EMBO/sd-character-level-ner huggingface dataset:
a) Giant unilamellar vesicles (GUVs) incubated with mCherry–Atg19.figf4(a) Giant unilamellar vesicles (GUVs) incubated with mCherry-Atg19. The membrane was marked by incorporation of an Oregon Green labelled lipid. (b) GUV coated with GFP-Atg8 incubated with mCherry-Atg19. (c) Schematic representation showing the proteins and reaction resulting in GFP-Atg8 conjugation to the GUV shown in b. (d) GUVs containing nickellipids incubated with GFP-Atg8-6×His and wild-type mCherry-Atg19. (f) Quantification of membrane binding by the indicated mCherry-Atg19 proteins. The experimental set-up is shown in e. The graph is based on three experiments. Shown are the averages and s.d. of these three experiments (N = 3, on the basis of 318 GUVs for Atg19, 171 GUVs for Atg19 W412A, 166 GUVs for Atg19 F376A, F379A, W412A, 188 GUVs for the enhanced GFP (eGFP) control). Scale bars, 5 μm. Abbreviations: 3, Atg3; 5, Atg5; 7, Atg7; 8, Atg8; 12, Atg12; 16, Atg16; 19, Atg19 redundant. Experiments shown in a,b have been conducted twice and the experiment in d three times.
Figure 4(A) Schematic illustration of the single-cell RNA-seq experiment and the isolated populations during germ cell maturation in rOvaries. The first 24h of culture are indicated as d0. rOvary = reconstituted Ovary, d = day of rOvary culture; XGFPint. = XGFPintermediate. (B) (i) Imaging of XGFP and XTomato reporters in rOvaries d5 aggregated with E13.5 gonadal and mesonephric cells. Scale bars = 50μm. BF = bright field. (ii) FACS gating strategy for single-cells sorted XTomato+ cells against XGFP intensities. Numbers indicate the percentage of gated live cells over the total population. Numbers in brackets indicate the percentage of gated cells over the XTomato+ population. (C) UMAP projection labelled with FACS sorted populations. XGFPint. = XGFPintermediate. (D) UMAP embedding based on Shared Nearest Neighbor (SNN) modularity clustering identified 5 clusters, termed Mitotic 1 (n = 77), Mitotic 2 (n = 97), Pre-meiotic 1 (n = 62), Pre-meiotic 2 (n = 90) and Meiotic (n = 53) labelled with different colours. Arrows indicating cell trajectories, inferred by RNA velocity analysis. (E) Marker gene expression projected onto the UMAP plot. (F) Heatmap of gene expression dynamics throughout germ cell maturation clusters. Selected genes belong to the category "early germ cell" and "meiotic". Zscore is shown. (G) Schematic representation of the IVDi (In Vitro Differentiation) maturation system. The stages of oogenesis in culture for 11 days are indicated. The condition of culture is indicated above. rOvary = reconstituted Ovary. Agg = aggregation day. AA = Ascorbic Acid. (H) Immunofluorescence images of SYCP3 (red), DAZL (yellow) and DAPI at agg11 of IVDi tissue maturation from XGFP- PGCLCs. IVDi = in vitro differentiation. White squares indicate the positions of the magnified section shown below. Top panel scale bar = 100 μm. Middle panel scale bar = 10 μm. Bottom panel scale bar = 50 μm. (I) Quantification of SYCP3+ cells (oocytes in cyst and primary follicles) in IVDi tissues at agg11. Each dot represents one IVDi tissue performed in 3 biological replicates.
CLU modulates Cdc25 expression in vitro.Figure 2(A) Real-time PCR for CLU and Cdc25C in three different prostate cancer cell lines. Target genes expression was calculated relative to GAPDH and normalized to siSCR. Error bars represent mean ± SD, n=3. (B) Western blot for CLU and Cdc25C after siSCR or si CLU transfection in the same prostate cancer cell lines as in A. Vinculin was used as loading control. (C) Western blot for CLU and Cdc25C in PC3 cells after transfection with siSCR or siCLU followed by transfection with CLU or empty expression plasmid for additional 48h. Vinculin was used as loading control
a) Immunoblot analysis of autophagy, ER stress- and mTOR-related proteins, and JNK in U2OS cells after 24 h glutamine deprivation with or without CCI-779.f6(a) Immunoblot analysis of autophagy, ER stress- and mTOR-related proteins, and JNK in U2OS cells after 24 h glutamine deprivation with or without CCI-779. (b) RT-PCR analysis of IL-8 mRNA expression in U2OS cells transfected with two different siRNAs targeting JNK (JNK #1 and JNK #2) and subjected to 24 h glutamine deprivation. (c) IL-8 enzyme-linked immunosorbent assay (ELISA) of conditioned media from the experiment described in b. (d) Immunoblot analysis of JNK protein expression in JNK siRNA-treated cells. e) RT-PCR analysis of IL-8 expression in U2OS cells after 24 h glutamine deprivation with or without the JNK inhibitor SP600125 (JNKi). (f) IL-8 ELISA of conditioned medium from the experiment described in e. (g) IL-8 ELISA of U2OS cells transfected with two different siRNAs targeting IRE1 (IRE-A and IRE-B) and subjected to glutamine deprivation for 24 h. (h) RT-PCR analysis of IL-8 mRNA expression from experiment described in g. (i) RT-PCR analysis of CHOP mRNA expression from experiment described in g. (j) RT-PCR analysis of IRE1 mRNA expression from experiment described in g. (k) Immunoblot analysis of experiment described in g showing phosphorylation of JNK. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.
TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress.Figure 1TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress. (A) ROS levels in U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) left untreated, after 6 h of nutrient starvation or 24 h of metabolic stress. ROS levels were measured by flow cytometry after DCF treatment. The results are expressed as the mean DCF fluorescence (and standard deviation), from three independent experiments. TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress.(B) Basal, nutrient starvation-induced (5 h) or metabolic stress-induced (18 h) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, measured by flow cytometry after DCF treatment. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05).
a) Immunoblotting.f6(a) Immunoblotting. Jurkat T cells overexpressing ATG5 or control cells were cultured in the presence and absence of 3-MA for 48 h. 3-MA blocked the formation of LC3-II and p62 degradation, indicating that it indeed inhibited ATG5-induced autophagy. Results are representative of three independent experiments. (b) Cell cycle analysis. Normal Jurkat T cells and cells overexpressing ATG5 were cultured for 48 h in the presence and absence of 3-MA. Induction of G2- or M-phase arrest was seen as a consequence of ATG5 overexpression independent of treatment with 3-MA, as normal Jurkat T cells exhibited no G2/M arrest following 3-MA treatment. Representative flow cytometry diagrams are shown (n=3). Moreover, induction of autophagy by Beclin 1 overexpression or starvation failed to produce cell cycle arrest (Supplementary Fig. S4). (c) Morphological analysis. Normal Jurkat T cells and cells overexpressing ATG5 were cultured for 48 h in the presence and absence of 3-MA and analysed. Representative examples of morphology are shown (n=3); large multinucleated cells and cells with abnormal nuclei were seen as a consequence of ATG5 overexpression independent of 3-MA. Neither induction of autophagy by Beclin 1 overexpression nor starvation resulted in such abnormal cells (Supplementary Fig. S4). Scale bar, 10 μm. ( (d) Viability assays. Jurkat T cells either overexpressing ATG5 following lentiviral gene transfer or treated with etoposide were cultured in the presence and absence of the indicated pharmacological inhibitors for the indicated times to block autophagy and caspase activity. All values are means±s.d. (n=3).
STAMP2 expression during PCa progression.Figure 1A Immunohistochemistry was used to assess STAMP2 expression in normal and malignant human prostate specimens. Tissue microarrays (TMAs) with normal prostate (n = 27) and early-stage (Gleason grade < 7) (n = 35) or late-stage prostatetumors (Gleason grade ≥ 7) (n = 149) were subjected to immunohistochemistry as described in Materials and Methods. Representative images are shown.B Quantification of cytosolicSTAMP2 staining in TMAs is shown in (A). One-way analysis of variance (ANOVA) with a post hoc test, *P < 0.0001. Error bars indicate SEM.C, D The details of cytosolic (C) and membrane (D) STAMP2 staining in the TMAs described in (A).
Figure 4A Left, transmission electron microscopy analysis of adipocytes from WAT and BAT of WT and FADD-D mice. Arrowheads indicate mitochondria and lipid droplets (L) (× 5,000 magnification). Right, quantification of mitochondria in WAT and BAT of WT and FADD-D mice, respectively. Data are expressed as mean ± SEM from three independent experiments (n = 6 total for each genotype). *P = 0.0007, **P = 0.0083 (Student's t-test). B Quantitative real time PCR for UCP1, Dio2, PPARd, UCP3, CPT1 and PGC1-α, using RNA from epididymalfat from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from three independent experiments (n = 6 total for each genotype). *P = 0.0014, **P = 0.0025, ***P = 0.0006, ****P = 0.0007, #P = 0.0003, ##P = 0.0008 (Student's t-test). C Quantitative real time PCR for UCP3, CPT1, PPARd and PGC1-α, using RNA from skeletal muscle from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from three independent experiments (n = 6 total for each genotype). *P = 0.0027, **P = 0.0329, ***P = 0.0092, ****P = 0.0085 (Student's t-test). D Quantitative real time PCR for UCP1, using RNA from BAT from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from three independent experiments (n = 6 total for each genotype). *P = 0.0018 (Student's t-test). E Immunoblotting of UCP1 and Tubulin (control) (top) with relative quantification (bottom), using total cell lysates of BAT from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from three independent experiments. *P = 0.0217 (Student's t-test). F Oxygen consumption in isolated BAT from 10-week-old male WT and FADD-D mice on a SD (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0025, **P = 0.0006 (Student's t-test). G Quantitative real time PCR for PGC1α, C/EBPα and PPARγ, using RNA from BAT from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from two independent experiments (n = 4 total for each genotype). *P = 0.0113 (Student's t-test). NS, not statistically significant. H β-oxidation analysis of white adipocytes isolated from WT (n = 5) and FADD-D mice (n = 4). 1-14C labeled oleic acid was added to the medium containing isolated adipocytes (5×105), and 14CO2 trapped by the filter paper soaked with hyamine hydroxide was measured after a 3 h-incubation by a scintillation counter. Data are expressed as mean ± SEM. *P = 0.0022 (Student's t-test). I Basal and stimulated (+ 100 nM isoproterenol) lipolysis, as measured by glycerol (left) and fatty acids (right) released from explants of epididymal WAT from overnight fasted 10-week-old male WT and FADD-D mice fed a HFD (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0078, **P = 0.0065, ***P = 0.0008, ^P = 0.0133, ^^P = 0.0053, ^^^P = 0.0006, ^^^^P = 0.0001, #P = 0.0082, ##P = 0.0051, ###P = 0.0009, *^P = 0.0151, *^^P = 0.0019, **^^P = 0.0005, ***^P = 0.0002 (Student's t-test). J Molar ratio of FFA to glycerol release from WAT explants (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0412, **P = 0.0033 (Student's t-test).
Figure 5(A) Experimental scheme. The selective expression of CRE-recombinase in PV-CRE and PV-CRE;Cox6a2fl/fl mice ensures the expression of PercevalHR only in PV+ interneurons. (B) Experiment timeline. AAVs expressing PercevalHR were injected in the somatosensory (SS) cortex of mice at postnatal day 15 (P15). Approximately, 3 weeks after the injections, the brains were sliced into coronal sections for live-cell imaging of ATP-to-ADP ratio by 2-photon microscopy. (C) PercevalHR is a ratiometric fluorescent protein that changes its excitation spectrum based on its binding to ATP (930 nm) or ADP (840 nm). (D) Representative images of PV+ interneurons from PV-CRE and PV-CRE;Cox6a2fl/fl mice showing PercevalHR fluorescence after excitation at 930 nm and 840 nm. White arrowheads indicate PercevalHR expressing cells. Scale bar: 20 μm. (E) PV+ interneurons from PV-CRE;Cox6a2fl/fl mice exhibited significantly lower ATP-to-ADP ratios than PV+ interneurons from PV-CRE mice throughout the entire duration of the recordings (23 cells from 4 PV-CRE mice; 15 cells from 6 PV-CRE;Cox6a2-/- mice).
Figure 1A - Three-dimensional imaging of WT and ChAT-eGFP IWAT using the Adipo-Clear method and lightsheet fluorescence microscopy. Whole IWAT was stained with Alexa Fluor 488-conjugated anti-GFP antibody to visualize ChAT-eGFP-expressing cells. High-magnification sections are shown to the right of each sample. Scale bars: 100 μm. B - UMAP plot displaying the profile of ChAT-eGFP+ cells from IWAT, analyzed by flow cytometry and combined from four biological replicates. ChAT-eGFP+ cell types are color‑coded with accompanying labels, and the percentage breakdown of ChAT-eGFP+ cells is featured in a bar chart to the right. MΦ: macrophages. C - Left: Percentage of total live cells expressing ChAT-eGFP derived from IWAT (including inguinal lymph node; n = 7), IWAT alone (n = 8) and lymph node alone (n = 8). Right: Proportion of ChAT-eGFP+ cells comprised by T cells, B cells and MΦ in each tissue type. D - Total abundance of ChAT-eGFP+ CD45+ hematopoietic cells in IWAT, VWAT and BAT SVF isolated from ChAT-eGFP mice housed at room temperature (RT) or exposed to 4°C (CE) for 4 h (n = 4). E - LC/MS-MS traces showing acetylcholine (Ach) levels in SVF isolated from ChATfl/fl and ChATfl/fl;Vav-iCre IWAT compared to an internal control (d4-Ach). F - Left: relative mRNA expression of Chat in ChATfl/fl and ChATfl/fl;Vav-iCre IWAT (n = 9). Chat expression was analyzed by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method. Right: quantification by LC/MS-MS of Ach concentration in SVF isolated from ChATfl/fl and ChATfl/fl;Vav-iCre IWAT (n = 6). G - Percentage of total IWAT SVF cells expressing ChAT-eGFP in male (n = 6) and female (n = 9) ChAT-eGFP mice housed at RT or CE (4 h). H - Percentage of total IWAT T cells, B cells and MΦ that express ChAT-eGFP, from male (n = 6) and female (n = 5 for T cells and B cells; n = 8 for MΦ) mice housed at RT or CE (4 h). I - Left: MΦ as a percentage of all ChAT-eGFP+ cells in IWAT at RT and 4 h CE (n = 15). Middle: Total number of ChAT-eGFP+ MΦ at RT and 4 h CE (n = 15). Right: ChAT-eGFP median fluorescence intensity (MFI) for ChAT-eGFP+ MΦ at RT and 4 h CE (n = 15). J - MFI for Ki67 in ChAT-eGFP+ MΦ from IWAT at RT and 4 h CE (n = 6). K - Schematic depicting the experimental strategy for transcriptomic profiling of ChAT-eGFP+ and ChAT-eGFP- MΦ from IWAT of mice housed at 4oC for 4 h. L - Relative expression (pseudocounts) heatmap of genes relevant to acetylcholine signaling in ChAT-eGFP- (n = 4) and ChAT-eGFP+ (n = 3) MΦ. M - Biological pathway analysis of significantly enriched genes in ChAT-eGFP+ MΦ.
The familial caPOT1 Y36H mutation leads to telomere elongation in somatic tissues A) A pedigree of a Hodgkin Lymphoma family with POT1 Y36H mutation (adapted from and previously published in McMaster et al.Figure 6A) A pedigree of a Hodgkin Lymphoma family with POT1 Y36H mutation (adapted from and previously published in McMaster et al., 2018). More details of the subjects are listed in Appendix Table S1. B) Telomere restriction fragment assay for the five subjects shown in (A). Mean telomere length is indicated.
Figure 1A, B. Representative images of H&amp;amp;E- (A) and PAS- (B) stained colon sections from wild-type and Angptl2-/- mice. Scale bar = 100μm.C, D. Representative images of colon crypts as assessed by Ki-67 (C) and pH3 (D) staining. Scale bar = 100 μm. E. mRNA levels of indicated proliferation and ISC markers in isolated crypts from wild-type (n=4) and Angptl2-/- (n=4) mice based on qRT-PCR analysis. F. Western blotting analysis of isolated colon crypts from wild-type and Angptl2-/- mice. HSC70 served as an internal control. Numbers below panels represent normalized expression of proteins. G. IHC for expression of active β-catenin in colon or wild-type and Angptl2-/- mice. Scale bar = 50 μm.
Flagellar assembly intermediates in H.Figure 3A-D) slices through electron cryotomograms of H. pylori (left panels) indicating the presence of various flagellar intermediates. Middle panels show central slices through STAs. Right panels show schematic representations. Question marks in the schematic in (C) indicate that it is unclear whether the densities surrounding the upper part of the C-ring are the same as the novel cytoplasmic rings seen in the MS-complex. Empty panels indicate that there were not enough examples to produce a STA. E & F) cryotomogram slices, STAs, and schematics of H. pylori fliP* cells showing the MS-complex (E) and the C-complex (F). The two novel cytoplasmic rings are highlighted with light and dark green arrows in the STA panel. White arrows in (A-F) point to the particles. G & H) central slices through the STAs of the MS-complexes of the motile H. pylori (G) and H. pylori FliP* mutant (H) with the diameters of their various constituent rings indicated.
GBP1 targets Salmonella vacuoles and mediates caspase-4-dependent pyroptosis (A) LDH release assay from indicated THP-1 cells untreated or treated with IFNγ and infected with Salmonella Typhimurium (STm) WT or a SPI-1 mutant ΔinvA for 4 h at indicated multiplicities of infection (MOI).Figure 5LDH release assay from indicated THP-1 cells untreated or treated with IFNγ and infected with Salmonella Typhimurium (STm) WT or a SPI-1 mutant ΔinvA for 4 h at indicated multiplicities of infection (MOI). Mean ± SEM of n = 3 experiments are shown. Propidium iodide (PI) uptake assay from IFNγ-primed primary MDMs transfected with non-targeting control siRNA (CTRL) or siRNA against GBP1 (left) or indicated lines of THP-1 (right) infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. PI uptake assay from IFNγ-primed primary MDMs transfected with indicated siRNA or non-targeting control (CTRL) and infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 4 independent experiments. PI uptake assay from IFNγ-primed or untreated THP-1 ΔCASP-1 cells transfected with indicated siRNA and infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. Schematic on right shows overview of the pathway leading to caspase-1 and caspase-4 activation. LDH release assay from indicated THP-1 cells treated with IFNγ and Dox and infected with STm-GFP for 4 h. Shown is the mean ± SEM of n = 3 experiments. Representative images from IFNγ-primed THP-1 ∆GBP1 cells stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or primary MDMs stained for GBP1. Cells were infected with STm-GFP for 2 h. Blue, Nuclei; Grey, STm; Red, mCH-GBP1 or Magenta GBP1. Scale bar, 5 μm. quantification (G) from IFNγ-primed THP-1 ∆GBP1 cells stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or primary MDMs stained for GBP1. Cells were infected with STm-GFP for 2 h. Blue, Nuclei; Grey, STm; Red, mCH-GBP1 or Magenta GBP1. Scale bar, 5 μm. Graph in (G) shows percentage of STm vacuoles targeted by GBP1. Quantification of GBP1 recruitment to STm-GFP containing vacuoles (SCV) in MDMs pre-treated with IFNγ or left untreated (UT). Mean ± 95% CI of GBP1 fluorescence intensity measured on the indicated number (n) of SCVs for each condition from three independent experiments are shown. Representative images from THP-1 ∆GBP1+Tet-mCH-GBP1 cells stably expressing YFP-CASP4C258S. Cells were infected with STm for 2 h. Blue, Nuclei & STm (Hoechst dye); Red, mCH-GBP1; Green, YFP-CASP4C258S. Scale bar, 5 μm. Quantification of THP-1 ∆GBP1+Tet-mCH-GBP1 cells stably expressing YFP-CASP4C258S and infected with STm for 2 h. Graph shows percentage of STm vacuoles targeted by GBP1, CASP4 or both from n = 3 experiments. n.d. not detected.
Figure 7(A, B) The growth curve of WT and two ftsj1 KO HEK293T cell lines under normal culture or in the culture condition with 0.2 mg/mL paromomycin assayed by cell counting kit-8 proliferation analysis. (C, D) The growth curve of WT with or without overexpressing tRNAPhe(GAA) under normal culture or with 0.2 mg/mL paromomycin. (E, F) The growth curve of ftsj1 KO cells with or without overexpressing tRNAPhe(GAA) under normal culture or with 0.2 mg/mL paromomycin. (G) The RNA reporter vector encodes F-luc as the primary reporter and R-luc on the same plasmid as the internal transfection control. The effect of tRNAPhe(GAA) in protein translation was revealed by a reporter assay. 6×TTC (Phe) or 6×TTT (Phe) were inserted after the PGK promoter region of F-luc as the positive reporter. (H, I) The translation efficiency of F-luc with the 6×UUC codons or 6×UUU codons. (J, K) The translation efficiency of F-luc with all UUC codons or all UUU codons. (L) The frequency usage analysis of UUU and UUC codons showed that in 488 genes at least one transcript was found to use the UUU codon for more than 80% of Phe codons. Among the 488 genes, 189 genes are involved in the nervous system and brain functions.
Figure 6A. Mislocalization of Bcl-2 is observed in MITOL KO cells in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin were transfected with FLAG-Bcl2 and treated with DMSO or CCCP (10 µM) for the indicated times. Cells were immunostained with indicated antibody. Scale bar, 1 μm. B. MITOL prevents caspase-dependent cell death in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were treated with DMSO or CCCP (10 µM) with or without Z-VAD-FMK (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. C. Cell death in mitophagy caused by MITOL deficiency depends on calcium leak. WT and MITOL KO HeLa cells stably expressing HA-Parkin or HeLa cells were treated with DMSO or CCCP (10 µM) with or without BAPTA-AM (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.