EMBO/sd-character-level-ner huggingface dataset:
IMCs promote growth and EMT of lung tumour cells.Figure 3CMT64 cells (1 × 104/well in 12-well plates) were co-cultured with 1.5-3 × 105 IMCs for 4 days. Cells were collected by trypsinisation to exclude most of the IMCs since they are resistant to trypsin due to their strong adhesion to the culture plate. Harvested cells were subjected to counting (left). Any contaminating IMCs were separately counted according to their morphological distinction from CMT64 cells. The bar graph (left) shows CMT64 growth (cell counting) data pooled from two independent experiments. Phase-contrast images of CMT64 cells cultured without IMCs (CMT64 only) or co-cultured with IMCs (CMT64/IMC co-culture) are also indicated in the middle and right. A higher magnification image of co-cultured CMT64 cells (right) highlights IMCs migrating into CMT64 cells (arrow heads). Scale bars are 200 μm, except for the high magnification image (right) in which the scale bar is 100 μm. (Left) Growth and EMT-like morphological changes of CMT64 cells cultured with 25% IMC-CM. CMT64 cells cultured for 4 days at low-density in DMEM/10%FCS without IMC-CM (-IMC-CM) or with 25% IMC-CM (+IMC-CM) were counted and normalised to the average of control as 100%. The data in the bar chart represent mean + SD of five cultures utilising IMC-CM obtained from three independent sources. (Middle) Phase-contrast images to indicate representative morphologies of CMT64 cells maintained without IMC-CM (control) or cultured with 25% IMC-CM (+IMC-CM) for 2 weeks. (Right) E-cadherin immunofluorescence of CMT64 cells cultured as above. Scale bars are 200 μm (phase contrast) and 10 μm (immunofluorescence). Co-culture of AT2 cells and autologous IMCs from BVE mice (8- to 10-week-old) for 48 h using the Transwell® culture system. In vitro BrdU incorporation (flow cytometry, left and middle) and EMT marker expression (RT-PCR, right) in co-cultured AT2 cells are indicated. Representative flow cytometry plots are indicated on the left, and the bar chart in the middle indicates % BrdU+ AT2 cell numbers normalised to control cultures without IMCs (n = 4, mean + SD). Gapdh serves as a loading control for the RT-PCR on the right. Morphological alterations and intracellular signalling in AT2 cells cultured with IMC-CM. On the left and middle, primary AT2 cells from 3-week-old BVE mice were cultured for 48 h and then incubated in serum-free IMC-CM (AT2 with IMC-CM) or DMEM (AT2 w/o IMC-CM, serving as a control) for another 48 h, followed by immunostaining for E-cadherin (red) and vimentin (green) and confocal laser scanning microscopy (CLSM) imaging. The 3× zoomed image in the middle right highlights dividing vimentin+ cells that show internalised E-cadherin (arrows). Scale bars, 50 μm. On the right, primary AT2 cells from 10-week-old BVE mice were cultured for 48 h, serum starved for 5 h and treated with or without serum-free IMC-CM for 30 min. Phosphorylation of AKT, SMAD3, MEK and ERK were analysed by immunoblotting.
JAK2 is critical for Mpl subcellular localization.Figure 4HEL (A) and K562 (B) cells treated with non‐targeting (NT) or anti‐JAK2 siRNA analyzed by western blot. C) Confocal live cell (upper panel), or fixed cells (lower left panel), imaging of K562 cells expressing MpleGFP or MplmKO2 treated with NT or anti‐JAK2 siRNA showing a difference of distribution of intracellular Mpl in the siRNA treated cells compare to the control cells. After treatment with anti‐JAK2 siRNA, Mpl strongly colocalizes with the ER marker, calnexin. This colocalization in treated vs non‐treated cells is quantified and shown in Figure C lower right panel. Nuclei are labeled with Hoechst and scale bars = 5 µm.
Figure 6A RNA-seq expression values of select genes in BT168FO GSCs along a 48 h time course of Omomyc induction (n = 3 independent biological replicates). The block on the left represents DUSP (dual specificity phosphatase) family genes. The middle block contains transcription factors (TFs): the upper thirteen (ASCL1-SALL1) are GSC-specific and the remaining ones are oncogenic. The right block contains genes, including well-known MYC targets, with functions in proliferation, neurodifferentiation, and gliomagenesis. The first column of each block represents the average expression (log2 FPKM) in untreated cells (0 h) in the colour scale illustrated by the lower bar: violet indicates low and blue high expression. The other columns depict relative expression versus untreated cells (average log2 FC) at different times (8-48h) of Dox treatment, according to the scale shown by the upper bar: red indicating low and green high expression. The table in the upper left summarizes the GSEA score of the set 19 GSC-specific transcription factors [40], showing a significant downregulation by Omomyc. B Enrichment plots obtained by GSEA (Gene Set Enrichment Analysis) of RNA-seq data from BT168FO cells. Left: genes downregulated by Omomyc in BT168FO identify the set of genes targeted by the GSC core TFs [40]. Middle: Omomyc downregulated genes identify a gene set upregulated by MYC in cancer cells [66]. Right: genes upregulated by Omomyc identify genes downregulated in small cell lung cancers carrying MYC amplification [2]. . C The tables show Enrichment Analyses of various examples of MYC-regulated gene sets present in MSigDB (http://www.broadinstitute.org/gsea/msigdb) and of the gene set targeted by the GSC core TFs [40], versus genes repressed (left table) or induced (right table) by Omomyc in the RNA-seq dataset. NES: Normalized Enrichment Score; FDR: False Discovery Rate.
Figure 2(A) Estimation of the intrinsic force that the vacuolar SNARE complex can exert on a stalk-like fusion intermediate via its TMD C-termini (Nyv1 and Vam3). At this stage, the distance between the C-termini is about 8 nm. The force that the C-termini exert on the membrane, about 9 pN, is estimated from the difference in work required to induce a small indentation in, respectively, the presence of structured or unstructured Nyv1 and Vam3 linker regions (the slope of this profile defines the force). The additional presence of a structured Vti1 linker doubles the exerted force from 9 pN to 18 pN. (B) The inherent elastic response of a lipid membrane (POPC) was studied by enforcing indentations via the isolated SNARE C-termini in the presence or absence of a short linker peptide, or the linker plus a fluorescent protein tag (FP). We define "relative indentation" as the change in C-terminus (Nyv1) - C-terminus (Vam3) distance that occurs when the SNARE complex performs mechanical work. It reflects the depth of the formed well upon squeezing. The slope of the obtained work profile estimates the required force (thin black line). Perforation (red arrow) requires a force of 80 pN for the wild-type C-termini. The plateau within the work profile indicates that the membrane gives up its elastic response (perforation transition). C-terminal tags oppose such a perforation transition but do not significantly affect the forces observed within the indentation regime. Furthermore, if the unstructured peptide-spacer would be alpha-helical - and thus much shorter - indentation forces would still be only slightly affected.
a) Molecular structure of ATRA (top) highlighting three different regions: the hydrophobic ring (black), the polyene linker 'connector' (blue) and the carboxylic acid moiety (red).figf4(a) Molecular structure of ATRA (top) highlighting three different regions: the hydrophobic ring (black), the polyene linker 'connector' (blue) and the carboxylic acid moiety (red). The basic structure of the four families of compounds generated through modifications of ATRA using structure-based chemical design strategies are shown. Numbering is shown in the retinoic acid and the α-aminonitrile retinoid backbone to indicate how these positions have been conserved in the new molecules. (b-d) Molecular docking of AR7 (b), GR1 (c) and GR2 (d) in the RARα-binding pocket. A close view of the RARα-binding pocket in ribbon (gray) and interacting residues in stick (magenta) for each compound docked in the lowest-energy conformation for docking pose I is shown. Compounds are docked to a hydrophobic region of the RARα-binding pocket formed by h3, h10 and h12. Hydrogen bonds are formed from the guanidinium group of GR1 and GR2 to side chain hydroxyls of Ser229 and Thr233 and to backbone carbonyl oxygen of Pro407 (yellow dotted lines).
Figure 1IFM images of KIF13A-YFP transfected control and Rab-knockdown HeLa cells. TN: average tubule number (mean±SEM, n=3). B, C Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 1A (mean± SEM). n=3. nc: total number of cells. nt: total number of tubules.arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm. IFM images of KIF13A-YFP and mCherry-Rab7A/11A/22A transfected HeLa cells arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm Live cell imaging of GFP/mCherry-Rab22A with respect to mCherry-Rab11A or GFP-Rab7A in HeLa cells. Magnified view of insets (at 0, 20, 40 seconds) are shown separately ), arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm Pull-down of different His-KIF13A domains using HeLa cell lysate and then probed with indicated Rab proteins. The bead bound His-KIF13A in each pull-down was shown as coomassie gel. *, non-specific bands.
Figure 3ST2 wild type or TBK1/IKKε DKO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (A) The heatmap shows the row-normalized Z-Score calculated from log2 transformed iBAQ intensities from five independent experiments. ST2 wild type or TBK1/IKKε DKO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (B) The ratio between iBAQ intensities of selected IL-17RSC components to iBAQ intensity of IL-17RC. Mean + SEM from five independent experiments is shown, statistical significance was determined by two‐tailed Mann-Whitney test. C. ST2 wild type or TBK1/IKKε DKO cells were stimulated with SF-IL-17 (500 ng/ml) for 15 minutes or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC and samples were analyzed by immunoblotting. ACT1 phosphorylation detected as upper band in WT cells is absent in TBK1/IKKε DKO cells. D. Cells were pretreated with TAK1 inhibitor 7-oxozeanol (2 µM) and/or TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) for indicated time points and lysates were analyzed by immunoblotting. E. ST2 wild type or HOIP-deficient cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) as indicated, solubilized, and analyzed by immunoblotting. * indicates unspecific band. F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 were stimulated with SF-IL-17 for 15 minutes or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated and analyzed via immunoblotting.
Figure 5Male, 12wk wildtype (WT; C57Bl/6J) or obese/diabetic (db/db; BKS.Cg-m+/+ Lepr DB/J) with (AD-G45b OE) or without (AD-NC) prior liver-restricted GADD45β over-expression were fasted and blood glucose ( A) and serum insulin (B) were measured from which HOMA-IR was calculated (C) (n = 4-6/group). Data are mean ± SEM. Effect of genotype/viral manipulation, * p < 0.05, ** p < 0.01, *** p < 0.001. Effect of nutritional state: # p < 0.05, ## p < 0.01, ### p < 0.001. Liver GADD45B mRNA expression from men with (T2D) or without (NGT) type 2 diabetes (n = 14-23/group). Scatter plots fasting plasma triglycerides (E) and glucose (F) in correlation with liver GADD45B mRNA expression (n = 37). Inserts show r2 values and p values from Spearman's correlation test. The statistical test used and respective p-value outputs can be found in Appendix Table S1.
is upregulated by TGF-β1 in GSCs and is clinical relevant in GBM Heatmap of RNA-Seq analysis of differentially expressed lncRNAs (≥ 2-fold change and FDR< 0.Figure 1Heatmap of RNA-Seq analysis of differentially expressed lncRNAs (≥ 2-fold change and FDR< 0.05) in mesenchymal (MES) 1123 and proneural (PN) 528 GSCs. GSCs were pre-cultured for 16 h in DMEM/F12 medium with EGF (2 ng/ml) and bFGF (2 ng/ml) and then followed by co-culturing with or without 20-μg/ml TGF-β1 for 3 h. Representative images of LINC00115 expression in clinical GBM tumors and adjacent tissue using RNAscope analysis. Images are representative of two independent experiments. Scale bars: 50 μm. Kaplan-Meier analysis of patients with high versus low LINC00115-expressing GBM tumors from B. Median survival (in months): low, 13.9; high, 8.1. Black bars, censored data. Data information: , *, p < 0.05, by log-rank test. Expression levels of LINC00115 are higher in LGG (low grade gliomas) versus normal brain tissues and GBM versus LGG. Expression data of LINC00115 were downloaded from the REMBRANDT dataset. Data are presented as mean ± SEM. **, p < 0.01, ***, p < 0.001, by two-tailed t test. E to G. Kaplan-Meier analysis of patients with high versus low LINC00115-expressing Grade III and GBM tumors from the REMBRANDT (E) or the TCGA (G) 26[], and GBM from GSE83300 50[] (F) datasets. Median survival (in months) in E: low, 20.2; high, 15.9; in F: low, 19.4; high, 13.0; in G: low, 33.2; high, 25.4. Black bars, censored data. Data information: , *, p < 0.05, by log-rank test.
Figure 2Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. On the schemes above the charts in (A) and (C), chromosomes are red with green centromeres. Changes in the distance to the centre for all chromosomes in a representative oocyte undergoing transition from anaphase I to metaphase II from Fig 1. The black arrow on the scheme above the chart indicates chromosome-centre distance. Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. Changes in the speed of all chromosomes observed for the oocyte from Fig 1. Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. On the schemes above the charts in (A) and (C), chromosomes are red with green centromeres. (C) Changes in inter-centromere distance (indicated on a scheme by the black arrow), observed for the oocyte from Fig 1. Kinetochore-MT attachments at the prometaphase II stage were visualized using an anti-tubulin antibody (white), an anti-centromeric ACA antibody (green) and DAPI to label chromatin (red). The image represents maximum intensity projection through all z-planes containing MTs. Representative chromosomes displaying syntelic, amphitelic or merotelic/lateral attachments are enclosed in white frames and their enlarged single z-plane images are shown to the right. PB: polar body. Scale bar, 10μm. Chromosomes with syntelic (red), amphitelic (green) and merotelic/lateral (grey) attachments were plotted according to their inter-centromere distance (on the horizontal axis) and distance to spindle equator (vertical axis). Data shown for 156 chromosomes taken from 8 prometaphase II oocytes.
Membrane-associated TRBP expression blocks the BDNF-induced decrease of miR16 and BDNF-induced dendritogenesis.Figure 7More Dicer binds to membrane-targeted GFP-TRBP (mGFP-TRBP) compared to GFP-TRBP. HEK293T cells were transfected with GFP, GFP-TRBP or mGFP-TRBP and cell lysates were processed for co-IP using anti-GFP antibody. Dicer binding was normalized to respective input lanes in each condition (n=4, p=0.02; t-test type 2, error bars are s.d.). B) mGFP-TRBP but not GFP-TRBP blocks the BDNF-induced decrease in miR-16-5p activity in dual-fluorescence sensor assay. Hippocampal neurons were transfected with 200ng miR16- or control-sensor (see Fig 5A) plus 50ng GFP, 400ng GFP-TRBP or 400ng mGFP-TRBP. Neurons were treated with BDNF or vehicle control prior to fixation and immunostained with anti-GFP antibody. BDNF led to a decrease in miR-16-5p activity in GFP-transfected neurons (p=0.03, n=4, t-test type 3), which was significantly different from mGFP-TRBP-expressing neurons (p=0.04, n=4, t-test type 2, error bars are s.d.). C&D) BDNF-induced dendritogenesis is blocked by mGFP-TRBP overexpression. C) Hippocampal neurons (4DIV) were transfected for three days with 200ng dsRed and 50ng GFP, 400ng GFP-TRBP or 400ng mGFP-BDNF. Neurons were treated with BDNF or vehicle control 24 hours before fixation. Pictures were obtained on a confocal microscope and threshold was set for dsRed to distinguish individual dendrites (scale bars are 10μm). D) The total number of intersections was calculated in Sholl analysis, and values were normalized to control treatment in each of three independent experiments. BDNF led to a significant increase in dendritic complexity (p=0.04; t-test type 3), which was significantly different in cells transfected with mGFP-TRBP, but not GFP-TRBP (n=3, p=0.03; t-test type 2, error bars; s.d.).
Figure 3A. Ectopic expression of NLK attenuates interaction between YAP and 14-3-3. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-HA antibody and immunoblotted with anti-GFP antibody (top two panels). Whole cell lysates (WCL) were immunoblotted with antibodies shown in the figure (bottom three panels). B. Deficiency of NLK leads to increased interaction between YAP and 14-3-3. Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti-YAP antibody and immunoblotted with anti-14-3-3 antibody (top two panels). WCL were immunoblotted with antibodies indicated in the figure (bottom three panels). C. Phosphorylation status of Ser127 or Ser128 determines interaction between 14-3-3 and YAP. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-HA antibodies (top two panels). Lysates were immunoblotted with anti-HA or anti-Flag antibodies (bottom two panels). "n.s." arrow indicates a non-specific band also visible in the non-transfected lane. D. Phosphorylation status of Ser127 or Ser128 determines subcellular localization of YAP. Immunofluorescence analysis was performed in HeLa cells transfected with EGFP-YAP, EGFP-YAP-S127A, EGFP-YAP-S128A, or EGFP-YAP-S128D at high cell density. Figure shows a representative of multiple areas. Nuclei were stained with Hoechst 33342. Scale bars: 20 μm. E. NLK knockout leads to increased cytoplasmic localization of endogenous YAP. Immunoblot analysis shows significant reduction of NLK level in NLK knockout HEK293 cell pools (top panel). E. NLK knockout leads to increased cytoplasmic localization of endogenous YAP. To determine subcellular localization of YAP indirect immunofluorescence analysis was performed in NLK knockout HEK293 cell pools (bottom panel). Figure shows a representative of multiple areas. Nuclei were stained with DAPI. Scale bars: 20 μm.
Chemical inhibition of DOT1L results in aberrant plasma cell differentiation in response to in vitro stimulation.Figure 5A. Representative flow cytometry plots showing gating strategy to identify and compare plasma cells (Sca-I+CD138+) after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4). B. Representative flow cytometry plots showing gating strategy to identify and compare activated B cells (CD138+ Blimp1+), Pre-PB cells (CD138- Blimp1+) and plasma blast (PB) cells after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4). C. Representative flow cytometry plots showing the relative surface density for B220 (CD45R) after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analysis of MFI of B220 for each treatment (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4). D. Representative histograms showing the relative surface density for CD19 on total live cells after four days of stimulation with LPS (left panel) and statistical analysis of MFI of CD19 for each treatment (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).
Figure 3A,B) Scatterplots showing that the wPRS correlates with A) adjusted baseline performance on the ECAS ALS-Specific, Total, Executive Function, and Language scores, and B) rate of decline on the ALS-Specific, ALS-Non-Specific, and Total scores.
Figure 6WT or STX16KO HeLa cells were starved in EBSS for 1 h, followed by starvation with the presence of LysoTracker Red DND-99 (LTR) (100 nM) for additional 30 min, and subjected to HCM analysis of LTR. Scale bar: 20 µm.   Quantifications of LTR count per cell or total area per cell in WT and STX16KO HeLa cells treated as in (A). Data shown as means ± SEM of LTR puncta per cell or object total area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant (two-way ANOVA).   WT or STX16KO HeLa cells were starved in EBSS for 1 h, followed by starvation with the presence of LTR (100 nM) for additional 30 min, and subjected to HCM analysis of the colocalization between LTR and TGN46. Masks: white, cells identified based on nuclei; red, LTR puncta; green, TGN46 puncta; yellow, overlap between LTR and TGN46. Scale bar: 20 µm.   Quantifications of overlaps between LysoTracker Red and TGN46 in WT and STX16KO cells treated as in (C). Data shown as means ± SEM of LTR and TGN46 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).   WT or STX16/STX17DKO HeLa cells were starved in EBSS for 2 h, and subjected to ultrastructural analysis of the Golgi apparatus with electron microscopy. VF of Golgi: volume fraction of Golgi relative to total cytoplasmic area. Scale bar, 2 μm. Red arrows indicate Golgi apparatus.
Model of ER-phagy.Figure 7During micro-ER-phagy, portions of the ER (blue) are reorganized to form stacks (1). ER stacks are bent, recognized by the vacuole, taken up into vacuole membrane invaginations, converted into whorls and detached from the remainder of the ER (2). The order of these events is uncertain. ESCRT machinery is recruited to sites of ER whorl uptake and mediates scission of the vacuole membrane (3). In parallel, macro-ER-phagy can also selectively target the ER, which is mediated by the core autophagy machinery.