EMBO/sd-character-level-ner huggingface dataset:
Figure 4A) From the quantitative proteomics experiment, average abundance of catalytic proteasome subunits was calculated and normalised to WT means. Trypsin-like (β2), chymotrypsin-like (β3) and caspase-like (β1) catalytic subunits are shown. The average was calculated from all 24 time points of the proteomics experiment. Mean±SD, 2-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001. B) Representative Western blot using an antibody that recognises all 7 α subunits of the 20S proteasome, with anti-histone H3 as loading control. C) Quantification of the blots in B), using all replicates (n=3), normalised to WT mean. Mean±SD, one-tailed Student's t test with Welch correction - prior prediction from experiment in A) that CKO abundance would be lower, *p≤0.05. D) Proteasome activity measured using the ProteasomeGlo Assay (Promega), normalised to WT means. Mean±SD, 2-way ANOVA with Holm-Sidak's multiple comparisons. N=6 experiments, representative experiment shown (n=4 technical replicates), ****p≤0.0001. E) Translation rate was measured using 35S-methionine labelling and imaging with phosphor screens. The quantification values were normalised to the total protein concentration as measured using Coomassie stain, and then normalised to WT mean. Mean±SD, Student's t test with Welch correction. N=3 experiments, representative experiment shown (n=4 technical replicates), **p≤0.01. F) Total protein mass per cell in confluent WT and CKO cultures. Cells were grown in two 12-well plates; one was used for cell counting and the other was used for lysis in RIPA buffer prior to protein quantification by BCA assay. Quantification shown, normalised to WT mean. Mean±SD, Student's t test with Welch correction, n=6 technical replicates.
The Mpc1/Mpc3 hetero-complex transports pyruvate.Figure 3Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in liposomes (n=8) in comparison to empty liposomes (n=6) at a ΔpH of 1.6. Kinetic analysis of pyruvate homo-exchange at ΔpH of 1.6 (n=3). The hetero-complex was assayed for initial rates of uptake in the concentration range of 25-600 μM. The calculated KΜ was 299 μΜ in this experiment and 318 and 409 μΜ in two additional biological repeats. Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in physiological pH (n=4) compared to empty liposomes (n=4). In the absence of a ΔpH, the time course of pyruvate homo-exchange was similar for the Mpc1/Mpc3 proteoliposomes and the empty liposomes (n=4). Inhibition of [14C]-pyruvate homo-exchange by UK5099 (1-100 μM), Zaprinast (1-1000 μM), lonidamine (10-10000 μM) and 7ACC2 (5-500 μM). Data points represent the mean of three technical replicates of a typical experiment. The IC50 measurements have also been independently replicated, three times for UK5099 Zaprinast and 7ACC2 (average IC50 9 ± 7 μM, 18 ± 8 μM and 27 ± 13 μM, respectively) and two times for lonidamine (average IC50, 118 ± 24 μM). [14C]-pyruvate homo-exchange inhibition by the TZDs, pioglitazone and rosiglitazone (n=6).
Proteome analysis of tumor growth patterns in pulmonary Adenocarcinoma (ADC) FFPE tissue.Figure 4A) Schematic illustration of the sample collection. Samples were collected from eight different patient tumors. For each tumor, sections were processed with hematoxylin & eosin (H&E) staining to locate different growth patterns of lepidic (low-grade; group 1), acinar and papillary (intermediate grade; group 3), and solid and micropapillary (high-grade; group 3). Two to four growth patterns per tumor were selected and sectioned in two consecutive 5 μm iterations, to provide replicates with highest possible similarity, resulting in a total of 51 samples (one iteration was missing). B) t-distributed stochastic neighbor embedding (t-SNE) analysis of the proteome data corrected via a linear regression model. The different growth patterns are color-coded as in panel A. C) Volcano plot showing differential expression analysis using Limma moderated t-statistics for the comparison of lepidic samples against all other samples. Proteins passing significance thresholds of -log10 p-value < 0.05 (Benjamini-hochberg adjusted) and an absolute log2 fold change of >1 are highlighted in orange. D) Summary of significantly expressed proteins in the comparison of each growth indicated pattern against all others.
Figure 7A. Schematic of drug pulse and wash-out experiment. PeTa cells were treated with 100 nM GSK-LSD1 or DMSO. After 24h, a cell sample was harvested (T0, ON-drug) and the remaining cells were washed and changed into fresh medium without drug. Cell samples were taken 24h, 72h, 6 days (D6) and 8 days (D8) after drug wash-out (OFF-drug) for downstream readouts. B. RT-qPCR quantification of neuronal genes at the different time points and conditions depicted in Fig 7A. Data are relative to the housekeeping gene HPRT1 and normalized to the respective DMSO control. n = 4 technical replicates. Bar graphs represent mean ± SD. C. Immunoblot probing for sustainability of LSD1 activity inhibition at the different time points and conditions depicted in Fig 7A. α-Tubulin serves as loading control. pSMAD1/5/9, phospho-SMAD1/5/9. D. Immunoblot probing for LSD1-CoREST complex members at the different time points and conditions depicted in Fig 7A. α-Tubulin serves as loading control. E. Representative FACS readout of EdU/Propidium iodide cell cycle staining of PeTa cells 8 days after drug wash-out. F. Quantification of EdU/Propidium iodide staining depicted in Fig 7E. n = 3 biological replicates. Data are represented as means ±SD. *p<0.05, **p<0.01; ***p<0.001 (G1 phase: DMSO vs GSK-LSD1 p = 0.0104; S phase: DMSO vs GSK-LSD1 p = 0.0001; G2/M phase: DMSO vs GSK-LSD1 p = 0.0062; unpaired Student's t-test). G. Quantification of FACS Annexin V/DAPI staining of PeTa cells 8 days after drug wash-out. n = 3 biological replicates. Data are represented as means ±SD. ****p<0.0001 (unpaired Student's t-test). H. Schematic depicting the experimental setup of the in vivo tumor growth of pre-treated cells. PeTa cells were pre-treated in vitro with 100 nM GSK-LSD1 for 1, 3 or 6 days, or DMSO treated for 6 days prior injection. I. Subcutaneous tumor growth of pretreated cells (untreated, n = 8; D1, n = 9; D3, n = 9; D6, n = 4) Data are represented as means ±SEM. *p<0.05, ***p<0.001 (untreated vs D1 p = 0.023; untreated vs D3 p = 0.023; untreated vs D6 p = 0.0007; Kruskal-Wallis test).
Figure 7HP1b null mutant did not show ectopic vein in the wing and failed to genetically interact with HP1c and Notch. Scale bars, 500 μm. HP1b null mutant did not show ectopic vein in the wing and failed to genetically interact with HP1c and Notch. Scale bars, 500 μm. HP1b null mutant did not show ectopic vein in the wing and failed to genetically interact with HP1c and Notch. Scale bars, 500 μm. HP1b null mutant did not show ectopic vein in the wing and failed to genetically interact with HP1c and Notch. Scale bars, 500 μm. HP1b null mutant did not show ectopic vein in the wing and failed to genetically interact with HP1c and Notch. Scale bars, 500 μm. HP1b null mutant did not show ectopic vein in the wing and failed to genetically interact with HP1c and Notch. Scale bars, 500 μm. The notum bristle phenotype caused by HP1c depletion can be rescued by human homolog HP1γ OE, as indicated by the enlarged views in the left bottom panes. Scale bars, 100 μm. The notum bristle phenotype caused by HP1c depletion can be rescued by human homolog HP1γ OE, as indicated by the enlarged views in the left bottom panes. Scale bars, 100 μm. The notum bristle phenotype caused by HP1c depletion can be rescued by human homolog HP1γ OE, as indicated by the enlarged views in the left bottom panes. Scale bars, 100 μm. The ISC differentiation defect produced by HP1c KD can be rescued by human homolog HP1γ OE. Scale bars, 30 μm. The ISC differentiation defect produced by HP1c KD can be rescued by human homolog HP1γ OE. Scale bars, 30 μm. The ISC differentiation defect produced by HP1c KD can be rescued by human homolog HP1γ OE. Scale bars, 30 μm. (M) Statistical results of the number of ISCs and ee cells per unit area (200 μm × 200 μm) in (D-F). More than twenty-five midguts were examined for each group. Error bars indicate SEM. Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01).
Other Histological Characteristics in the npc1 Mutant and Chimeric Mice.Figure 4(A-C) Microglia characteristics. Sections from 50-d-old npc1 mutants were stained with anti-Calbindin (green) to visualize PCs and anti-F4/80 (red) to mark microglia. (A) Very few microglia are present in lobule X, where no PC degeneration has yet occurred. (B) Lobule VIII demonstrates an infiltration of microglia in the granule cell layer and white matter tract. (C) In lobule II, nearly all PCs are gone and numerous microglia are present throughout the cerebellum. The dashed line indicates the edge of the granule cell layer. (D) Microglia infiltration is not sufficient to induce PC degeneration. C1.4 demonstrates numerous microglia marked by anti-F4/80 (red) in all layers of the cerebellum, including immediately adjacent to wild-type (GFP-positive) PCs (arrows). Despite this close approximation, no loss of wild-type PCs was detected by cell counting. The GFP transgene is apparently poorly expressed in microglia, as all microglia appear GFP-negative regardless of their genotype. E) Mutant PCs are lost even when surrounded by wild-type Bergmann glia. A stretch of the PC layer in C4.8 is shown where no npc1−/− (GFP-positive) PCs remain despite the presence of numerous wild-type (GFP-negative) glia (arrowheads) marked by anti-S100β (red). The space occupied by two wild-type PCs is indicated (arrows) with two npc1−/− (GFP-positive) glia directly underneath.(F) Wild-type PCs do not degenerate even when surrounded by mutant Bergmann glia. Three wild-type PCs (GFP-positive; arrows) in C1.4 have not degenerated despite only mutant glia (arrowheads) being in the immediate vicinity. (G) Oligodendrocytes in chimeric mice are a mixture of wild-type and mutant cells. Oligodendrocyte cell bodies are stained with anti-CC1 (red). Wild-type (GFP-positive; arrows) and npc1−/− (GFP-negative; arrowheads) oligodendrocytes are interspersed in C1.4.
Schematic model of the differences between a cell expressing one or two actins to perform two cellular functions.Figure 7Schematic model of the differences between a cell expressing one or two actins to perform two cellular functions. (Top) A model of the molecular mechanisms by which two actin isoforms may segregate to different actin networks. On the left, a system carrying wild-type actin is able to generate both the branched- and linear-networks. On the two central panels, defective interactions of an actin isoform with one or several ABPs, affect branched- or linear-network assembly. On the right, combining these two actin variants in one cell should trigger a natural segregation of actins and rescues the wild type actin organization. (Bottom) Effect of perturbing an actin assembly pathway for cells using one or two actin variants. On the left, when one actin is shared for two actin functions, the inhibition of one actin assembly pathway (for example branched-networks with CK-666) leads to a reinforcement of the other actin assembly pathway. On the right, when two actin variants are used for two different actin functions, this effect is limited as both actin networks assemble more independently. In other words, having a system with two actin variants can buffer against the addition of the drug.
Post-transcriptional regulation of MAKR5 by CLE45 treatment.Figure 5(A) Increased expression of BAM3-CITRINE fusion protein under control of the BAM3 promoter in developing protophloem sieve elements (asterisks) of 5-day-old bam3 roots upon CLE45 treatment. (B) Expression of BAM3-CITRINE fusion protein under control of the BAM3 promoter in 5-day-old Col-0 roots or makr5 roots. (C) Expression of MAKR5-GFP fusion protein under control of the MAKR5 promoter in 5-day-old Col-0 or bam3 roots. (D) GUS reporter gene expression driven by the MAKR5 promoter in roots of 5-day-old Col-0seedlings upon CLE45 treatment. (E) Expression of MAKR5-GFP fusion protein under control of the MAKR5 promoter in 5-day-old roots upon CLE45 treatment (3 h). Note accumulation of MAKR5-GFP in protophloem sieve elements (magnified) and its dependence on functional BAM3 and CLE45. The frequency of representative observations per total observations is indicated. (F) Close up view of MAKR5-GFP accumulation in the protophloem cell file upon CLE45 treatment. (G-H) Accumulation of MAKR5-BKI1 fusion proteins in protophloem cells upon CLE45 treatment. The frequency of representative observations per total observations is indicated. (I-J) Quantification of fusion protein abundance in developing protophloem cells upon expression under control of the MAKR5 promoter, showing mean total fluorescence (I) and plasma membrane to cytosol signal ratio (J).
Cargo velocity on GaMTs and non-GaMTs.Figure 2Representative trajectories of E-cad cargos tracked over 120 s in an HRPE cell. Colors represent different average velocity ranges. Red: > 400 nm/s; Purple: 200~400 nm/s; Green: 100~200 nm/s; Yellow: 0~100 nm/s. Scale bar: 20 μm. Total number of cargos within different velocity ranges in the 4 quadrants, respectively (Data were pooled from 3 independent experiments and n= 45 cells). Proportions of cargos with different velocity in the 4 quadrants. Red: the 1st quadrant; Dark grey: the 2nd quadrant; Light grey: the 3rd quadrant; White: the 4th quadrant (Data were pooled from 3 independent experiments and n= 45 cells). Flow chart shows the method of combining STORM imaging with single particle tracking in live cells. Red lines indicate the cargo trajectories. Lower right, MT networks (Grey) were extracted and present in purple. Representative time series show one fast cargo moving on a GaMT and a slow cargo moving on a non-GaMT. Upper, conventional imaging; lower, STORM imaging. Red line: GaMT; Green line: non-GaMT; White circle: cargo position. Scale bar: 2 μm. Marginal distributions show the velocity on GaMT (red dots) or non-GaMTs (green dots). Red curve and green curve, Gaussian fitting curves of velocity distribution on GaMT or non-GaMT, respectively. (Data were pooled from 3 independent experiments and n= 69 MTs). Peak of red curve (431.14 ± 9.7 nm/s) and peak of purple curve (188.96 ± 22.03 nm/s). ***p < 0.001, unpaired t-test. Upper panel: time series show an example of several fast cargos present on GaMT. Lower panel: time series show one slow cargo on non-GaMT switching onto a GaMT and becoming faster. Red line: GaMT; Green line: non-GaMT; White circle: cargo position. White line, cargo trajectory. Scale bar: 2 μm. Box-whisker plot presents the averaged acetylation level of fast or slow MT tracks. (Data were pooled from 2 independent experiments and n= 63 MTs). The ends of the whiskers are set at 10% and 90% of the entire population, ns, no significant difference, unpaired t-test. Marginal distributions of the mean velocity of cargos on segments of Ac-MTs (orange dots) and non-Ac-MTs (purple dots). Orange curve and purple curve represent the Gaussian fittings (Data were pooled from 2 independent experiments and n= 63 MTs). ns, no significant difference, unpaired t-test.
The mitochondrial apoptosis pathway contributes to cytokine secretion during infection HeLa cell lines with an intact (CTRL) or a deficient (Bax/Bak-double deficient or Bcl-XL-over-expressing) mitochondrial apoptosis apparatus were infected with pathogens; supernatants were analysed for cytokines.Figure 3A Infection with MVA (MOI=10 for 16 h; IL-6). B infection with HSV-1 (MOI=1 for 16 h; IL-6). Infection with Chlamydia trachomatis (Ctr; MOI=1 for 30 h; IL-6 Infection with Chlamydia trachomatis (Ctr; MOI=1 for 30 h; , IL-8). Infection with Salmonella Typhimurium (STy; MOI=50 for 24 h; IL-6 Infection with Salmonella Typhimurium (STy; MOI=50 for 24 h; CXCL-1). Infection with Toxoplasma gondii (MOI=5 for 16 h; IL-6 Infection with Toxoplasma gondii (MOI=5 for 16 h; , IL-8 Infection with Toxoplasma gondii (MOI=5 for 16 h; CXCL-1 Infection with Toxoplasma gondii (MOI=5 for 16 h; FGF-2).
Figure 7Presence of IgG antibodies detecting dsDNA, cardiolipin, SSA (Ro-52) and SSB (La) in the serum of Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice at the age of 2, 4 and 12 months. The titers of the different IgG antibodies were determined in the serum of male and female mice by ELISA and are displayed as arbitrary units (AU). The serum of 6-month-old Faslgld/gld mice was used as positive control Representative images of anti-nuclear antibody (ANA) staining obtained with serum from 12-month-old Prdm1ihCd2/+ or wild-type mice (right), as detected by indirect immunofluorescence assay using HEp-2 cells and an Alexa488-conjugated anti-mouse IgG detection antibody. The serum of Faslgld/gld mice was used as positive control. The presence of ANA-IgG antibodies was analyzed for 22 Prdm1ihCd2/+ and 12 wild-type mice, as summarized in the pie-chart (right) Periodic acid-Schiff (PAS) staining of paraffin-embedded kidney sections of Prdm1ihCd2/+ and wild-type mice at 12 months of age. The mean score of kidney pathology was determined for each mouse by evaluating 40 individual glomeruli to assess the severity of glomerulonephritis as shown in Table EV2 and described in the Appendix Supplementary Methods. Representative pictures of glomeruli of Prdm1ihCd2/+ kidneys with different pathology scores are shown to the left, and dot plots with average scores for male and female mice (Table EV2) are shown to the right. Arrows denote intracapillary hyaline "thrombi", asterisks indicate mesangial sclerosis with obscured capillary loops, and the arrowhead points to an obsolescent glomerulus with collapse of the glomerular tuft architecture. Statistical data (A,C) are shown as mean value with SEM and were analyzed by the Mann-Whitney test (A) or the Student"s t-test (C); *P < 0.05, **P < 0.01, ***P < 0.001. Each dot correspon Proposed model explaining the autoimmune phenotype of Prdm1ihCd2/+ mice. Precocious Blimp1 expression in Prdm1ihCd2/+ mice results in decreased numbers of developing B cells (red arrows) and increased differentiation of immature and mature B cells to plasmablasts (blue arrows) and plasma cells (green arrow). As a consequence, autoreactive immature B cells may differentiate to plasmablasts prior to their elimination by central tolerance mechanisms (receptor editing or clonal deletion; Nemazee, 2017), and autoreactive mature B cells may escape their anergic state, imposed by anergy-induced BCR desensitization (peripheral tolerance; Theofilopoulos et al., 2017), by premature differentiation to plasmablasts, which leads to an increase of autoantibody-secreting plasma cells in Prdm1ihCd2/+ mice
Figure 4A-C. Wild type embryos injected with control MO or with shox MO. (A) Lateral views of the embryos at 55 hours post fertilization (hpf). (B) Dorsal view and magnification on the lateral view of the embryos. Dotted line, pectoral fins. shox morphants show smaller fins compared to controls (n = 30 embryos). A-C. Wild type embryos injected with control MO or with shox MO. C. Expression of col2a1 at 55 hpf was examined by in situ hybridization in wild type embryos injected with control MO and with shox MO. Arrow and dotted line indicate the pectoral fin. A-C. Wild type embryos injected with control MO or with shox MO. D. Pectoral fin area was measured by imageJ (n = 30 embryos).
EHD1 localization is dependent on cellular PS levelsThe effect of various lipids on the ATPase activity of the purified ATP8A1‐CDC50A complex.Figure 4A The effect of various lipids on the ATPase activity of the purified ATP8A1‐CDC50A complex. The ATPase activity was measured in the presence of 5 mM ATP and 10 mol % of one of the following lipids: 1,2‐dioleoyl‐phosphatidycholine (PC), 1,2‐dioleoyl‐PS, 1,2‐dioleoyl‐PE, 1,2‐dioleoyl‐phosphatidylglycerol (PG), 1,2‐dioleoyl‐phosphatidylinositol (PI), 1,2‐dioleoyl‐phosphatidic acid (PA), sphingomyelin (SM), cholesterol (Chol), or brain polar lipid (BPL) and 90% PC. The activity of the ATP8A1 (E191Q)‐CDC50A in the presence of 1,2‐dioleoyl‐PS was also determined. B NBD‐labeled PS or PE transport (flippase) activity of ATP8A1 (WT and E191Q)‐CDC50A complex. The percentage of NBD‐lipid flipped was shown. C Schematic of the biosynthetic pathway of PS in mammalian cells. The impaired pathways in PSA‐3 and R‐41 cells are indicated. D, E PSA‐3 and R‐41 cells were transiently transfected with GFP‐EHD1 and then cultured under the indicated conditions for 72 h. Cells were then fixed, and the nuclei were strained with DAPI. Arrowheads indicate the perinuclear RE localization of GFP‐EHD1. Scale bars, 10 μm.
Figure 4Cideb co-precipitates with SCAP. Flag-tagged Cideb was co-expressed with different HA-tagged proteins (PLIN2, Insig, SCAP, SREBP-1 or SREBP-2) in 293T cells and immuno-precipitated with an anti-Flag antibody, and the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE. Interaction between the endogenous SCAP and Cideb. Endogenous SCAP was immuno-precipitated from primary hepatocytes under normal medium, 25-HC depletion or addition conditions. And the presence of Cideb in the immune-complex was detected by IB. SREBP-1 was detected as the positive control. Grp94 was detected as the negative control. Membrane topology of SCAP. The dashed line labels the portion of SCAP (a.a.416-800) that interacts with Cideb. Cideb interacts with the 416-800 portion of SCAP. Flag-tagged Cideb was co-expressed with the indicated HA-tagged SCAP truncation mutants in 293T cells and immuno-precipitated with an anti-Flag antibody. Levels of the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE. Sterol-dependent interaction switch of SCAP. SCAP interacts with Insig in the presence of sterol, while switches to Cideb binding upon sterol deprivation. Flag-tagged Insig-1 or Cideb was co-expressed with HA-tagged SREBP-2 and SCAP or SCAP-Y298C in CHO-K1 cells in sterol-depleted medium. When indicated, 10 µg/ml 25-HC were supplemented before cells were immuno-precipitated with an anti-Flag antibody, and the levels of co-immunoprecipitated proteins were detected by IB following SDS-PAGE. The dotted blue vertical line was drawn to separate the result between SCAP-WT and SCAP-Y298C. A "Ping-Pong" scheme of sterol-regulated SCAP/Insig and SCAP/Cideb interactions. SCAP switches from Insig binding to Cideb binding in the absence of sterol, while the sterol-insensitive SCAP-Y298C consistently interacts with Cideb. Cideb relieves sterol-dependent suppression of SREBP processing. Hepatocytes infected with adenovirus expressing GFP or Flag-Cideb were cultured in sterol deprived condition. When indicated, cells were treated with different doses of 25-HC before subjected to IB with the indicated antibodies following SDS-PAGE. Quantification of IB data from 3 independent experiments. Data represents Mean ± SEM. Cideb promotes Golgi localization of SCAP. HepG2 cells transfected with GFP-SCAP alone or with HA-Cideb were cultured in sterol deprived conditions or treated with 1 µg/ml 25-HC. Cells were fixed and subjected to confocal microscopy following staining with antibodies against GM130 or HA. Scale bars represent 10 µm (Merge), and 2 µm (Inlay). Bottom: quantification of the percentage of SCAP localized in Golgi-positive region per cell (left) and quantification of the percentage of cells containing Golgi-localized GFP-SCAP (right) under indicated conditions, from 3 independent experiments; Data represent Mean ± SEM; **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.