red) and cultured in starved conditions for 40'. Wide-field live-cell imaging of starved cells were taken by cellSens microscope. Representative images of lifespan of ATG13 (B) are reported. Images of ATG13 (B) forming association with LC3B-II are also shown. The appearance of LC3B on ATG13 (B) (bottom right) (C) puncta were quantified and reported. All values are expressed in seconds (sec) as mean ± SEM. Yellow arrowheads point at the DFCP1 and LC3B particles in the first frame from their onset, white arrowheads and white arrows show omegasomes and autophagosomes as indicated. Statistical analyses were performed by unpaired Student's t‐test. *, P<0,05; **, P<0.01.(n=3 independent experiments, n=50 events/condition analysed). Scale bar 1 μm.
E. Catheterization in Ctrl or FIJs-treated mice 21 days post-MI under baseline or inferior vena cava occlusion condition.
A) HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR); after culturing cells in fed (FED) or starvation conditions (STV) for 1h, cells were fixed and labelled with the indicated antibodies. Co-localization analysis was performed by using ImageJ Jacop plugin and mean ± SEM of Mander's coefficients of ULK1, expressed as percentage, is reported (bottom). White arrowheads point at co-localization events between ULK1 and LC3B; white arrows track puncta not co-localizing; N, nucleus. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. *, P<0,05; **, P<0.01; ****, P<0.0001. (n=3 independent experiments, n≥16 fields analysed). Scale bar 5 μm.
LIN28A-RHA co-IP assays in HEK293 cells transfected with pFLAG-WT-LIN28A and pFLAG-LIN28A R192G.
(J) Left panel: Electron micrographs of the posterior pituitary gland from Shank2+/+ and Shank2-/- mice in lower (left panel) and higher magnification. Scale bar (500 nm). Right diagram: No significant difference was detected in the number of vesicles in the posterior pituitary gland between Shank2+/+ and Shank2-/- mice, Mann-Whitney test, P=0.507, Shank2+/+ n = 3, Shank2-/- n = 3. (K-M) Additionally, Shank2-/- mice display no significant difference in: (K) Estradiol-, Mann-Whitney test, P=0.475, Shank2+/+ n = 6, Shank2-/- n = 7, (L) Progesterone, unpaired, two-tailed Student's t-test, P=0.570, Shank2+/+ n = 5, Shank2-/- n = 5 or (M) Prolactin- plasma concentrations, unpaired, two-tailed Student's t-test, P=0.980; Shank2+/+ n = 3, Shank2-/- n = 3. Data information: All data are presented as mean ± SEM, NS: not significant. E, endothelium; CL, capillary lumen; M, mitochondria; NG, neurosecretory granule; AE, axonal endings, HYP, Hypothalamus; PIT, Pituitary gland.
(I) Additionally, no significant difference between Shank2+/+ and Shank2-/- was evident in the spontaneous alternation behavior during a Y Maze task, unpaired, two-tailed Student's t-test, P=0.146. Shank2+/+ n = 10, Shank2-/- n = 10. Data information: All graphs are presented as mean ± SEM, NS: not significant.
D-E, Violin plots depicting the number (D) and size (E) of condensed chromatin spots from cells electroporated as in A-C. Note that Trnp1 overexpression is sufficient to significantly increase the size of condensed chromatin spots, while the ∆1-16Trnp1 acts as a dominant negative reducing the number and size of heterochromatin spots. N=10 cells per region from 5 embryos for GFP and Trnp1 and 8-15 cells from 4 embryos for ∆1-16Trnp1 of at least 4 litters. Each dot represents a cell.
(B) BT474ccells were treated with inhibitors as indicated either as monotherapy or in combination and cell confluency measured on the Incucyte ZOOM every 4 hours for up to 4 weeks.
(D) Impact of PI‐3K inhibition using LY294002 on LC3B conversion after PMA challenge (lane III), (lane I: control cells, lane II: PMA, lane IV: LY294002 only). One representative out of four independent experiments is shown.
D) HEK293 cells were genetically engineered to knockout RIG-I, MDA5, or both, and subsequently subjected to retroviral transduction to stably express FLAG-LGP2 or an empty vector (EV). Correct gene editing and intact type I IFN responsiveness was validated by SDS-PAGE and immunoblotting using the indicated antibodies (n=2). *, non-specific band.
(F) Internalization of endogenous p75NTR in wild type hippocampal neurons (30min time point). Counterstaining for MAP2 and DAPI are also shown. Scale bar, 10μm.
E Embryonic myosin expression: immunostaining showing its localization along with laminin (green) on transverse cross-sections from diaphragm of WT, mdx and mdx/CD38-/- mice. Scale bars: 50 µm. Histogram showing the relative proportion of embryonic myosin area (% total area) in diaphragm of WT (n=7), mdx (n=8) and mdx/CD38-/- (n=8) mice. Data information: Each dot of the graphs represents a mouse. E one value/mouse. After normality and variance comparison tests, significance was assessed using: E: Welch's ANOVA followed by Welch's t-tests. Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.
(D) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-WT and TFEB-S3A/R4A mutant upon incubation with DMSO (vehicle) or 250 nM Torin-1 for 1 h. Cells were fixed, permeabilized with 0.2% Triton X-100, and stained with antibodies against FLAG (used to detect TFEB).
D, E ISH of miniature pig canine frontal sections showing expression patterns of β-catenin at E60 and E90. (D', E') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.
(A) Immunofluorescent staining of AEP (red) and C/EBP β (green) in CA1 region from hippocampus of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. Scale bar: 20μm. (B) Quantitative analysis of AEP positive cells and C/EBP β positive cells, respectively. The density of both AEP and C/EBP β positive cells were significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. **P =0.016 (**P < 0.01), ****P<0.0001, one-way ANOVA.
(D) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. (E) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units.
The ABA-dependent regulation of the PP2Cs ABI1 (A,D,G), ABI2 (B,E,H), and HAB1 (C,F,I) by RCAR12 and RCAR13 were analyzed in yeast (A-C), Arabidopsis protoplasts (D-F), and in vitro (G-I).
C: Tumors were measured using a slide caliper and tumor volumes were calculated. Arrow indicates starting point siELDR treatment. n=5 animal per group and tumor volume presented as the mean ± SD
F. Representative images of different growth cone morphological categories: fan-like, torpedo-like and bulb-like. Scale bar = 5µm. G. Quantifications of the ratios of different subtypes (fan-like, torpedo-like, bulb-like) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. H. Quantifications of the average area (µm²) of different subtypes (fan-like, torpedo-like, bulb-like) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. n = 5-55 growth cones in 3 independent experiments. Da Data information: Chi-square-test including post-hoc analysis with Bonferroni correction (G), One-way ANOVA including Sidak's post-hoc analysis (H). *** p<0.001, ** p<0.005, * p < 0.05, ns p≥0.05
H Western blot analysis of Viperin protein in RAW264.7 treated with mIFNβ (100 and 500 IU/ml, 12 hrs) immediately after addition of NaCl (+34 mM). I Western blot analysis of Viperin protein in HT1080 treated with IFNα (1,000 IU/ml) and NaCl (+34 mM) as (H). J Western blot analysis of Viperin protein in MEF cells infected with VSV (MOI=0.5 and 1.0, 24 hrs) immediately after addition of NaCl (+34 mM). K Western blot analysis of Viperin protein in THP1 infected with H1N1 (MOI= 1.0) immediately after addition of NaCl (+34 mM). Data information: Data are representative of at least two biological replicates
a, LAMP1/LC3NRK cells were starved for 4 h and treated with GTPγS for 6 h of further starvation.
RT-PCR analysis of SPPL2c expression in different murine tissues. Total RNA from the indicated tissues was either transcribed into cDNA prior to PCR amplification (+RT) or, as negative control, used directly as template (-RT). A fragment of the actin ORF was amplified as control.
A. HeLa cells transfected or not with indicated siRNAs were synchronized in early S phase by double thymidine block, released and pulse-labeled 5,5 h later with 5-azadC for 30 min in the presence or absence of SUMOi. Following 5-azadC removal, cells were subjected to live-cell imaging analysis, and the duration of mitosis (nuclear envelope breakdown (NEBD) to anaphase onset) was quantified (red bars, median; at least 72 cells, pooled from three independent experiments, were analyzed per condition; ****p<0.0001, ns: not significant, Mann-Whitney test).
The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. General cell survival (calcein-AM)/death (EthD-1). (B) Cleaved caspase-3+ apoptotic cell death. (C) Cellular senescence (β-gal staining). (D) DNA damage estimated by γH2AX staining. Cells with >4 γH2AX foci in the nucleus were counted. (E) Mitochondrial ROS (MitoSox). (F) Mitochondrial membrane potential (MitoID).
C Full-length structures of CbpCT4 (PDB ID: 2V04), CbpJ (PDB ID: 6JYX), and PcpC (homology model).
(F) RNA ISH for circLMP2A. U6 and 18S are mainly localized in the nucleus and cytoplasm, respectively, and were used as controls.
(f,g) Gel-filtration analysis on the membrane fraction of mouse brain lysates. Fractions obtained were subjected to immunoblot analysis (f), or immunoprecipitation followed by immunoblotting (g). Uncropped images of blots are shown in Supplementary Fig. S8.
(A) Representative immunoblots showing co-immunoprecipitation with pIRE1. After 5 hr of Tm treatment, immunoprecipitation was performed on HeLa cell lysates with an anti-pIRE1 antibody (lanes labeled 'pIRE1') or control anti-rabbit IgG (lanes labeled 'IgG'), and the immunoprecipitates were analyzed by Western blotting with a panel of antibodies against molecules involved in the ER-Golgi vesicular transport pathways or against ER- (Sec61α, HSP47, and calnexin), ERGIC- (ERGIC53) and cis-Golgi- (GM130) resident proteins.
E: Initial DHE transport rate measured with cSTD3 or pS209 cSTD3 in the presence or absence of VAP-A HIS6 or VAP-A KD/MDHIS6. Data are represented as mean ± S.E.M. (n = 3 for cSTD3-VAP and n=4 for all other data). Mann-Whitney test (*P < 0.05).
D) Representative images of HeLa cells transfected with control, H2A.Z.1 or H2A.Z.2 siRNA and stained for senescence-associated β-galactosidase (blue) Sclae bar 200μm. At the bottom-left, quantification of the percentage of β-galactosidase-positive cells. At least 500 cells were counted for each experiment. Error bars show the standard deviation (SD) from two biological replicates. **=p<0.01; ***=p<0.001 (Student's t Test).
c, Confocal images of HeLa cells infected with S. Typhimurium for 1 h and stained with antisera against NDP52 and galectin 8. Arrowheads, bacteria shown in insets.
(A) Coimmunoprecipitation of Atg11 and Atg8 with Atg36. IP was performed in either C13-Atg36-PtA atg1Δ or C13-Mvp1-PtA atg1Δ cells containing plasmids expressing either HA-Atg11 or HA-Atg8 under control of the TPI1 promoter. Cells were grown for 24 h to post‐log phase in glucose medium followed by growth in oleate medium for a further 18 h. Cells were then grown under non‐starvation or starvation conditions for a further 2 h. IgG Sepharose beads were used to immobilise Atg36-PtA or Mvp1-PtA from spheroplast yeast lysates. After extensive washing, bound material was eluted by Tev protease cleavage. SDS-PAGE gels (percentage indicated) were probed with anti‐HA (monoclonal 12CA5) and PAP. TL, total lysate; IP, immunoprecipitate (Tev eluate).
Ramos cells were infected with wild type EBV-WT or BRLF1-deficient EBV-ΔBRLF1 virus at a high titer (MOI=100). After being transduced with empty control or BZLF1-expressing lentivirus, the supernatants and cell pellets of EBV-WT- or EBV-ΔBRLF1-harboring Ramos cells were collected after 12 h in serum-free culture, and then the levels of EBNA1, BRLF1, BZLF1, pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-1β, mature IL-1β, pro-IL-18, mature IL-18, GSDMD and cleaved N-GSDMD in the supernatants and whole-cell lysates were analyzed as indicated (d).
(M, N and O) Statistical analysis of the relative levels of Ace-H3-K9I n WT and MZsinhcaf -/- EV (M), MV (N) and FG (O) follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III)
(h) Western blot against HIF1α in Pink1 KO MEF incubated with (or without) GSH-EE, or transfected with empty vector, mitoGCL or mitoCAT, showing reduction in HIF1α protein abundance by the treatments that remove mitochondrial ROS; α-TUBULIN was used as loading control. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).
C) SH-SY5Y cells without (nt) or with stable PINK1 knockdown (kd) were kept in medium with 5% FCS and either untreated or treated for 2 h with CCCP to stabilize PINK1. Afterwards the PINK1 63 kDa protein (arrowhead) and actin protein levels were determined by western blotting (see representative gel on the right). The quantification revealed a reduction of PINK1 protein under both conditions in PINK1 kd cells.
(E) FRAP analysis of mCherry-IPO4 and mCherry-sNASP-∆NLS bound to tethered EGFP-H3. Scale bars represent 10 μm
B. TBK1/IKKε phosphorylate mTOR S2159 directly in vitro: In vitro kinase (IVK) assays with recombinant (re) active TBK1 or IKKε [50 ng] (Invitrogen) and recombinant GST-mTOR substrate [200 ng] for 30 min at 30oC. Reactions were pre-incubated on ice 30 min with amlexanox [500, 250 or 50 μM], BX-795 [10 μM] or MRT-67307 [10 μM] and immunoblotted (IB) as indicated.
Protein-binding microarrays (PBM) were probed with PIF4-His, MBP-BES1 and the PIF4-His/MBP-BES1 complex. For PIF4-BES1 complex purification, both proteins were co-expressed in E. coli using the double expression pCOLADuet-1 vector. An anti-MBP antibody was used for BES1 and BES1-PIF4 signal detection, while PIF4 was detected with an anti-His antibody Electrophoretic mobility shift assays (EMSA) showing interaction of the PIF4, BES1, and PIF4-BES1 proteins with the conserved G-box, PBE-, and BRRE-elements in the PIL1 (At2g46970), PRE5 (At3g28857), and DWF4 (At3g50660) promoters. Increasing amounts of protein were used for the assay
(H) miR-122 levels in the blood serum of pCIneo (control) or HA-HuR expression plasmid injected mice were measured. The Ct values of miR-122 and CAT-1 and Aldolase mRNAs obtained for each individual animal liver and blood serum were plotted (mean+/- s.e.m., n=5).
(I) Correction in abnormal granule shape following high cholesterol feeding (bar = 1μm), with I' showing magnification of the boxed region indicated.
D Overexpression of dominant-negative Rab5 (Rab5DN), but not constitutively active Rab5 (Rab5CA), rescues the progressive accumulation of Synaptophysin-pHluorin on the surface of VPS34IN1-treated hippocampal neurons stimulated and analyzed as in A-C. N = 3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
E - F Confocal images of olfactory sensory neuron axons coalescing into glomeruli where they synapse with dendrites of olfactory bulb neurons. The axons of olfactory sensory neurons can be readily visualized with PFA staining (green) in the superficial olfactory nerve layer and terminating in glomeruli located below (green arrows). While sections fixed with either PFA or glyoxal display adequate staining levels, the signal distribution of the Immunostaining-fixed tissue appears more irregular (E), seemingly lacking the neurofilimentary morphology that appears preserved in the glyoxal-fixed sections (F). The glomeruli themselves are neuropil structures comprised primarily of olfactory sensory neurons forming synapses with dendrites of mitral/tufted cells as well as PFA of periglomerular neurons. Immunostaining with Vesicular Glutamate Transporter 2 (VGLUT2) allows visualization of these structures and is easily seen in the glyoxal-fixed section (F), while it appears the antigen was masked by PFA fixation as no signal above background can be seen in the PFA-fixed panel (red arrows in E). Note that a different polyclonal antibody for VGLUT2 from the same provider does provide signal with glyoxal, albeit weaker versus Glyoxal (inset). Staining with ß3-tubulin touts the benefits of glyoxal both due to the signal improvements in the case of the mild staining in the axons of the olfactory nerve layer that is only visible in the glyoxal-fixed tissue, but also in preserving tissue morphology as demonstrated by the dendritic processes inside glomeruli (blue arrows) and in the external plexiform layer located below the glomeruli.
Microscopy images show increased DPR fluorescent intensity levels from medium and high frequency stimulation of ChR2 in primary cortical neurons transfected with the C9 DPR reporter.
HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (G) LAMP1 (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15) is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.
C ULK1+/+, ULK1−/− FUNDC1‐KD, or ULK1−/− FUNDC1‐KD cells rescued by introduction of HA‐ULK1 and FUNDC1‐Myc were cultured under hypoxic (1% O2) conditions for 24 h or with FCCP (20 μM) for 24 h. Samples were analyzed by electron microscopy. Scale bar, 2 μm. The yellow asterisks mark mitochondria. The red arrows indicate double‐membraned autophagic structures. The yellow arrow denotes autolysosomes.
D. Neurofilament/CtBP2 labelling shows swollen and retracting terminals (white arrows), especially in the 24kHz regions in the mutants at 8 weeks (arrows). Scale bars: 10μm.E. Counts of pre-synaptic ribbons in the 8, 18 and 24kHz regions, showing no difference in their number per IHC in the mutants compared to littermate controls at 8 weeks.
D. Log-log plot of L-asp KD as a function of Na+ concentration. Empty circles are KD values (n=20) at pH=9.0 in the absence of UCPH101 (blue), and solid line is the fit of a straight line. For comparison, average L-asp KD at 1 mM Na+ in the presence pf of UPCH101 is also shown (n=3; red).
G. Inhibitory effect of low doses of prazosin (0.15 mg/Kg) on glioblastoma growth in vivo. Left panel: Bioluminescent in vivo images of tumors in mice treated with vehicle or prazosin (PRZ) for 45 days. Tumors were initiated with GBM44 GICs (compare with panel B). Middle panel: Quantification of the bioluminescent signals. *P=0.0007, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice demonstrating a significant survival benefit of a treatment with low doses of prazosin, log-rank Mantel-Cox test. The treatment period is shaded in gray.
A) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). Nuclei were counterstained with DAPI (blue). MuSCs were cultured alone (-) or with EVs (EVs FAPs) isolated from FAPs in vivo exposed or not to TSA (CTR and TSA) and transfected or not with Antagomir-206 (- and A-206). (n=7 for all samples, but antagomir's samples n=3, biological replicates). Scale bar =50 μm.
C BN‐PAGE confirms the formation of MPCFERM and MPCOX complexes after constitutive plasmid‐based protein expression. Non‐specific reactivity of the antibody is denoted by an asterisk (*). It has to be noted that all complexes migrate slightly faster than in Fig C, most likely because MPC proteins are tagged with a single epitope tag as opposed to the 3HA‐tag in Fig C.
C, D) Correlation analyses of the levels o ErbB4 and SypCFP (F) staining in vGlut1/2+ synapses; every dot corresponds to one synapse. Cultured Nrg3-/- neurons were transduced wit SypCFP/Nrg3∆EGF (red in D) lentiviruses
(H) Eosin-hematoxylin-stained sections of the lung and liver of a Pax5Jak2/+ tumor mouse. Infiltrating and blasting tumor cells are indicated by an arrow.
Heat map (left) and tSNE plot (right) (F) of digested day 21 BALO cultures depicting four distinct clusters (C1, airway, blue; C2, alveolar, purple; C3, MYO, orange, and C4, LIF, green).
(F) Western blot analysis of Aurkb in total lung lysates from saline and bleomycin treated mice. Quantification was performed using phosphor imager software and normalization was done using loading control Gapdh. **P < 0.005, unpaired t-test, (n=4-5).
Schematic design of the KLF5 3'-UTR. To test the potential miR-9 binding on KLF5 3'-UTR, four vectors were generated: WT (Wild Type containing both the seed sites), mut A or mut B (mutated for one single binding site) and mut A+B (mutated in both seed site). The predicted seed sites for miR-9 binding sites are shown in red when present and in blue when mutated.
C. Changes in gene expression of 359 detected genes in Grn-/- microglia and Trem2-/- microglia. Note that from the 38 genes significantly altered in both genotypes, 32 genes are regulated in opposite direction.
(f,g) Striatal cells from 7Q-htt and 111Q-httmice untreated or treated with vinblastine were co-stained for LC3 and BODIPY 493/503 (g). Arrows, colocalization events. Extended study in Supplementary Figure 16.
(C) Gram and Giemsa stains analyzed neutrophil phagocytosis and bacterial clearance in mice surviving on day 9. White arrows indicate phagocytosed S. aureus in neutrophils.
(A) Glycan microarray analyses of mClec4g, mCD209c and hCLEC4G. Terminal GlcNAc, Gal or Man are colour-coded along the X-axis. Miscellaneous epitopes could not unambiguously be grouped to either terminal GlcNAc, Gal or Man. Mucin type O-glycans, characterized by a reducing-end GalNAc, are highlighted. The identified main binding epitope of each lectin is shown in red.
B Interaction of HA-tagged AGO2 (wild-type or pocket mutants) with endogenous TNRC6A in HEK293T cells.
C qRT-PCR analysis of AGO6 and AGO4 mRNA transcript levels in inflorescence and juvenile leaf tissue.