EMBO/sd-character-level-ner huggingface dataset:
Figure 5Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(a) Spinning disk confocal microscopy images acquired at 160x magnification. Left panels show a maximum intensity projection imaged of a Z-stack, sampled according to the Nyquist criterion and deconvolved. Right panels shows a 3D volume rendering of the same data. Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(b) A projection of 4 planes from a structured illumination microscopy super-resolution Z-stack of the same cells. Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(c) A thin section electron microscopy image of the peri-nuclear region of a GFP-Rab34 transfected HeLa cell. * indicates lysosomes. Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(d) A cryo-immuno-EM image of a GFP-Rab34 transfected HeLa cell. GFP-Rab34 (dark black dots) is identified by immunostaining for GFP and detection with gold conjugated protein A. The lysosome is intimately associated with Rab34 positive membranes. Sites of contact are highlighted by arrows.
Figure 2(A) Left panel: IκBα western blot of U2-OS cells. Lane 1, untreated (ut); Lanes 2-4, single IR (20 Gy) at indicated time points prior to harvest; Lane 5, IR at 5 days prior to harvest, plus 1.5 hours prior to harvest. Right panel: Samples treated as in the left panel, analyzed by RT-qPCR. Significance from n = 3 replicates per condition determined by ANOVA analysis with Tukey multiple comparisons test. SD shown. *** = p < 0.001. (B) NFKBIA knockdown by dox-inducible shRNA in U2-OS cells. Cells were pre-treated with Dox to induce knockdown. For knockdown efficiency see Fig. S2F. Cells were irradiated (20 Gy) or not, as indicated and described above. NF-κB activity was analyzed by EMSA. ns, non-specific band. A representative gel from n = 3 biological replicates is shown. (C) Venn diagram of RNA-Seq analysis of genes activated following irradiation in the first and second NF-κB activity phases or following shRNA-mediated NFKBIA knockdown in non-irradiated cells (as in B). Numbers of overlapping genes are shown in red. Hypergeometric probability calculation showed significance between overlaps (adjusted with Bonferroni correction for multiple testing). SD shown. *** = p < 0.001. (D) Top GO terms obtained using DAVID Functional Annotation Bioinformatics Microarray 6.7 for first and second phase genes and genes activated upon NFKBIA knockdown, as in (C), as indicated. (E) GSEA was performed comparing the IκBα IEC‐KO gene list (n = 4 villin-Cre x floxed IκBα mice and n = 4 littermate controls (GSE 139251) Mikuda et al, 2020(), with a gene signature based on a list of SASP factors Coppe et al, 2010(). NES: normalized enrichment score. FDR: false discovery rate. (F) RT-qPCR analysis of indicated genes was performed using RNA extracted from the duodenum of 5 and 8 weeks old control villin-Cre (n = 6) or villin-Cre x floxed IκBα mice (n = 6) both male and female. Expression is shown as fold change between littermates, paired t-test. SD shown. * p < 0.05 ** = P < 0.01.
Figure 5(A) Representative fluorescence images of Mock, SARS-CoV-2 infected (Ctrl), and Salmeterol-treated wells analyzed with the Multiwavelength Cell Scoring application in MetaXpress. Grayscales of the images were adjusted to enable direct comparison of the relative levels of fluorescence among the treatments: Segmentation images show how cells were segmented and identified as spike positive. Purple, nuclei; cyan, spike. Scale bar, 100 μm. (B) Violin plots of Vero-E6 cells labeled for Spike protein. The Multi-wavelength Cell Scoring algorithm in MetaXpress was used to determine the integrated fluorescent signal in individual cells as a measure of the amount of Spike protein within each cell. The plots show the population distribution of the integrated signal for all of the treatments. The Boxes in the plot show the interquartile range (IQR) with the top and bottom edges marking the 75th and 25th percentiles, respectively. The horizontal line in the box is the median value, and the whiskers are defined to be 1.5 IQR. The ordinate is a log scale. The effect of the treatment is assessed quantitatively by changes in the median signal level, and qualitatively by observing changes in the modes. The dashed line is 3 standard deviations above the mean signal in the Mock samples and is used as a cut-off to quantify the number of cells that are positive or negative for the Spike signal. The statistics table below the plots shows the number of cells counted in each treatment group, and the median of the population. (C) Pie charts showing the effect of treatment on the preventing infection of Vero-E6 cells. The number of cells above and below the cut-offs for being positive for Spike were counted and the percent cells in each category were determined. All analyses were done in Tibco Spotfire.
Figure 5(A, B) Primary neurons were exposed to Fib-Tau-ATTO550 (0.36nM, red) for 10 (top row) or 60 min (bottom row) and excitatory (Homer, green) and inhibitory synapses (Gephyrin, blue) were immuno-labelled. Fib-Tau clusters co-localized and/or apposed to Homer (arrowheads) but not Gephyrin (A). Quantitative analysis shows that 10 to 20% of Fib-Tau clusters are localized at Homer containing synapses (B). Box-plot represents: median, interquartile range and 10-90% distribution, Unpaired t-test to compare difference between 10 and 60 min, n is number of images analyzed from 3-experiments (10 min: 49; 60 min: 50). Scale bar, 5µm. Exposure of neurons to Fib-Tau-ATTO550 (0.36nM, 60 min, red) and immuno-labelling of excitatory synapse (Anti-Rabbit-Homer or Anti-Mouse-PSD, blue) and α3-NKA or GluA1-AMPA or GluA2-AMPA or GluN1-NMDA or GluN2B-NMDA subunits (green, post-permeabilization). Arrows indicate excitatory synapses where Fib-Tau and α3-NKA/AMPA/NMDA are co-localized (C). Mann-Whitney-test, n is number of images analyzed from 3-4 experiments (α3-NKA: 75; GluA1: 95; GluA2: 50; GluN1/N2B: 45). Exposure of neurons to Fib-Tau-ATTO550 (0.36nM, 60 min, red) and immuno-labelling of excitatory synapse (Anti-Rabbit-Homer or Anti-Mouse-PSD, blue) and α3-NKA or GluA1-AMPA or GluA2-AMPA or GluN1-NMDA or GluN2B-NMDA subunits (green, post-permeabilization). Quantification of the fluorescence intensity (indicating size of clusters of synaptic α3-NKA/AMPA/NMDA spots (obtained after thresholding) following exposure to Fib-Tau showed a reduction in the size of α3-NKA and increase in the size of GluA2-subunit containing AMPA receptors (D). Box-plot represents: median, interquartile range and 10-90% distribution, **p<0.01; ns= not significant. Scale bar, 5µm.
Comparison of protein turnover measured by pSILAC and CHX-chase.Figure 8A. Jurkat cells were treated with 100 µg/ml CHX for 0, 0.5, 1.5, 4, 8, 12 or 24 hours. Protein degradation was monitored by Western blotting and stabilities of several short-lived endogenous proteins were confirmed using antibodies (including lamin B, securin, β-catenin, GCIP interacting protein p29) and compared to stable proteins (such as GAPDH and actin). Proteoform-specific bands were used to calculate protein half-lives which were in a good agreement with turnover times obtained from pSILAC B-C. Validation of the differential turnover time of the dbTIS and aTIS derived proteoforms of MARE2 and AN32E. Selective C-terminal V5-tagged proteoforms were overexpressed in HCT116 cells for 24 hours and CHXpulse-chase experiments were performed as described for Jurkat cells. Degradation of overexpressed proteoforms was monitored by an anti-V5 antibody and compared to the turnover of stable proteins such as actin and tubulin.
Vacuoles and autophagosome-like structures in the degenerating epithelial glia.Fig 2(A-E) Horizontal sections of the adult head lamina cartridge. (A) In repo>GFP.nls flies, two lamina neurons L1, L2 axons (L) and R1-6 axons (blue area) were surrounded by the electron-dense cytoplasm of the epithelial glia (G). The section was examined at three different depths, and the size of a single cartridge is not significantly different at different depths. (B-D) repo>Shits1 adults maintained at 29°C for 4 days. Axons were enlarged but intact. A large number of small and medium vacuoles were identified in the epithelial glia. (C) Small vacuoles in the glia that contained a double-membrane structure (arrowhead). (C') Higher magnification of the boxed area in (C). (D) A double-membrane autophagosome-like structure (arrowhead) in the glialcytoplasm. (E) In repots>DERDN adults maintained at 29°C for 2 days, large vacuoles were identified in between the lamina cartridges. N: glialnuclei. (E') Higher magnification of the boxed area in (E), which shows autophagosome-like vesicles (arrowhead) in the gliacytoplasm.
Figure 1A. Immunoblotting of c-MYC and ATF4 in melanoma cells treated with NT-siRNA (-) or siASNS (#1 or #2) for 72 hr. Reverse transcription with quantitative PCR (RT-qPCR) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS (#1 or #2) (B) C. Reverse transcription with quantitative PCR (RT-qPCR) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS, siATF4, or both (C). D. Immunoblotting of ATF4 and ASNS in melanoma cells 72 hr after treatment with siASNS, siATF4, or both. E. Proliferation of melanoma cells over indicated times after treatment with siASNS, siATF4, or both. F. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS (#1 or #2). Immunoblotting of c-MYC in melanoma cells treated for 72 hr with siASNS, L-Asn, or both (G) H. Immunoblotting of c-MYC in A375 cells treated as indicated for 72 hr (H).
Figure 7(A) Live-imaging of MyoII-GFP in a control wing disc epithelium. Asterisk indicates the location of a single mitotic cell about to initiate rounding. Scale bar ~1µm. n > 5 independent biological replicates. (B) Live-imaging of MyoII-GFP upon treatment with the dual AurA/B inhibitor VX-680 after initiation of mitotic rounding, which rapidly arrests progression of rounding and prevents cytokinesis. Scale bar ~1µm. n > 9 independent biological replicates. (C) Live-imaging of E-cad-GFP upon treatment with the dual AurA/B inhibitor VX-680 after initiation of mitotic rounding, which rapidly arrests progression of rounding and prevents cytokinesis. Scale bar ~1µm. n > 6 independent biological replicates. (D) Working model for mitotic rounding and junctional downregulation in pseudostratified Drosophila epithelial cells.
Mitochondrial ribosomes.Figure 2(A-J) The small 28S (A) and large 39S (D) subunits are found in control (143B, blue lines) but absent in ρ0 cells (red lines), while subassemblies highlighted in color in the structural surface model (PDB 3J9M; Amunts et al., 2015) persist in the absence of ribosomal RNA. The relative changes for the individual components of the 28S (B) and 39S (C) subunits and migration profiles of the subassemblies as averages of its components (E-J) are shown using the same color code. Note that peaks at 30 - 60 kDa result from individual proteins rather than subassemblies. In (B-C) data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001, ns, not significant (Student's t-test).
Figure 1A. RGFP966 (966)-dependent HDAC3 inhibition resulted in significant and robust neurite outgrowth (24h in culture) compared to HDAC class II, HDAC1/2 and HDAC1 or 3 inhibitions via 69A, 963 and 233 respectively. Data information: Data expressed as mean fold change N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle ANOVA followed by Bonferroni test). B, C. Specific pharmacological inhibition of HDAC3 facilitates DRG neurite outgrowth (average neurite outgrowth after 24h in culture, Tuj1 positive neurons) on PDL-laminin or Myelin substrates. Scale bars, 100 μm. Data information: Data expressed as average neurite length ± s.e.m. N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle Student's t-test) D, E. Immunoblotting shows significantly increased H3K9 acetylation after 966 vs vehicle. Data information: Data expressed as mean fold change N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle Student's t-test) F, G. AAV-mediated mutant HDAC3 (Y298H, V5) infection of DRG neurons induces neurite outgrowth (Tuj-1/V5 double positive neurons) after 36h in culture, which is repressed by wildtype HDAC3 overexpression. Scale bars, 100 μm. Data information: Data expressed as average neurite length ± s.e.m. N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus V5 vs AAV-GFP ANOVA followed by Bonferroni test).
The activation state of Rags determines localization and activation of TFEB.Figure 5.(A and B) ARPE-19 cells were transfected with either active or inactive RagB/D heterodimers and infected with adenovirus expressing TFEB-FLAG-WT (A) or TFEB-FLAG-S211A (B). After 12 h, cells were double stained with antibodies against FLAG and GST (used to detect TFEB or Rag proteins, respectively). (C) Quantification of TFEB-WT nuclear localization from A. (D) Quantification of TFEB-S211A lysosomal localization from B. (E and F) ARPE-19 cells were cotransfected with plasmids expressing TFEB-GFP together with inactive (E) or active Rag heterodimers (F). Cells were then incubated with 250 nM Torin-1 for 1 h (E) or starved in serum- and amino acid-free medium for 3 h (F). Anti-GST antibodies were used to detect Rag proteins. Arrows point to cells that do not express Rag heterodimers.
Long-term transgene expression in the brain mediated by AAV BR1 vector.Figure 3AAV-BR1 vector harboring the luciferase gene under control of the CAG promoter was administered intravenously (5x1010 genomic particles/mouse, age 8 weeks). Long-term transgene expression was analyzed by in vivo bioluminescence imaging at 16 time points during a 665-day period (n = 2 animals). Original images of analyzed animals (above) and quantification of luminescence in the brain as the region of interest = ROI (bottom).
Figure 4(A) Flg22-induced ROS burst in Gmlmm1-1, Gmlmm1-2 and Williams 82. The indicated leaves (aged 2 weeks) were subjected to flg22-induced ROS examination, and the peak relative luminescence unit (RLU) value was recorded (Mean ± SEM, n ≥ 12, n represents sample number). Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data. (B) Flg22-induced ROS burst in the CRISPR lines C9-24-13 and DN50. The indicated leaves (aged 2 weeks) were subjected to flg22-induced ROS examination, and the relative RLU value was recorded (Mean ± SEM, n ≥ 9, n represents sample number). Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data. (C) Suppression of flg22-induced ROS production in Arabidopsis by GmLMM1. The GmLMM1 gene was introduced into Arabidopsis WT (Col-0) plants by Agrobacterium-mediated transformation. Two independent T2 lines were subjected to flg22-induced ROS examination (Mean ± SD, n ≥8, n represents sample number, ***, p < 0.001, Student's t-test). The protein expression is shown. Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data. Suppression of flg22-induced (D) ROS production in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed by Agrobacterium-mediated transient expression for 2 days and subjected to flg22-induced ROS examination. GmLMM1K564E contains a mutation at the predicted ATP binding site without kinase activity. GFP was used as a control (Mean ± SD, n = 6, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The protein expression is shown in the lower panel. Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data. E) Suppression of chitin-induced (E) ROS production in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed by Agrobacterium-mediated transient expression for 2 days and subjected to ROS examination. GmLMM1K564E contains a mutation at the predicted ATP binding site without kinase activity. GFP was used as a control (Mean ± SD, n = 6, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The protein expression is shown in the lower panel. Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data. (F) Inhibition of XEG1-induced cell death in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed in N. benthamiana 1 day before infiltration of Agrobacterium containing the XEG1 or GFP vector. The cell death phenotypes were visualized 5 days later. Protein expression of GmLMM1-HA and GmLMM1K564E-HA is shown in the lower panel.
Figure 4Effects of compounds that target histamine receptor family members. The outline shows the compounds (listed in Dataset EV3) that target histamine receptor family members as antagonists (HRH1, HRH2, HRH3 and HRH4) or agonists. These include the compounds originally present in the Prestwick library as well as additional compounds selected from the litterature. Cells treated with this small library of compounds were analysed by automated microscopy after labeling with DAPI and antibodies against LBPA. Compounds that increased LBPA staining intensity, like thioperamide (Fig 1) or pitolisant (Fig 4B) are green, while compounds without effects are red. (n=2 independent experiments with 96 images acquired and analysed automatically). Effect of Pitolisant on LBPA. HeLa MZ cells treated or not with pitolisant 10µM for 18h at 37°C were labeled with DAPI and anti-LBPA antibodies and analysed by fluorescence microscopy (quantification in fig EV3A). LBPA intensity in a mixed population of cells expressing HRH3-GFP. After transfection with HRH3-GFP, uncloned stably expressing cells (C) were labelled with anti-LBPA antibodies and analysed by automated microscopy (C). Unbiased quantification (D) shows the inverse correlation between HRH3-GFP expression (high expressing cells in green) and the endosome integrated intensity of LBPA staining (high LBPA labelling in red). (n=2 independent experiments with 500,000 cells analysed automatically, error bars = SD). LBPA intensity in cells expressing or not HRH3-GFP. HeLa MZ cells stably expressing HRH3-GFP grown in 96-well plates were separately treated, with 4 different siRNAs that target HRH3 (siHRH3#1; siHRH3#2; siHRH3#3; siHRH3#4) or with control non-target (siNT) siRNA (E). HRH3-GFP was analysed by fluorescence microscopy in 4 rows of cells per condition; micrographs are stitched together in the montage. Panel F shows an example of cells treated with siHRH3#2 or siNT, and then labelled with DAPI and antibodies against LBPA. Cells treated as in (E) and labelled with DAPI and antibodies against LBPA as in (F), were analysed by automated microscopy and the integrated intensities of LBPA (left panel) and HRH3-GFP (right panel) signals are compared (G). (n=2 independent experiments with 192 images acquired and analysed automatically, error bars = SD).
Fig.Figure 5HEK293 cells were transfected with α11.2 with the K1647E mutation, α2δ-1, and β2A plus α-actinin-1 WT or E847K/E851K before cell attached patch recording. Representative single CaV1.2 channel recordings of CaV1.2 K1647E with WT or E847K/E851K α-actinin-1. Mean assemble averages for all experiments for both combinations. Dot plots and means + SEM for availability (c), NPo (d), Po (e), unitary current amplitude i (f), and mean of the current of a single channel calculated from the ensemble averages of each experiment (g) (**p<0.01; unpaired t-test; n = 16 to 30; see Suppl. Fig. 8 and Table 6 for more details).
Figure 4A, Coimmunoprecipitation (Co-IP) analysis was performed in A549 cells with antibodies against p53 or USP7. B, H1299 cells were transfected with an empty vector (Cont.), a Flag-tagged p53, a USP7-specific shRNA, or the last two plasmids together for 48 h. Western blot analysis was then performed. C, Nuclear and cytosolic extracts were differentially prepared from H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.). Western blot analysis was then performed. D, H1299 cells transfected with a Myc-tagged USP7 or a Myc-tagged USP7 together with a Flag-tagged p53 were subjected to IF analysis (upper). A549 cells transfected with a p53-specific siRNA or a control siRNA (siCont.) were examined by IF analysis, and the RNAi efficiency is shown (lower). Representative images are shown (magnification, ×40). E, Diagrams showing the interaction status of USP7 and its truncated mutants (USP7-△N, lacking the N-terminal region; USP7-△M, lacking the middle region; USP7-△C, lacking the C-terminal region) with p53, based on previous reports F, IF analysis of A549 cells transfected with a Myc-tagged wild-type USP7 (Myc-USP7-WT) or three Myc-tagged truncated mutants. Representative images are shown (magnification, ×40). G, H1299 cells were transfected with a Flag-tagged p53 or an empty vector together with a USP7-specific siRNA or a control siRNA as indicated for 48 h. Cells were then harvested and subjected to a MNase digestion assay.
Figure 6The in vitro suppressive potency of wild type- and K262R Foxp3-expressing T cells. Naïve CD4+ T cells were purified from Thy1.1+ BALB/c mice and subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262. These engineered "Tregs" were then co-cultured with naïve responder CD4+ T cells stained with CFSE at the indicated ratios and activated with anti-CD3/CD28 antibodies. Dilution of CFSE signal by the responder populations (i.e. proliferation) was assessed by flow cytometry. The in vitro suppressive potency of wild type- and K262R Foxp3-expressing T cells. Naïve CD4+ T cells were purified from Thy1.1+ BALB/c mice and subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262. These engineered "Tregs" were then co-cultured with naïve responder CD4+ T cells stained with CFSE at the indicated ratios and activated with anti-CD3/CD28 antibodies. Dilution of CFSE signal by the responder populations (i.e. proliferation) was assessed by flow cytometry. C In vivo suppression of colitis by wild type- and K262R mutant-Foxp3 expressing T cells. 2x105 naïve CD4+ transductants from A were mixed with 1x106 colitogenic naïve CD4+ T cells from wild type mice and transferred i.v. into Rag2-/- mice. Rag2-/- mice receiving no cell transfers, naïve CD4+ T cells alone, or purified wild type Tregs served as controls (n= 6/group). Recipient mouse body weights were monitored weekly for each group. D, E After 8 weeks, colons were excised, fixed in buffered formalin, and processed for H and E staining (D) and histopathology scoring (E). Scale bars shown in 1 mm).