EMBO/sd-character-level-ner huggingface dataset:
HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cell viability was assessed by Crystal Violet (B) after 16 hours. Data are means ± SEM of three independent experiments. *P< 0.1, ****P<0.0001 (ANOVA).
(A) BMDMs derived from WT and Atg5−/− mice were infected with L. pneumophila JR32 or legA9 mutant at an MOI of 0.5. Data are shown as mean ± SD of n = 3 and are representative of two independent experiments. Asterisks indicate significant differences (*p < 0.05; two−tailed t−test.
Schematic describing the measurement of spindle angle (θ) (Kikuchi et al., 2010). Z-stack images were obtained from 0.45 mm-thick sections of metaphase cells and the angle between the axis of metaphase spindle and that of the substrate surface, which is termed the spindle angle, was calculated. Results presented are scatter plots of the angle (θ) of the mitotic spindle (n=28 for all samples of vector, HA-WT, -3D, -3A, -R899X, -Q671X, and -R603X INVS stably transfected MDCK cells). Three independent experiments showed similar results. Statistical significance was determined by Mann-Whitney's non-parametric median test.
(A) 293 cells were transfected with bag1, bag3 or nonsense (nons) siRNAs, as indicated. After 48 h, cells were transfected with d2GFP expression plasmid together with half the amounts of the indicated siRNAs. After additional 24 h, levels of indicated proteins were detected by immunoblot analysis.
Tumor burden (left panel), tumor number (middle panel) and average tumor size (right panel) per mouse assessed during colon resection at the end of treatments. αCD4/αCD8: n=10; IgG: n=9.
D. Sensitivity of LC3C specific sensor mCh-AS3_67 is increased upon fusion of membrane-tethering FYVE domain or PB1 oligomerization domain.
F Structural docking model of CUL3Δ403-459 based on the structure of full-length CUL1 (1LDK) using Chimera (see Materials and Methods). The NTD is coloured green, while the CTD is red, and the four panels sample possible positioning of the CTD relative to the NTD in CUL3Δ403-459, assuming full flexibility of the linker after deletion of three helices encoded by exon 9.
(B) Aliphatic part II. Phospholipid metabolites and the α-methylene groups of amino acids and lactate are visible.
(F) Representative images of IL-33 and p-SMAD2/3 immunostaining on adjacent sections of matched normal pancreas, chronic pancreatitis and PDAC collected from pancreatic cancer patients (scale bar: 100 μm).
O-T Functional effects of in vivo inhibition of microglial phagocytosis. Representative dark-adapted ERG recordings at P26 (following intravitreal injections at P20 and P23) in a cRGD-injected eye (red) relative to the contralateral control eye (black) (O) showing that mean a- (P) and b-wave (Q) amplitudes were significantly greater in the cRGD-injected vs. control eyes at higher flash intensities. Representative light-adapted responses (R) illustrating that while mean light-adapted a-wave amplitudes (S) were slightly but not significantly greater in cRGD-injected eyes, mean b-wave amplitudes (T) were significantly greater at higher flash intensities (n = 13 animals, two-sided paired t-test, *P < 0.05, with exact values alongside).
F. Maximum amplitude (-dB) of each syllable in YFO and AFO. Data information: Data are presented in the box plot or pie chart format. The central band in each box represents the median; boxes indicate the middle quartiles, and whiskers extend to the minimum and maximum values. *p<0.05, determined using Student's or Welch t-test.
MCF-10AT1k.cl2 cells were treated with 20 µM importazole or an equivalent volume of DMSO as a control for 24 hours and stained with an antibody against lamin B1. Representative images are shown.
Simulated kymograph of a less polarized cell with a narrow PI(3,4) P2 enriched region. The white dashed lines correspond to the periphery of the back regions which spanned on average 30% of the cell perimeter.
B Cell lysates from wild‐type (WT) or GIW1KO cells at 3 hpm were incubated with radiolabeled double‐stranded 27‐nt RNAs and with (+) or without (−) ATP and the ATP regeneration system. The Twi1p‐containing complex was immunoprecipitated with an anti‐Twi1p antibody, co‐precipitated RNAs were separated on a denaturing gel and detected by autoradiography. The intensities of the bands in the different conditions relative to the sample with wild‐type cell lysate and without ATP (1st column) are shown on the top. The standard deviation (SD) between technical replicates is indicated. A result of a representative experiment is shown on the bottom.
D. Structure of the miRNA 3' tail suggests tension in the miRNA may restrict PAZ domain movments and opening of the supplementary chamber (target RNA not shown for clarity).
(A) Images of colonic sections stained for LC3β (green) from control and Atg5VC mice. The white‐dashed lines indicate the border between the basal surface of the crypt epitheliumand the mesenchyme. White asterisks denote non‐specific plasma cell staining from the mouse monoclonal anti‐LC3β antibody. Bars=50 μm.
Representative IF staining of γH2AX and Ki67 in the regions of frontal cortices from WT and FTLD-TDP Tg mice. Nuclei were stained with DAPI (upper panel) or NeuN (middle panel). Scale bar: 50 μm. Subregions, scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX and Ki67 immunoreactivity from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, ***p = 0.0004 by t-test.
(d) Phosphorylation of Ulk1 Ser 317 and Ser 777 are induced by AMPK. Wild-type HA-Ulk1 or S317/777A mutant were co-transfected with AMPK into HEK293 cells as indicated. HA-Ulk1 was immunoprecipitated (IP) and phosphorylation of Ser 317 and Ser 777 were determined by western blotting. Uncropped images of blots are shown in Supplementary Fig. S5.
Representative IF staining of Prom1 using antibodies directed either its extracellular loop (clone 13A4, green) or cytoplasmic C-terminal end (Biorbyt, Orb129549, red) on transit amplifying cell regions of the WT vs. Prom1 KO mice. Samples are counterstained with DAPI (blue). Note the lack of Prom1 labeling in Prom1 KO mice.
M (left) Long-term viability of melanoma cells treated as indicated for 8 days. (right) corresponding quantification showing change in viability. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
d, Quantifications of GFP-positive and Npas4-positive cells 1h post cocaine treatment. Data were normalized so that the number of GFP- and Npas4-positive cells was set to 100%. 20 fields of view were analysed from n = 3 mice in the case of AAV-shscr-GFP and 30 fields of view from n = 3 mice for AAV-shNpas4-GFP. Mean + SEM; unpaired Student's t-test; p* < 0.05, n = 3 mice per group.
C, Interspecies chimera assays in which the 8C-stage mouse embryos indicated in (A, B), injected with one single human iPSCs and cultured in vitro for 48 h to reach the blastocyst stage, were then transfected to 2.5 days post coitum pseudo-pregnant females. The post-implantation mouse conceptuses were dissected at the E9.5 developmental stage and analyzed by immunofluorescence. Representative images showing the integration of control human iPSCs (top) or miiPSCs (bottom) into mouse E9.5 embryos. Anti-human HuNu antibody was co-stained with Td-Tomato direct fluorescence to detect human cells in E9.5 mouse embryos. Scale bars, 20 μm. D, E, Quantification of the Td-Tomato-(D) or HuNu-(E) positive area in 40-50 different cryosections of these embryos randomly and blindly selected for IF analysis. Data are mean ± s.e.m. **P<0.01; ***P< 0.001 (Student´s t-test).
F MEFs were infected with S. pneumoniae R6 WT or ΔcbpF for the indicated periods, and the intracellular survival of bacteria expressed as the number of CFUs.
G Quantification of E where total EGFR is normalized to Hsc70. Mean +/- SD n=3 for all treatment groups from 3 independent experiments
(C) Volcano plots of proteins in close proximity to Apl5-TurboID versus control cells, from a label-free proteomics analysis of streptavidin-biotin pulldowns. The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student's t-test, equal variance, performed from n = 3 independent experiments. The red dashed line (significance, 0.05) separates specifically identified proteins (top right portion of plot) from background. Selected top hits are indicated with black dots,
F, Heat-scatterplots representing the mean methylation over all transcripts (transcription start sites, TSSs) in mm10, pairwise for "t=10 vs. t=0" (left panel) and "t=25 vs. t=0" (right panel). Note that especially for "t=25 vs. t=0" most of the points are below the diagonal, indicating broad hypomethylation across most of the genome. G, Heat-scatterplots representing the mean methylation over the TSS of the 282 genes included in the 2-cell signature (as in Fig. 1D). Analysis as in (F).
Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) or high fat diet (HFD) (n=3). Organoids isolated from ND or HFD mice were cultured in collagen for 2 days. Representative images of organoids cultured in collagen for 2 days. The number of invasive protrusions per organoid is higher for organoids isolated from mice on HFD compared to mice on ND. Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse (labelled with a different symbol shape).
Proliferation of primary MEFs. Cells were treated o/n with 0.2 µg/ml doxorubicin (Doxo) and pulse-labeled with 32 µM 5-Bromo-2-deoxyuridine (BrdU), fixed and processed for flow cytometry analysis. n=4.
D Immunoblotting of phosphorylated HSL (p-HSL), HSL, p-PKA substrate, desnutrin and Tubulin (control) (left) with relative quantification (right). Data are expressed as mean ± SEM from three independent experiments. *P = 0.0032, **P = 0.0011 (Student's t-test). NS, not statistically significant.
(a) Flow cytometry of circulating LCMV-specific CD8+ T cells among peripheral blood mononuclear cells from Atg5fl/fl and Atg5fl/flGzmb-Cremice on days 8, 15 and 50 after infection with LCMV Armstrong strain. Numbers adjacent to outlined areas indicate percent H-2Db-gp33+ T cells, gated on CD8+ T cells. (b) Longitudinal kinetics of the appearance of H-2Db-gp33+ T cells in the peripheral blood of mice as in a (left), and percent H-2Db-gp33+ CD8+ T cells remaining on day 15 (right) (all presented as in Fig. 4h).
(F) Immuno-EM showing p62-positive aggregates containing dense clusters of vesicular-tubular structures in cells transfected with myc-CHMP2BIntron5. Bar, 200 nm.
a and b, Schematic representation of the potential acetylation domain organization of ISX and its lysine mutants (AC1‒AC3). Data information: Each experiment was repeated at least three times.
(C) Classification of the CBMSO cohort according to the Score generated from S/EGFR data. The Broken line indicates the 0.024 threshold above which samples are considered positive.
(a) SK-N-SH cells were pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 4 h. Calpain activity was detected by immunoblotting with anti-calpain small subunit antibody. Densitometry analysis is relative to actin. Error bars show s.e.m.
(A) A scheme of the experimental design. Triangles represent the time points (in minutes) of cell harvesting for RNA purification, after EGF stimulation. (GF, growth factor).
B: Cotransfection of zDHHC9+21 reduced vacuolation in prion-infected Gt1 cells. Each dot represents a separate experiment (1000 cells/experiment, χ2: p<0.001). **: p<0.01; unpaired t-test. Error bars represent s.e.m.
(D) Control or CITK-depleted cells were immunostained for γ-tubulin (red) and DNA (blue) and imaged in z (0.3 µm-thick sections). Upper panel: maximum intensity projections of confocal z-stacks are shown. Lower panel: cross-section (XZ) through the two poles of the same cell.(E) Distribution of spindle angles (°) in control and in CITK-depleted cells. The values represent the angles between the axis crossing the two poles of metaphase spindles and the coverslip. (n ≥ 150 cells, 6 independent experiments).
a. Representative synthetic dataset of nuclei (black) and mRNAs, where each color represents a different gene.
(B) Cas9-based approach for conditional disruption of the MSA180 gene. Sequential modification by marker-free gene editing was used to achieve: (I) insertion of a C-terminal HA3 epitope tag and a loxP site just downstream of the stop codon; followed by (II) insertion of a loxPint module just upstream of the central conserved region. Modification I produced a P. falciparum clone called MSA180-HA3 [B11], which was subsequently modified (II) to generate the MSA180-HA3:loxP parasite line. Conserved regions (black bars), sgRNA targeting sites (break), loxP sites (arrow heads), recodonised segments of MSA180 (hatched) and corresponding homology arms (regions linked by grey dotted lines) are shown. Half-arrows, primers used for diagnostic PCR to confirm gene editing and excision of floxed sequences (see Table EV1 for primer sequences).
F Histological analysis of NeuN and GFAP staining was performed in spinal cord tissue from ERp57WT and ERp57Nes-/- mice in three animals per group using indirect immunofluorescence. The nucleus was stained with Hoechst. Representative images from one mouse per group are shown. Scale bar: 50 μm.
Knockdown of USP36 reduces Nop58 and Nhp2 SUMOylation in cells. Cells infected with scr or USP36 shRNA lentiviruses were assayed by IB. The SUMOylated Nop58 (D) and Nhp2 (E) are indicated.
(K) qRT-PCR analysis of ZIKV RNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.
Immunofluorescent staining for LC3 and p62 in TCHP knock-down and control cells. Scale bars, 25μm. Lower panel: quantification. (n=90 cells, unpaired t-test; LC3: **p<0.0001 vs control; p62: **p<0.0001 vs control).
Steady state levels of different metabolites obtained from metabolomics were used to derive activity curves for (A) Adenylate Kinase (AK) (B) Uridylate Kinase (PyrH) (C) Adenylosuccinate Lyase (PurB) and (D) GAR Transformylase (PurN). AK, PyrH and PurB appear to follow hyperbolic MM kinetics in vivo, while PurN shows Hill-like dependence with cooperativity. In panel (A), the red data points were acquired during growth in amino acid supplemented M9 medium in the absence of external AMP and are derived from WT and various stabilizing and destabilizing point mutations of AK (Adkar et al., 2019) as well as DHFR mutants used in the present study, while black points were derived exclusively from cultures of AK mutants supplemented with 1mM external AMP (Adkar et al., 2019). Data points for all other panels (B-D) are derived from mutant DHFR strains in the present study. Since SAICAR, AICAR, GAR and FGAR levels were undetectable in WT, all data points in (C-D) are relative to those observed in I91L+W133V mutant following 4 hours of growth at 40°C. Data points in all panels represent metabolite levels for individual biological replicates without averaging.
(C) Co-immunoprecipitation between HA-VP30 and wildtype (WT) or mutant RBBP6 peptides fused to GFP. GFP alone served as a negative control. IP was performed for GFP and blots were probed with anti-GFP and anti-HA antibodies. Anti-β-tubulin served as a loading control for the whole cell lysates.
Cryotomographic slices (9.7 nm) showing side views of TssJLM. In these examples, the basal parts of TssJLM (red arrows) could be seen extending into the cytoplasm. Scale bar 10 nm
I Bar graph showing the fractional release following caffeine application in cardiomyocytes from WT (n=24 cells), mdx (n=33 cells) and mdx/CD38-/- (n=32 cells) mice. Data information: experiments performed in 3 mice of each group. After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; ; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.
(g) Clearance of soluble EGFP-HDQ74 in stable PC12 cells as in Figure 1b, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for the 48 h switch-off period. Error bars show s.e.m.
(A) Immunoblotting analysis demonstrated a large accumulation of α-tubulin acetylated at Lys40 (Ac-α-tubulin) in NDST3 KO RPE1 cells, while the total α-tubulin relative to GAPDH remained unchanged (n = 3 independent cultures, ***P = 0.0008). Data information: Error bars represent ± standard deviation. ns means non-significant.
Transcriptional activity of wide type N-terminal MYRF, S259A, or S261A with the MBP promoter reporter in HEK293 cells was detected using a luciferase assay. Compared to wide type MYRF or S261A, S259A (non-phosphorylated) remained transcriptional activity that was not inhibited by N-terminal mutant HTT. The ratios were obtained from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P< 0.001. Data are mean ±SEM.
Cross-talk between different ciliary modules and distinct classes of ciliopathies. Most ciliary modules are associated with a specific class of ciliopathies; for example, IFT-A and IFT-dynein are linked to skeletal ciliopathies, BBS proteins are associated with Bardet-Biedl syndrome, and transition zone proteins are associated with Joubert Syndrome and Related Disorders (JSRDs), MKS, NPHP and eye-related disorders, but not skeletal ciliopathies. However, a subset of proteins are associated with ciliopathies generally typical of a different module, indicating potential overlap in function. The module-disease connections are shown as solid lines, with the width correlating to the number of causative genes currently identified (1 to 10+, as indicated). MKS, Meckel syndrome; JBTS, Joubert syndrome; COACH, Cerebellar vermis hypoplasia/Oligophrenia/Ataxia/Coloboma/Hepatic fibrosis; OFD, Oral-Facial-Digital syndrome; SLSN, Senior-Løken syndrome; NPHP, nephronophthisis; RP, Retinitis Pigmentosa; CRD, Cone-Rod Dystrophy; LCA, Leber Congenital Amaurosis; BBS, Bardet-Biedl Syndrome; CED, CranioEctodermal Dysplasia; SRTD, Short-Rib Thoracic Dysplasia; JATD, Jeune Asphyxiating Thoracic Dystrophy. SUPPLEMENTARY FIGURE
B: Silver nitrate staining of proteins pulled-down using the control peptide (left) or the FFAT peptide (right), after SDS-PAGE. The two major differential bands are highlighted by arrows
Weights of innervated and denervated TA muscles electroporated either with control or Myoparr shRNA. Muscle weights were measured 7 d after denervation. n = 5 for each group, mean ± SEM. ***p < 0.001. n.s., not significant (unpaired two-tailed Student's t-test).
C) ROC curve for the XGBoost-based model when trained on one cohort and tested on the other cohort. Random performance is indicated by the dotted diagonal line.
Confocal images of CAD cells transfected with GFP-βCaMKII WT plasmid (green) and treated or not with Wnt7a. Cells were fixed and then stained with phalloidin (red) and DAPI (blue). The insets (right panels) show a magnified image of the area depicted in the expanded field images (left). 3D reconstruction shows co-localization of GFP/phalloidin signals. Scale bars represent 10 µm.
3A) Regular, non-transduced SH-SY5Y cells were cultivated either in RPMI+10% FCS or starved in HBSS medium for 24 and 40 h. The LAMP-2 and actin content were determined by western blotting and the relative LAMP-2 content of untreated cells was set as 1. Starvation enhanced LAMP-2protein levels; n = 3, p<0.05.
(A-B) Overexpression of Bch1 displaces Chs6 from the TGN and impairs plasma membrane localization of Chs6-dependent cargo Chs3. Fluorescence images of Chs6-GFP (A) and Chs3-GFP (B) in WT and GPD-BCH1 strains. Bar, 5 µm. Arrows point to Chs3-GFP signals at the bud neck. The inset represents a twofold magnification.
B Quantification of pWOX5::GFP GFP fluorescence in wild-type (WT) and seu-3 mutant embryos. Fluorescence intensity at the early globular stage of WT embryos was set to 1. Data Information: In (B) data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).
(B) Murine bone marrow-derived macrophages (BMDMs) were infected with BCG at an MOI of 5 for the indicated times, and the expression levels of miR-155 were measured by real-time PCR.
Pharmacological action profiling of the entire compound library and confirmed hits.The 54 final hits were selected and sorted into 43 pharmacological action categories. Gray and black bars indicate the distribution of all screened compounds and confirmed hits with a therapeutic index (TI) >6, respectively. The vertical axis displays counts of each compound on a log scale with +1 added to the count to prevent negative values.
A. PCR amplification of CALR exon 9 from patients confirming heterozygous del52 mutation in sorted CD34+ cells obtained from CALR mutated myelofibrosis samples.
(A) NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 strain at MOI 10 and 0.01, respectively. At the indicated time points, the culture supernatants were removed and the cell lysates were prepared for Western blot analysis. Relative protein levels were determined by normalizing against β-actin and compared to mock-infected cells
Representative immunoblot analysis of samples from (D). Full arrow indicates migration of the EGFP-FAM110A-WT, empty arrow of endogenous FAM110A.
(B) Inhibition of the GAPC activity redirects the glycolytic pathway towards PPP, resulting in the production of NADPHRalser et al., 2007(). While oxidative inactivation of GAPC is a fast event Ralser et al., 2009Zaffagnini et al., 2019(; ), we speculate that Tyr-Asp inhibition becomes critical under prolonged stress to maintain high levels of NADPH production. In contrast to GAPC, GAPN is unaffected by Tyr-Asp and less sensitive to oxidation.
Dot plots depicting the abundance of aNSCs and TAPs (BrdU-, Ki67+, DCX-) in the SEZ 7 days after aCSF or AntimiR204 injection.
(b) Western blot analysis of HeLa cells treated with a second independent siRNA targeting ATG5, ATG7 or NDP52. GAPDH and total protein stained with Coomassie blue serve as loading controls.
(C) MYD88 but not TRIF expression reduces dendrite outgrowth. HA-tagged MYD88 and TRIF were co-transfected with a GFP construct into cultured wild-type (WT) neurons at 4 DIV, and cell morphology was analyzed at 6 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by one-way ANOVA with Bonferroni's multiple comparison test.
A. Overrepresentation of Aβ APRs in amyloid plaques of APP/PS1 mouse brains (mean ±SD) from two biological replicates. (statistics: hypergeometric test with Bonferroni correction)
Time-lapse micrographs of agarose-embedded cells were taken at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Representative images of characteristically reoccurring fluorescence localization stages are displayed. In all combinations, fluorescence foci localize to areas of membrane outgrowth in the bud. Often, another prominent fluorescent spot is found in the mother cell on the side of the bud neck. Scale bar 5 µm. Kymographs of all time-lapse datasets shown in this Figure are presented in Fig EV3A.H. COPI•COPI (α-COPVN•α-COPVC) BiFC signals showed one spot that localized to the bud tip and, at later budding stages, to the bud neck (compare with A-G). Additionally, multiple spots with no apparent polarization tendency were observed, which are reminiscent of a Golgi pattern.
Diagrams of the reporter assay vectors DRGFP and EJ5GFP for measuring HDR and Distal-EJ, respectively.