EMBO/sd-character-level-ner huggingface dataset:
Basolateral α-β Spectrins are required to restrict cell proliferation in the Drosophila intestinal epithelium.Figure 7Basolateralα-β Spectrins are required to restrict cellproliferation in theDrosophila intestinal epitheliumA Control myo1A.Gal4UAS.GFP adult midgut stained for phospho-histone H3 to mark mitotic stem cells.B-E RNAi knock-down of βH-spectrin/karst (B) or crumbs (C) does not increase stem cellproliferation, while RNAi knock-down of β-spectrin (D) or α-spectrin (E) strongly stimulates stem cellproliferation.F Overexpression of Yki strongly stimulates stem cellproliferation.G Quantification of the number of PH3-positive mitotic cells. N > 13 samples. Statistical analysis performed using the two-tailed Student's t-test. Error bars show standard deviation. ***P-value ≤ 0.001.H-K A Hippo reporter DIAP1-HRE-GFP is normally expressed in sporadic stem cells. Its expression is not affected by RNAi knock-down of βH-spectrin/karst (I), but is up-regulated in many stem cells by RNAi knock-down of α-spectrin (J) or by overexpression of Yki (K).Data information: Scale bars, 100 μm.
Figure 2A Schematic illustration of the experiment. C57Bl/6 mice (7 weeks old) received AAV expressing PLTP or GFP (as control) at the viral titer of 5x1012 GC/mouse via tail-vein. High fat diet (HFD) started one week after AAV administration for both groups. Metabolic studies and euthanasia were performed after 10 weeks of HFD. B Changes in body weight of mice in (A) during 10 weeks of HFD. n=9 for GFP and n=8 for PLTP (cohort 1). *P<0.05, **P<0.01, ***P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test. C Changes in food intake of mice in (A) during 10 weeks of HFD (cohort 1). n=9 for GFP (cage 1 = 5 mice; cage 2 = 4 mice) and n=8 for PLTP (cage 1 = 4 mice; cage 2 = 4 mice) D Body composition of mice in (A) at 10 weeks of HFD determined by EchoMRI. n=9 for GFP and n=8 for PLTP (cohort 1). *P<0.05 relative to GFP by two-way ANOVA followed by Tukey's test. N.S., not significant. E Whole-body energy expenditure of mice at 3 weeks of HFD. Area under the curve (AUC) of VO2 is shown in the right graph. n=7 for GFP and n=8 for PLTP (cohort 2). *P<0.05 relative to GFP by Student's t-test. F Physical movement of mice at 3 weeks of HFD as determined by metabolic cage analysis. n=7 for GFP and n=8 for PLTP (cohort 2). N.S., not significant by Student's t-test G OCR in the interscapular BAT and iWAT of control and PLTP-treated mice. One week after the treatment, BAT and iWAT were isolated from mice (7 weeks old) that received adenovirus for GFP or PLTP. n=5 for both groups. *P<0.05 relative to GFP by Student's t-test. N.S., not significant. H Gene expression of indicated thermogenic genes in (A) from interscapular BAT for GFP (n=8) and PLTP (n=8) groups (cohort 1) except n=7 for Ucp1 of the PLTP group. Tbp was used as an internal control. *P<0.05, **P<0.01 relative to GFP by Student's t-test. N.S., not significant. I H&E staining of interscapular BAT from mice in (A) at 10 weeks of HFD (cohort 1). Representative images from n=3 mice for both groups. Scale Bar, 50µm.
Figure 3schematic showing the immobilization of McjD to the surface: labeled McjD reconstituted in biotinylated DOPE lipid was immobilized to a PEG-biotin-coated surface in a flow cell via a neutravidin-tag on the proteoliposome. A typical surface scan is shown in false-color representation on the right (orange, double-labeled McjD; green, McjD with donor fluorophore only; red, McjD with acceptor fluorophore only) Representative fluorescence time traces (blue, donor signal; red, acceptor signal; black, FRET signal; yellow, fit) of McjD labeled with Alexa555 and Alexa647-maleimide with indicated concentrations of corresponding substrate: (B) McjD L67C with 10 mM ATP and 200 µM MccJ25, McjD Y64C with 10 mM ATP and 200 µM MccJ25, and McjD C547 with 10 mM ATP. The respective right panels are the projections of accumulated time traces in the absence and presence of substrate for each variant. We note that differences between the setup dependent apparent FRET values are not influencing our interpretations since only relative changes of FRET efficiency are interpreted. Experimental support for proper reconstitution is provided in Figure EV3A showing longer bursts and distinct TEV cleavage for liposomes compared to detergent
Atg36 expression and cleavage correlates with Pex11–GFP degradation.Figure 6(A) WT cells expressing genomic Atg36-PtA and Pex11-GFP on a plasmid were grown in glucose for 22 h, transferred to oleate medium, and then shifted to SD‐N medium. Samples were taken at the times indicated and processed for western blot with peroxidase anti‐peroxidase complex to detect Atg36-PtA (top panel) or anti‐GFP to detect Pex11-GFP pexophagy (bottom panel). GFP* indicates the relative protease‐resistant degradation product and reflects vacuolar breakdown. (B) Western blot analysis of genomic Atg36-GFP under oleate and starvation conditions for the indicated time points in the strains indicated. (C) Western blot analysis of genomic Atg36-GFP in WT, pex14Δ, atg1Δ and pex3Δ cells grown for up to 3 days in oleate medium. Samples were taken at 24 h intervals. (D) Pex3 level in total lysate was determined from WT cells grown in glucose medium for 22 h, transferred to oleate medium and then shifted to SD‐N medium. Samples were taken at the times indicated and processed for western blot with anti‐Pex3. A pex3Δ glucose 22 h sample was used to determine the specificity of the antibody. **Indicates non‐specific band. (E) Western blot analysis of pex3Δ and atg36Δpex3Δ cells expressing Pex3-GFP from its own promoter after growth in glucose, oleate‐containing medium and after shifting to SD‐N medium for the times indicated.
Figure 3Immunohistochemical detection of Aβ40 (left column), Aβ42 (medium column) and Aβ43 (right column) in consecutive frontal cortex paraffin sections of two FAD cases with PS1 L435F mutation (cases #1 and #2, 1st and 2nd rows), another FAD case with different PS1 mutation (case #3, 3rd row) and one SAD case (case #4, forth row). In both PS1 L435F mutation cases the numerous Aβ plaques mainly contain Aβ42 and Aβ43 but less Aβ40. This is in contrast to Aβ plaques of the control FAD and SAD cases, in which solely Aβ42 predominates and Aβ43 is sparse; even levels of Aβ40 seem to be lower than in the PS1 L435F cases. Note that in both PS1 L435F cases several plaques are larger than those seen in cases #3 and #4, representing cotton wool plaques. Scale bar = 500 µm. Magnification is identical in all pictures.
Figure 5A) Identification of Cdc14 phospho-targets during the DNA damage response by mass spectrometry analysis. Wild-type and cdc14-1 cells were grown overnight and blocked in G2/M by using nocodazole to avoid cell cycle-dependent changes in protein phosphorylation between both strains. After the block was attained, cells were transferred to 37ºC for 45 minutes to deplete Cdc14 activity prior HO induction by galactose addition for 4h. Differential phosphorylation of phospho-peptides detected between the wild-type and cdc14-1 were grouped into broad categories depending on the molecular function of the proteins. The table includes the DNA damage and checkpoint-related proteins with Cdc14-dependent hyper-phosphorylated status and the relative ratio between the wild-type and cdc14-1 during the DNA damage response. Red and blue indicate relative amount of the residue phosphorylation between both strains (Red, high; Blue, low). B) Cdc14 dephosphorylates Spc110 in response to a DSB. Spc110 was tagged with the 6HA epitope in wild-type, cdc14-1 and spc110S36-91A backgrounds. Cells were grown overnight in raffinose-containing media and supplemented with galactose to induce HO expression. Samples were collected at each time-point indicated. Proteins were TCA extracted, separated on Phos-Tag containing gels and blotted. Coomassie staining is shown as loading control. C) Lambda-phosphatase treatment of protein extracts in the absence of DNA damage reduces Spc110 phosphorylation levels to the same extent as samples taken during the DNA damage response. Cells were grown overnight in raffinose containing media (line 1) and incubated with galactose for 2.5 hours to induce HO expression (line 2). Extracts from raffinose-containing cultures were treated with mock (line 3) or λ-PPase (line 4). All samples were separated in a Phos-Tag containing gel and blotted. Coomassie staining is shown as loading control. D) The canonical FEAR pathway is required for Spc110 dephosphorylation during the DNA damage response. Spc110 was labelled with the 3HA tag in both wild-type and esp1-1 background strains. Cells were grown overnight in raffinose-containing media and supplemented with galactose to induce the HO expression. Samples were collected at each time-point indicated. Proteins were TCA extracted, separated on Phos-Tag containing gels and blotted. Coomassie staining is shown as a control for loading.
Figure 4A Simplified photomicrographs of proximity ligation studies in WT and Nox4KO MEFs, showing cell borders, nuclei (blue) and yellow dots corresponding to co-localization of proteins. Quantification of the number of dots/cell in each condition is shown to the right. Proximity was tested for the following protein couples: FACL4/Nox4, InsP3R/Nox4, and FACL4/InsP3R. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment). B Simplified photomicrographs of proximity ligation staining in rat cardiomyocytes transfected with Nox4 siRNA or a scrambled control (siScr), with quantification as in (A). Proximity was tested for the FACL4/Nox4 protein couple. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment). C Simplified photomicrographs of proximity ligation staining in hiPSC-CM (human cardiomyocytes) transduced with Nox4 shRNA or scrambled control, with quantification as in (A). Proximity was tested for the FACL4/Nox4 protein couple. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment).
Figure 2A Schematic of the liposome floatation assay. In the assay, liposomes of defined lipid composition were floated through a dense sucrose gradient by ultracentrifugation. Protein that associates with the liposomes are enriched in the supernatant. B Upper panel. SDS-PAGE stained with Coomassie blue in which Bem1 is indicated. Lower panel. The liposomes were composed of 100% phosphatidylcholine (PC), 80% PC and 20% phosphatidylethanolamine (PE), 95% PC and 5% phosphatidic acid (PA), 95% PC and 5% phosphoatidylinositol (PI), 95% PC and 5% PI(4,5) P2 (PI(4,5) P2), 95% PC and 5% PI4P (PI4P), 80% PC and 20% phosphatidylserine (PS) or 75% PC 20% PS 5% PI4P (PS+PI4P). (S) supernatant, (P) pellet. C Liposomes composed of either 100% PC ± 40% ergosterol or 75% PC, 20% PS, 5% Bem1 ± 40% ergosterol replacing PC were incubated with Bem1 and treated as in panel (B). D Scheme of full-length Bem1 with its domains and the Bem1 deletion constructs used to identify the anionic lipid interacting sequences. E Percentage of the indicated bem1 constructs associated with liposomes containing 75% PC, 20% PS and 5% PI4P. F Lipid ordering determined by 2H solid-state NMR analysis of liposomes containing POPC-d31/POPS (4:1 molar ratio) in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/PI4P (19:1 molar ratio). Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/POPC/PI4P (15:4:1 molar ratio). Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/POPS/Yeast lipid extract/ergosterol (1:1:1:2 molar ratio).
Figure 1(A) qRT-PCR analysis for Islr, Chd1 and Vim in total intestinal tissues, intestinal epithelium and lamina propria. n = 4. (B) Western blotting for Islr and E-cadherin in total intestinal tissues, intestinal epithelium and lamina propria. α-Tubulin was used as a loading control. (C) qRT-PCR analysis for Islr in CD45+, CD45-Epcam+, CD45-Epcam-CD90+ and CD45-Epcam-CD90- cells sorted from intestinal tissues. n = 3 technical replicates. (D) In situ hybridization for Islr with RNAscope probe in intestine and colon from 8-week-old mice, showing that Islr is primarily expressed in stromal cells. Negative and positive controls were shown in Appendix Figure S1A. Scale bar: 25 μm. (E) In situ hybridization for ISLR with RNAscope probe in human mucosa, inflamed mucosa from CD and UC patients. Scale bar: 25 μm. (F) qRT-PCR analysis showing the dynamic changes of Islr in colonic tissues from mice upon DSS treatment and after DSS removal. n = 4 at each time points. (G) In situ hybridization for Islr with RNAscope probe in mouse colons without or with 3-day or 5-day DSS treatments. Scale bar: 50 μm. (H) The cancer genome atlas (TCGA) RNA-seq analysis showing that ISLR is upregulated in human rectal adenocarcinoma relative to normal rectal tissues. The horizontal lines between the box limits represent the median, the box limits indicate the interquartile ranges and the whiskers indicate limit superior and limit inferior respectively. n = 65. (I) Kaplan-Meier survival curve of 270 CRC cases. P = 0.02 (log-rank test). (J) ISLR level increased with nodal metastasis in colorectal cancer patients. N0, no regional lymph node metastasis; N1, metastases in 1 to 3 axillary lymph nodes; N2, metastases in 4 to 9 axillary lymph nodes; N3, metastases in 10 or more axillary lymph nodes. The solid horizontal lines represent the median, the box limits indicate the interquartile ranges and the whiskers indicate limit superior and limit inferior respectively. (K In situ hybridization for ISLR/Islr with RNAscope probes in normal human colon, human malignant adenocarcinoma, metastatic adenocarcinoma (K), t indicating tumor; a indicating adjacent tissues of tumor. Scale bar: 50 μm. L) In situ hybridization for ISLR/Islr with RNAscope probes in mouse colon tumors from AOM-DSS model (L). t indicating tumor; a indicating adjacent tissues of tumor. Scale bar: 50 μm.
Figure 1A Schematic diagram of humanINPP5E. PRD, proline-rich domain; IPPc, inositol polyphosphate phosphatase domain. B Levels of INPP5Eprotein 72 h after transfection of N1E-115 cells with control (siControl) or INPP5E-specific siRNAs (siINPP5E #1 or #2), as determined by immunoblot. C N1E-115 cells treated with siControl or siINPP5Es were cultured for 2 h in growth medium with or without 125 nM bafilomycin A1 (Baf.A1), and then analyzed by immunoblot using anti-p62, anti-LC3, and anti-GAPDH antibodies. D Quantitation of protein signal intensities from immunoblots in C showing LC3-II (left) or p62 (right) levels, following normalization to the control proteinGAPDH. Because Baf.A1 inhibits autophagosome-lysosome fusion and degradation within lysosomes via inhibition of vacuolar-type H+-ATPase, it is possible to measure autophagic flux, i.e., the degradation of autophagic substrates such as p62 and LC3-II, by calculating the difference of the signal intensities of these proteins in the presence and absence of Baf.A1. Results represent the mean ± s.d. of three independent experiments. **, P < 0.01. E N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LC3 antibodies and analyzed by immunofluorescencemicroscopy. Bar, 10 µm. F Quantitation of the number of LC3puncta per cell, as described in E (mean ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01. G Electron micrographsin N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Arrowheads indicate autophagosomes. Bar, 1 µm.
Inhibition of mitochondrial division prevents mitochondrial fragmentation caused by OPA1 deficiency in DOA+ patient-derived fibroblasts (A) Schematic of supervised machine learning (ML) mitochondrial morphology imaging and quantification pipeline.Figure 1Schematic of supervised machine learning (ML) mitochondrial morphology imaging and quantification pipeline. Fibroblasts plated in 384 well plates are stained for mitochondria (anti-TOM40, green), nuclei (DAPI, blue), and cell body (CellMask, blue). Supervised ML training performed on cells with fragmented (OPA1 or YME1L siRNA), normal (non-targeting NT siRNA), and hypertubular (DNM1L siRNA) mitochondria. Automatic single-cell trinary classification of control (CTL-1, 2, 3) and OPA1S545R patient fibroblasts by supervised ML. Representative confocal images of control (CTL-1, 2, 3) and DOA+ patient fibroblasts carrying indicated monoallelic mutations imaged as described in A. Scale bar=20μm. Passage number between P12-15. Mitochondrial morphology quantification of B. Data represent mean ± SD of two independent experiments, (195-2496 cells per cell line), One-way ANOVA; ** p < 0.01, ****p < 0.0001, ns; not significant Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with OPA1, DNM1L, or non-targeting (NT) siRNAs for 72 hours and imaged as described in A. Scale bar=20μm. Passage number between P12-14. Mitochondrial morphology quantification of D. Data represent mean ± SD of three independent experiments, (3219-5857 cells per cell line), One-way ANOVA; ****p < 0.0001, ns; not significant. Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with 50μM cycloheximide (CHX) where indicated for 6 hours. Imaging as described in A. Scale bar=20μm. Passage number between P14-P15. Mitochondrial morphology quantification of F. Data represent mean ± SD of two independent experiments, (879-4154 cells per cell line), One-way ANOVA; ****p < 0.0001, ns; not significant.
Dll4-ligand expression in the IAHC varies with cluster size (A) Detail images of Dll4 staining in Kit+ cells of IAHC of different sizes.Figure 3Detail images of Dll4 staining in Kit+ cells of IAHC of different sizes. Dll4 expression inversely correlates with the size of the cluster: 2-5 cell IAHCs are uniformly Dll4-positive (upper and middle panel), >5 cell IAHCs are partially Dll4 positive (lower panel). Multistack reconstruction of confocal images. Scale bars: 10 μm. Quantification of Kit+ Dll4+ cells using confocal images. Bars represent the percentage of Dll4 expressing cells within a cluster in each category of cluster size. Mean±SD (total 17 clusters analysed, n=2). Overview of Dll4-ligand expression in the embryonic aorta at E10.5. Dll4 is expressed in the majority of the endothelial layer. Scale bar: 30μm
Destabilising Kinesin-8 accumulates behind the stabilising Tea2 kinesin complex to ensure microtubule disassembly at the cell end.Figure 5  Figure 5 - Destabilising Kinesin-8 accumulates behind the stabilising Tea2 kinesin complex to ensure microtubule disassembly at the cell end. The MT stabilising Tea2 complex (Tea2/Tip1/Mal3) binds to MT plus ends containing GTP-β-tubulin (green and yellow subunits) as they grow towards the cell end. The MT destabilising Kinesin-8 (Klp5/Klp6/Mcp1) complex walks along the MT lattice and accumulates behind the Tea2 complex, particularly as the MT dwells at the cell end, but is prevented from accessing the plus end by the presence of Tea2. The Kinesin-8 complex may then either displace the Tea2 complex from the plus end, triggering MT catastrophe, or initiate MT depolymerisation resulting in the loss of the Tea2 complex and the GTP cap. An alternative, but not necessarily exclusive, possibility is that displacement of Tea2 from the MT plus end initiates Kinesin-8 mediated MT disassembly. In the absence of the MT destabilising Klp5/Klp6/Mcp1 complex (second column), MTs dwell for longer at the cell end and the Tea2 complex remains at the plus end during this period. Klp5/Klp6/Mcp1 is therefore essential for regulating timely dissociation of the Tea2 complex from plus ends. In the absence of Tea2 (third column), Kinesin-8 accumulates at, and has unrestricted access to, the plus end resulting in frequent MT catastrophe before reaching the cell end. When both stabilising and destabilising kinesin complexes are absent (fourth column), MTs reach and dwell at the cell end and may undergo catastrophe as a consequence of compressive force encountered at the cell end.  Expanded View Figure Legends
ATG-5 was upregulated in chemoresistant cells.Figure 3(A) The level of ATG5 was detected in cell lines using western blot analysis. β -actin was used as internal controls. (B) The IC 25, IC50 and IC75 of SGC-7901 and SGC-7901/DDP cells were tested using the MTT assays after DPP treatment.
TRF2 overexpression triggers an immunosuppressive tumor microenvironment A B16F10 melanoma cells with either mTRF2 overexpression or knockdown were inoculated into the backs of immunocompetent mice.Figure 1A B16F10 melanoma cells with either mTRF2 overexpression or knockdown were inoculated into the backs of immunocompetent mice. The percentages of intratumoral CD11bHi GR1Hi MDSCs (n = 10 mice), CD25+ Foxp3+ regulatory T cells (Tregs), or CD25+ Foxp3− activated T cells (n = 5 mice per group) among CD45+ cells were analyzed by fluorescence-activated cell sorting (FACS) and represented as box plots Data information: data are presented by Box plots Min to Max showing all points with median. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; Mann-Whitney test. Schematic representation of the experiment (B). TRF2-overexpressing or -knockdown BJcl2 cells were inoculated into the backs of immunodeficient mice in the presence of Matrigel. Five days later, the percentage of immune cells infiltrating the Matrigel plugs of CD11bHi GR1Hi MDSCs (C) were determined by FACS (n = 5 mice per group). TRF2-overexpressing or -knockdown BJcl2 cells were inoculated into the backs of immunodeficient mice in the presence of Matrigel. Five days later , the intranuclear staining of pSTAT3 (D) were determined by FACS (n = 5 mice per group). TRF2-overexpressing or -knockdown BJcl2 cells were inoculated into the backs of immunodeficient mice in the presence of Matrigel. Five days later, CD3− NKp46+ and IFN-γ+ among CD3− NKp46+ NK cells (E) were determined by FACS (n = 5 mice per group). Linear correlation plot of the percentage of IFN- γ + NK cells, the mean of fluorescence intensity (MFI) of pSTAT3 for MDSCs, and the expression of TRF2 (r2= 0,5177; Pearson's correlation, p = 0.003) (F) Data information: data are presented by Box plots Min to Max showing all points with median. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; Mann-Whitney test. Pearson correlation and p values are indicated. the mRNA expression level in the matrigel plugs of arg1, pSTAT3 MFI and TRF2 expression (r2= 0,7077; Pearson's correlation, p < 0.0001) (G); the mRNA expression level in the matrigel plugs of tgfβ, pSTAT3 MFI and TRF2 expression (r2= 0,3669; Pearson's correlation, p = 0.0046) (H) and the mRNA expression level in the matrigel plugs of il-10, pSTAT3 MFI and TRF2 expression (r2= 0,7418; Pearson's correlation, p < 0.0001) (I). Data information: data are presented by Box plots Min to Max showing all points with median. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; Mann-Whitney test. Pearson correlation and p values are indicated.