EMBO/sd-character-level-ner huggingface dataset:
Late endocytic compartments associate with elongated membrane clusters.Figure 8A) Hepatoma cells infected with UIS4−mCherry−expressing Plasmodium berghei parasites were fixed 22 h post−infection and stained with antibodies against host cell protein markers of distinct organelles: anti−EEA1 to label early endosomes, anti−CD63 and anti−LAMP1 to label late endosomes and lysosomes, anti−Calnexin to label the endoplasmic reticulum, and anti−Golgin97 to label the Golgi. In all samples, either anti−mCherry or anti−UIS4 was used to amplify the UIS4−mCherry signal. Imaging was carried out by epifluorescence microscopy. In the right panels, fluorescence is merged, and selected regions with areas of interest are shown enlarged. At least 20 infected cells possessing LS−TVN features were visually examined for each condition, and representative images of UIS4−positive elongated membrane clusters (left) and UIS4−positive tubules (right, and indicated with yellow arrows) are displayed. Scale bars, 10 µm. B) Profiling of the red (UIS4) and green (CD63, left or LAMP‐1, right) signals along the indicated line within the insets from the images in (A) above.
Figure 1(A) Scheme of the STC recording paradigm. The currents elicited by extracellular stimulation in layer 1 were measured in a voltage-clamped layer 1 astrocyte located at 200 µm from the stimulating electrode in an acute slice of mouse barrel cortex. (B) Time constants of decay, τdecay, of the STC isolated pharmacologically in WT and FHM2 KI mice. Top, superimposed representative traces of the inward current evoked in an astrocyte (held at -90 mV) by a single pulse stimulation (indicated by the arrow) in a WT slice, before (trace a) and after (trace b) application of a saturating concentration of the GluT inhibitor TFB-TBOA (TBOA). The STC was obtained by subtracting the residual current remaining in the presence of TBOA from the total inward current (trace a-b); the decay of the STC was best fitted by a single exponential function with τdecay = 6.53 ms (in red). The bar plot shows the average values of τdecay of the STC isolated pharmacologically in cortical slices (n=13) from P22-23 WT (N=7) and KI mice (n=9; N=3). STC τdecay is 17% higher in KI compared to WT astrocytes (unpaired t-test: P = 0.003). Hereafter, n indicates the number of slices and N indicates the number of mice. (C) τdecay of the STC isolated using an exponential waveform approximating the average TBOA-insensitive current in WT and KI mice. Top trace, average normalized TBOA-insensitive current obtained by pooling the normalized TBOA-insensitive currents recorded in 16 WT and KI cells. This current was best fitted by an exponentially rising function (1-exp (-t/τrise)) with τrise= 2.35 ms (in green). The STC was obtained by subtracting from the total current elicited in the astrocyte (a: same representative trace as in (B)) the exponential function A(1-exp (-t/τrise)) with τrise = 2.35 ms and A equal to the maximal current measured in the astrocyte at about 60 ms after stimulation (trace c); the decay of the STC (trace a-c) was best fitted by a single exponential function with τdecay = 6.49 ms (in red). The bar plot shows the average values of τdecay of the STC isolated as shown in the top panel in cortical slices from P22-23 WT (n = 28; N=11) and KI mice (n = 27; N=9). STC τdecay is 21% higher in KI compared to WT astrocytes (unpaired t-test: P < 0.0001).
SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells.Figure 1.SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells. (A-C) Human colon carcinoma HCT 116 cells were left untransfected (Co, control) or transfected with an irrelevant siRNA (UNR, unrelated) or siRNAs specific for ATG5, ATG7, or SIRT1 and then retransfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and left untreated or treated for 4 h with 100-µM resveratrol (Resv) or spermidine (Spd). The same experiment was performed in the presence of bafilomycin A1 (BafA1), which inhibits the fusion between lysosomes and autophagosomes, to evaluate the autophagic flux. (A) Representative images. (B) Quantitative data. SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells. (A-C) Human colon carcinoma HCT 116 cells were left untransfected (Co, control) or transfected with an irrelevant siRNA (UNR, unrelated) or siRNAs specific for ATG5, ATG7, or SIRT1 and then retransfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and left untreated or treated for 4 h with 100-µM resveratrol (Resv) or spermidine (Spd). The same experiment was performed in the presence of bafilomycin A1 (BafA1), which inhibits the fusion between lysosomes and autophagosomes, to evaluate the autophagic flux. (A) Representative images. (B) Quantitative data. (C) Representative immunoblots of HCT 116 cells transfected either with an unrelated siRNA or with a SIRT1-specific siRNA showing LC3 lipidation after treatment with 100-µM spermidine in the presence or absence of bafilomycin A1. (D-F) HCT 116 cells were left transfected with a GFP-LC3 plasmid, cultured in complete medium for 24 h, and treated with either vehicle (Co), 100-µM resveratrol, or 100-µM spermidine in the presence or absence of the SIRT1 inhibitor EX527 for 4 h. (D) Representative images. (E) Quantitative data. (B and E) Bars depict the percentages of cells showing accumulation of GFP-LC3 in puncta (GFP-LC3vac; means ± SEM; n = 3; *, P < 0.05). (F) Representative immunoblots showing LC3 lipidation in HCT 116 cells treated with 100-µM spermidine in the presence or absence of EX527. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 6The effects of Nbr1-ZZ1 mutations on its Ams1-binding ability. Nbr1-Ams1 interactions were examined by the Pil1 co-tethering assay. Mutations were introduced into the bait protein Pil1-mCherry-ZZ1-ZZ2. Scale bar, 3 μm. The effects of Nbr1-ZZ1 mutations on its Ape4-binding ability. Nbr1-Ape4 interactions were examined by the Pil1 co-tethering assay. Mutations were introduced into the bait protein Pil1-mCherry-ZZ1-ZZ2. Scale bar, 3 μm. Surface plasmon resonance (SPR) analysis of the Nbr1-Ams1 interaction. GST-tagged ZZ1-L1 fragment of Nbr1 purified from E. coli was captured on a CM5 sensor chip using the GST Capture Kit. Ams1 purified from S. pombe (two-fold serial dilutions from 1000 nM to 62.5 nM) was flowed through the chip surface. The effects of Nbr1-ZZ1 mutations on its ability to mediate the vacuolar targeting of Ams1. Mutations were introduced into Nbr1-Δ(ZZ2-ZZ3), which was expressed in the nbr1Δ mutant background. Scale bar, 3 μm. The effects of Nbr1-ZZ1 mutations on its ability to mediate the vacuolar targeting of Ape4. Mutations were introduced into Nbr1-Δ(ZZ2-ZZ3), which was expressed in the nbr1Δ mutant background. Scale bar, 3 μm. SPR analysis of the Nbr1-Ape4 interaction. GST-tagged ZZ1-L1 fragment of Nbr1 purified from E. coli was captured on a CM5 sensor chip using the GST Capture Kit. Ape4 purified from S. pombe (two-fold serial dilutions from 1000 nM to 62.5 nM) was flowed through the chip surface.
Figure 4(A) The stages of paralysis in C57BL/6 mice post CHIKV infection (MOI 1x107 PFU/mouse, 6 dpi) with scoring according to the severity of the disease. (B) The graph depicts percentage change in body weight of CHIKV infected irgm1+/+ and irgm1-/- neonates for duration as indicated (C) The graph depicts total paralysis scores of CHIKV infected irgm1+/+and irgm1-/-neonates (n=12) until 5 days post-infection. (D) Kaplan-Meier survival graph depicts percentage survival of mock and CHIKV infected irgm1+/+ and irgm1-/- neonates during the course of infection (E, F) Total RNA isolated from muscles of mock and CHIKV infected (MOI 1x107 PFU/mouse, 6 and 11dpi) irgm1+/+and irgm1-/-mice and was subjected to qRT-PCR to quantitate the viral load (G) Total RNA isolated from the brain of mock and CHIKV infected (MOI 1x107 PFU/mouse, 6 dpi) irgm1+/+ and irgm1-/-mice and was subjected to qRT PCR for quantitation of viral load The total RNA used for qRT PCR with the brain is four times more than muscles. (H, I) Total RNA isolated from muscles and brains of mock and CHIKV infected (MOI 1x107 PFU/mouse) (6 dpi) irgm1+/+ and irgm1-/-mice and was subjected to qRT PCR with MX2 The total RNA used for qRT PCR with the brain is four times more than muscles.
Figure 4A. Cellular proliferation analysis of Mettl3 or Mettl14 depleted and control CT26 cells treated with indicated combinations of cytokines for 48h. The mean ± SD of three replicates is shown. * P < 0.05; * * P < 0.01 by Student's t-tests. B. BALB/c mice bearing Mettl3 or Mettl14 deficient and control tumors were treated with IFNγ -blocking antibody and PD-1 antibody as indicated. Tumor size was measured over time. n, the numbers of mice. Data are mean ± s.e.m of the indicated number of mice. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests. C. Quantitative RT-PCR was performed to identify transcriptional changes of the IFN-γ response gene expression (n=3). Data are shown as the relative fold change (RFC, color coded bar). D, E. mRNA stability of Stat1 and Irf1 were measured by qRT-PCR in tumor cells treated with IFN-γ and actinomycin D. Mean ± SD of n=3. * * P < 0.01; * * * P < 0.001 by Student's t-tests. F. Validation the effect of knock out of Ythdf1-3 using western blotting, Gapdh served as a loading control. G. qPCR analysis of Stat1 and Irf1's expression in the indicated depletion of CT26 cells with/without stimulation of IFNγ. Mean ± SD of n=3. * P < 0.05 by Student's t-tests. H. Western blot analysis of Mettl3 and Mettl14 in overexpressed CT26 cells. Gapdh served as a loading control for each. I, J. qPCR analysis of the mRNA stability of Stat1 and Irf1 in the indicated CT26 cells treated with IFNγ and actinomycin D. Mean ± SD of n=3. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests.
a, Immunoblot of Thr-172-phosphorylated and total AMPK, Ser-79-phospho-acetyl-coA-carboxylase (pACC) and &bgr-actin in +/−KRas cells for two independent tumours.f4a, Immunoblot of Thr-172-phosphorylated and total AMPK, Ser-79-phospho-acetyl-coA-carboxylase (pACC) and &amp;amp;bgr;-actin in +/−KRas cells for two independent tumours. b, Immunoblot of −KRas cells treated or not with oligomycin (Oli) probed with anti-Thr-172-phospho-AMPK and &amp;amp;bgr;-actin antibodies for three independent tumours. c, Immunoblot of LC3 and &amp;amp;bgr;-actin in +/−KRas cells for two independent tumours. d, Autophagic flux of +/−KRas cells stably expressing GFP-LC3. Bafilomycin treatment rescues GFP (−KRas+Baf) (n = 3); representative flow cytometry of two tumours. e, Lipid droplet quantification in +/−KRas cells (n = 4); representative flow cytometry of two tumours. f, Fusion between lipid droplets (red arrowheads) and autophagosomes (green arrowheads) in −KRas cells (TEM; ×25,000). g, Confocal microscopy for lipid droplets (Lipidtox; red) and lysosomes (Lysotracker; green) in −KRas spheres. Hoechst, blue. h, Oxygen consumption of +/−Kras cells pre-treated with bafilomycin (Baf), etomoxir (Eto) or vehicle (Ctrl) (n = 4). i, Effect of bafilomycin and etomoxir on spherogenic potential of +/−Kras cells (n = 6). Data are mean ± s.d.
Expression profiles of HS memory-related genes after a recurring HS show HSFA2-dependent transcriptional memory.Figure 6Figure 6: Expression profiles of HS memory-related genes after a recurring HS show HSFA2-dependent transcriptional memoryResponse of selected HS-inducible loci upon recurring HS as determined by qRT-PCR. Transcript levels of APX2 (A), HSP18.2 (B), HSP22.0 (C), and HSP70 (D) in Col-0 (blue bars) and hsfa2 (orange bars) at the indicated time points after a single HS or two HS separated by 2 d. Plants were subjected to 37°C for 60 min on either d 4, d 6 or on d 4 + d 6 after germination. NHS samples were harvested at the same time points as HS d 4 samples. Transcript levels of four biological replicates normalized to TUB6 are shown. Small brackets compare the two genotypes at the same condition, large brackets compare the same genotype across two treatments (at 1 h after treatment) with Col-0 indicated first (Col-0/ hsfa2). Error bars show SE over four biological replicates. *, p<0.05; **, p<0.01; Student´s t-test.
Pkm2 and Ldha are essential for miR‐290‐mediated reprogrammingqRT–PCR analysis of Pkm2 and Ldha in MEF cells on days 0, 2, and 4 post‐infection with 3F plus miR‐290 cluster or control mimics.Figure 3A qRT-PCR analysis of Pkm2 and Ldha in MEF cells on days 0, 2, and 4 post‐infection with 3F plus miR‐290 cluster or control mimics. Oct4‐GFP MEFs were introduced with the indicated factors, and Oct4‐GFP‐positive colonies were counted on day 16 after virus infection. AP staining of iPSC colonies was shown below. Colony morphology of ESCs and iPSCs derived from the indicated factors. Hematoxylin and eosin staining of teratoma sections derived from 3F plus Pkm2 showed differentiation of iPSCs into cell types of three germ layers: keratinized epithelium (i), cartilage (ii), and glandular tissue (iii). Scale bars, 100 μm. Oct4‐GFP MEFs were introduced with the indicated factors and Oct4‐GFP‐positive colonies were counted on day 16 after virus infection. AP staining of iPSC colonies was shown below.
Reducing nuclear export increases nuclear size.Figure 6Cell lysates from control and XPO1 siRNA transfected MCF-10AT1k.cl2 cells were analyzed by western blot and probed for XPO1 and tubulin. One representative western blot is shown. XPO1 band intensity was normalized to tubulin. Quantification from two biological replicates is shown. MCF-10AT1k.cl2 cells were transfected with control or XPO1 siRNA. Lamin B1 and lamin A/C nuclear staining intensities were quantified based on experiments using two different XPO1 siRNA sequences and two biological replicates. For each experiment, 108-865 nuclei were quantified per condition (416 nuclei on average). Average and SD values based on averages from these four experiments are plotted, normalized to the negative siRNA control. MCF-10AT1k.cl2 cells were transfected with the indicated siRNAs and nuclear cross-sectional areas were quantified. For each experiment, 203-604 nuclei were quantified per condition (403 nuclei on average). Average and SD values based on averages from four biological replicates are plotted, normalized to the negative siRNA control. MCF-10AT1k.cl2 cells were transfected with control siRNA or with siRNA against XPO1. Cells were stained with DAPI and quantification of DNA staining intensity was used to estimate the fraction of cells in various stages of the cell cycle by high-throughput imaging as previously described [123] (see Methods). The stacked bars represent the means of the fractions for each cell cycle phase calculated over 3 biological replicates. MCF-10AT1k.cl2 cells were transfected with control or XPO1 siRNA. For the bottom row of images, cells transfected with control siRNA were treated with 20 ng/ml leptomycin B for 24 hours. Cells were fixed and stained with 2 µg/ml FITC. Representative images are shown. Scale bar, 20 µm. For each experiment, nuclear cross-sectional areas and nuclear FITC staining intensities were quantified for 105-620 nuclei per condition (267 nuclei on average) and normalized to the negative control. Two biological replicates, data from one representative experiment shown.
Mutation of the histone-binding motif of Spt5N is lethal, even in the presence of wild type Spt5.Figure 2(A) Linearised versions of the indicated plasmids were transformed into SPT5-9MYC budding yeast cells and integrated at the ura3 locus. For each transformation, four colonies were analysed by immunoblotting and DNA sequencing as shown. Colonies transformed with plasmid expressing Spt5-3A either lost the plasmid (1), contained the SPT5-3A DNA but failed to express Spt5-3A protein (2+4) or experienced a gene conversion and thus expressed wild type Spt5 from the integrated plasmid DNA (3). (B) Serial dilutions of yeast cells expressing the indicated fragments of wild type Spt5 or Spt5-3A from the GAL1,10 promoter were grown on the indicated medium, together with a control of wild type cells. (C) Yeast cells were generated that expressed Protein A-tagged versions of wild type Spt5 (full length or the indicated truncations), or full length Spt5-3A, under the control of the GAL1,10 promoter. Cells were grown in medium containing galactose and the ProteinA-tagged Spt5 proteins were isolated from cell extracts on magnetic beads coated with IgG. Association of the tagged Spt5 proteins with the elongating form of Pol II was monitored by immunoblotting, using antibodies specific to the indicated phosphorylation sites in the C-Terminal Domain repeats of the Rpb1 subunit of Pol II. (D) Control and spt5-AID strains were grown at 30°C in rich medium, before addition of auxin (0.5 mM 3-indoleacetic acid or IAA) for 1 hour. Spt5 protein was monitored in cell extracts by immunoblotting. (E) Serial dilutions of control cells or spt5-AID cells, expressing the indicated versions of Spt5 from the GAL1,10 promoter, were grown on the indicated medium.
Figure 3A. Representative spinning disk confocal time series of MT-flux in bipolar spindles in control cells and cells undergoing mitosis with unreplicated genomes (MUGs). U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), labeled for chromosomes with SiR-DNA (grey) are shown, with 5 mM caffeine used as a control. Scale bar, 10 µm. Time, min:sec. B. Quantification of the MT-flux rates from indicated conditions. MT-flux values with mean ± SD are plotted. N (number of cells, number of independent experiments): Caffeine-only control (30, 3) and MUGs (34, 3). C. Representative point-scanning confocal maximum-intensity projected images of mitotic spindles in U2OS cells subjected to indicated conditions, immunostained with antibodies against KIF4A and α-tubulin. DNA was counterstained with DAPI. KIF4A in cyan and α-tubulin in red in merged image. Scale bar, 10 µm.
Figure 2(A) Representative pictures of WT and Nedd4l cKO mice treated with or without IMQ. (B) Erythema, scaling and thickness of the back skin were scored daily on a scale from 0 to 4, and the cumulative score (erythema plus scaling plus thickness) is depicted. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments. For back tissues, scale bar, 200 μm (100×) or 50 μm (400×). For ear tissues, scale bar, 200 μm (100×) or 200 μm (200×). Significant differences were tested using a two-tailed Mann-Whitney U test Control, n=3, IMQ, n=6. ** P＜0.01, *** P＜0.001. NS, no significance. (C) Ear thickness of the right ear was measured on the days indicated. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments. For back tissues, scale bar, 200 μm (100×) or 50 μm (400×). For ear tissues, scale bar, 200 μm (100×) or 200 μm (200×). Significant differences were tested using a two-tailed Mann-Whitney U test Control, n=3, IMQ, n=6. ** P＜0.01, *** P＜0.001. NS, no significance. (D) Representative images of HE staining of the back tissues are shown on the left. The epidermal thickness measured using the scale in the microscope is shown on the right. (E) Representative images of Ki67 staining in back tissues are shown on the left. The number of Ki67-positive cells per HPF in each mouse is shown on the right. (F) Representative images of HE staining of the ear tissues are shown on the left. The epidermis thickness measured using the scale in the microscope is shown on the right. (G) Representative images of Ki67 staining in ear tissues (same specimen as in F) are shown on the left. The number of Ki67 positive cells per HPF in each mouse is shown on the right. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments. For back tissues, scale bar, 200 μm (100×) or 50 μm (400×). For ear tissues, scale bar, 200 μm (100×) or 200 μm (200×). Significant differences were tested using a two-tailed Student's t test Control, n=3, IMQ, n=6. ** P＜0.01, *** P＜0.001. NS, no significance.
Inhibition of pre‐autophagosomal maturation by Listeria PlcA/B protects Listeria against autophagy in trans.Figure 7(A, B) HeLa cells infected for 4 h with Listeria WT expressing GFP (WT GFP), Listeria PlcA/B− expressing mCherry (PlcA/B− mCherry), or both Listeria strains at a 1:1 ratio, were analysed by IF using an antibody against NDP52 (A). (B) Quantification of the percentage of cells displaying NDP52+ PlcA/B− mCherry from the experiments described in (A), in cells infected with either PlcA/B− mCherry alone or WT GFP and PlcA/B−mCherry (co‐infection). Values are means±s.e.m. n=3. **P0.01. (C) Murine embryonic fibroblasts (MEFs) from WT or ATG16L1‐deficient (ATG16L1 KO) mice infected for 4 h with WT GFP or Listeria PlcA/B− Listeria, either separately (Single) or in combination (Mix) were lysed and colony forming units (CFUs) determined on agar plates containing appropriate selection markers. Each point represents the value from individual independent experiments. **P0.01; *P0.05. Scale bars: 2.5 μm.
Figure 6 Representative images of day 21 BALO after microinjection of SC35M-GFP IAV (green) into the central airway-like (*) after 0 and 12 h pi visualizes IVA spread to the alveolar-like regions. Dotted lines illustrate BALO-borders. Scale bars represent 100 μm  Quantification of plaque forming units in supernatants from mock or SC35M IAV infected BALO 48 h after infection (n=5 biological replicates). N.D.: not-detectable  Relative NP expression in PR8 IAV infected (HA+) or non-infected (HA-) epithelial cells isolated from BALO 48 h pi, FACS-sorted according to EpCAM and HA expression (n=3 biological replicates with pooled cells from 4 cultures per replicate)  Representative flow cytometric data showing the percentage of EpCAM+NP+ cells in mock- and SC35M IAV‑infected BALO at 48 h pi in n=3 biological replicates with pooled cells from 4 cultures per replicate  Representative fluorescence images of a day 21 distal region in BALO generated from mTmG reporter mouse and infected by SC35M-Cre IAV after 0, 10, 14 and 25 h. Arrows indicate cell death. Scale bars represent 25 μm and 10 μm in the insert  mRNA expression of Ifnb in mock and PR8 IAV-infected BALO at 48 h pi (n=3 biological replicates with pooled cells from 4 cultures per replicate)  Release of TNFα (6 h pi), IL-6 (48 h pi), IL-1β (48 h pi) detected by Bio-Plex® Multiplex Immunoassay in the supernatant of mock and IAV infected BALO cultures with and without TR-Mac (n=3 biological replicates)
DCA treatment had beneficial effects on metabolism and mitochondrial function of SOD1G86R mice.Figure 7A-I Relative mRNA levels of (A) Pdk4, (B) Pparβ/δ, (C) Foxo1, (D) Pfk1, (E) Acsf2, (F) citrate synthase, (G) PGC-1α, (H) Mfn2, and (I) Gpx1 were evaluated by qPCR in tibialis anterior of control (CT) or DCA-treated (DCA) WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from CT WT group. P-values versus WT: Pdk4 ***P = 0.002 and ##P = 0.0039, Pparβ/δ #P = 0.0178, Foxo1 **P = 0.0033 and ##P = 0.0038, Pfk1 ***P = 0.0002 and ##P = 0.0080, Acsf2 *P = 0.0437, citrate synthase ###P = 0.0004, Pgc-1α **P = 0.0084 and ###P = 0.0009, Mfn2 *P = 0.0272, $P = 0.0145 and ###P = 0.0002 and Gpx1 ***P < 0.0001 and ###P < 0.0001 (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).