EMBO/sd-character-level-ner huggingface dataset:
C-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. GAPDH was used as a loading control. *non‐specific band.
D RT-qPCR analysis of indicated transcripts following transfection of HEK-293 cells with indicated siRNAs and treatment with 1 µM thapsigargin for 6hr. Relative fold changes are in reference to vehicle-treated, NT siRNA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons
Analysis of hippocampal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. DCV pool size measured as number of ChgB puncta per neuron.
(E) Immunoblot of Flag-p65-linked ubiquitination promoted by truncated Myc-CDC20 fragments. HEK293T cells were transfected with Flag-p65, HA-Ubiquitin, with or without Myc-CDC20 171-499 fragment (containing WD40 domain), Myc-CDC20 1-170 fragment (WD40 domain deficient) and Vector plasmids. Transfected cells were then treated with 10 μM MG132 for 6 h before collection.
Representative immunohistochemistry staining of KLHL3 and CUL3 in humankidney sections (n = 6). KLHL3 shows preferential DCT/TAL cytosolic staining similar to the mouse despite no evidence of PCT apical staining as seen in (A). CUL3 exhibits preferential basolateral cytosolic staining of the PCT with similarly low levels of diffuse staining in the DCT/TAL to that of the mouse. Scale bar, 100 μm.
variations of IL-6 expression with the altered level of PP2A (black) and HuR (red). For both cases, the data were fitted to an equation, which is shown above. The parameter m denotes the expression level of PP2A/HuR which corresponds to the maximum IL-6 expression; w denotes the width of the distributions for both cases. For PP2A and HuR, the values of m were found to be 0.6 and 0.9 respectively and A is a constant (F).
Detection of 47S pre-rRNA transcription level with northern blotting in HCT116KO following different treatments as indicated. siPLXNB2-1 and siPLXNB2-2: PLXNB2 siRNAs
A. OT-II WT cells were activated with OVA323-339, alone or with TAK-779. After three days, antigen and TAK-779 were removed and lymphoblasts expanded in IL-2-containing medium. TCR nanoclustering was analyzed in anti-CD3ε-labeled surface replicas of day 10 lymphoblasts. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of WT CD4+ T cells alone or with TAK-779. Bottom, quantification of gold particles in clusters of the indicated size. Inset, distribution of gold particles between clusters of one, two, three, four or more than four particles in vehicle- (gray bars; n = 5 cells, 11266 particles) and TAK-779-treated cells (black; n = 6 cells, 5138 particles). B. OT-II WT cells were activated with OVA323-339, and TAK-779 was added at days 3, 5, and 7 after antigen removal. Analysis as above, untreated (gray bars; n = 5 cells, 6400 particles) and TAK-779-treated cells (black; n = 6 cells, 7153 particles). Inset show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.
KO1 cells transfected with vector (KO1-vec), wild-type OAS1 (KO1-OAS1 wt), or mutant OAS1 (D75A/D77A, KO1-OAS1 mut) were treated with 500 μM of H2O2 for the indicated times and analyzed by the Western method. Loading controls: β-actin and overall protein levels were visualized by stain-free technology. The experiment was repeated three times; a representative image is shown.
Real-time PCR analysis of several cardiac markers expression during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).
The distribution of the drugs based on their IC50 and predicted ICD score is depicted as density plot. Based on the properties of the standard ICD inducer mitoxantrone (blue), we selected negative (green) and positive (red) hits: agents that entered into clinical trials, having an IC50 < 1 μM (and therefore -log(IC50) > 6) and whose predicted ICD score is higher than the ICD score of mitoxantrone are potential ICD inducers (positive hits). Some agents that entered into clinical trials, whose IC50 > 1 μM and which have an ICD prediction score lower than 1 are negative hits.
Immunoblot analysis of active caspase-1 (p20) generation in HEK293 cells that were transfected with inflammasome components (Flag-tagged NLRP3, NEK7, ASC, and Caspase-1) along with EGFP, GFP-tagged PLK4, PLK4 kinase dead mutant (D154A), or PLK1 and stimulated with 10 µM Nigericin for 1h 24h post-transfection.
Representative immunoblots of ISR component eIF2α (B) and quantification of phospho-eIF2α (p-eIF2aSer51) relative protein expression (C) in skeletal muscle (Gastroc, WT day n=4, WT night n=5, TG day n=9, TG night n=9). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm).
qRT-PCR analysis of the indicated mRNAs in untreated or LPS-stimulated WT or Spata2-KO BMDMs. Bars and error bars represent the mean ± S.D. of triplicate experiments. Statistical analysis was performed using unpaired two-tailed Student's t-test.
(F) RNA‐immunoprecipitation of Red1‐GFP and Red1H637I‐GFP. Cell lysates from untagged, Red1‐GFP, and Red1H637I‐GFP strains were subjected to immunoprecipitation using anti‐GFP, and precipitated RNAs were analysed by RT-PCR. The values of relative enrichment are shown.
(I) Relative fold changes of PER15 in pCASP1::SGN1-Citrine and pCASP1::myrpalm-SGN1-Citrine compared to the expression level in WT. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test). (J) Relative fold changes of PER49 in pCASP1::SGN1-Citrine and pCASP1::myrpalm-SGN1-Citrine compared to the expression level in WT. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test).
(E) HeLa cells were incubated for 1 hr at 4°C with ice-cold NHS-LC-Biotin (Pierce) solution, rinsed, and lysed in RIPA buffer. Biotinylated proteins were precipitated using streptavidin-agarose beads (Pierce) and blotted for mATG9 to assess protein localization at the plasma membrane.
F, Immunoblot analysis of co-immunoprecipitated TFEB-FLAG, 14-3-3 and endogenous TFE3 from ARPE-19 cells over-expressing TFEB-WT or TFEB-C212A mutant. Immunoblots are representative of at least three independent experiments.
(a) NRK cells were transfected with nonspecific (NS)- or Cltc (clathrin heavy chain)-RNAi and starved for 0, 4, 8 or 12 h. Scale bar, 5 mm. Cells were stained with antibodies against LC3. (b) Cells from a were assessed for LC3 puncta in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.
Schematic representation of the genetic elements for lineage tracing and Lgr5 ablation during postnatal development and adult homeostasis and experimental design. Lactating females were tamoxifen (Tam)-treated between PN 6 and PN 8. Small intestines of treated pups were analyzed 10 days after the last injection (PN 18); adult mice received one Tamoxifen intraperitoneal injection and intestines were analyzed 10 days later.
(C) Below the schematic representation of the experiment are representative confocal images showing maximal intensity projections of 6 z-slices (covering 2 μm of thickness) of 24-hour co-culture of RD-YFP SH cells (acceptor cells) with RD-YFP SH cells expressing nls-Red and challenged with K18-ATTO 594 fibrils (donor cells). WGA (white) labels cell membrane and DAPI the nuclei in the merge panel. Stars label donor cells containing green and red aggregates, arrows indicate an acceptor cell with green and red aggregates, and the arrowheads show an acceptor cell with green aggregates but devoid of red fibrils. Below are two-fold enlargement of the respective framed cells. The apparent discontinuity in the picture corresponds to the boundary between two adjacent tiles. Scale bars are 10 μm, 5 μm in the enlargements.
(A) CFU in homogenates from the distal small intestines of infant rabbits infected with the indicated strains 18, 28 and 38 h following inoculation. Black horizontal line indicates mean CFU (n=7-10 animals/group). One-way ANOVA was used for statistical analysis. *P<0.05 for the indicated comparison.
(E) Heatmap representation of gene expression changes within the "Reactome Innate Immune System" (left panel) and "Reactome Extracellular matrix Organization" (right panel) gene set. Genes in heatmaps are shown in rows and samples are shown in columns. Expression level is represented as a gradient from high (red) to low (blue). V, P, B and C respectively indicate vehicle, PLX4720, bevacizumab and COMBO.
F) NGS plot showing H3K27ac comparative patterns in the experimental conditions described in B).
e Equal amounts of proteins from total lysates of CD4+ T cells left unstimulated (-) or stimulated for 120, 300 or 600 sec with anti-CD3 and anti-CD4 antibodies were analyzed by immunoblot with antibodies probing the indicated phospho-sites/proteins.
D. CFLAR FL promoter were co-transfected with p50 and Bclaf1 FL or its individual deletion mutant into HeLa-Bclaf1-KO cells. The experiments were performed in triplicate with a Renilla reporter in the transfection mixture for normalization. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant. One-way ANOVA test.
A. Commonly and exclusively quantified proteins in liver cell types (hHSC: hepatic stellate cell, hHEP: hepatocyte, hKC: Kupffer cell, hLSEC: liver sinusoidal endothelial cell).
IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM beraprost, 5 µg.ml-1 αIL-10R or isotype antibody. Data shown are representative of at least 3 independent experiments.
(E) TEM images of a single ommatidium from (i) wild type and (iii) dEsytKO photoreceptors of 0-1 day old flies reared in dark, scale bar: 1µm (red asterisk indicates R7 photoreceptor) (ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. Arrows indicate the SMC forming an MCS with the microvillar PM. (F) Quantification of the number of MCS per photoreceptor of wild type day 1 v/s dEsytKO day 1 old flies reared in dark, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor (i) n=30 photoreceptors from 3 separate flies (R1-R6) (ii) n=9 photoreceptors from 3 separate flies (R7).
ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ monomers (G) refers to the soluble fraction after Tris-buffered saline extraction
(E) U2OS (EGFP-BIR-5085) cells expressing PIF1 WT or L319P mutant with endogenous PIF1 silenced by shRNA were infected by lentiviruses to express I-SceI. The percentage of EGFP positive cells was quantified by FACS analysis 5 days after I-SceI infection. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.
A bar graph showing the functional annotation enrichment analysis by DAVID biostatistical plate form of the 54 commonly downregulated protein identified the indicated pathways, among them the ribosomal biogenesis pathway showed the highest enrichment. P-values are indicated in white.
C Quantitative real time PCR for PPAR-α and PPAR-α target genes, using RNA from epididymalfat from 10-week-old male WT and FADD-D mice fed a SD. Data are expressed as mean ± SEM from two independent experiments (n = 6 total for each genotype). *P = 0.0022, **P = 0.0083, ***P = 0.0107, #P = 0.0011, ##P = 0.0037 (Student's t-test).
(e) NRK cells grown on the SAMCell were transfected with FAM-RNAi by reverse transfection and the transfection efficiency was monitored by microscopy. Left panel, FAM-RNAi-positive cells; right panel, differential interference contrast (DIC) image of the cells in the left panel.
(e) We isolated RNAs from SBMA and control NPC lines (three different NPC lines per human subject) and then measured the expression levels of TFEB target genes including CYCS (cytochrome c), t(4) = 2.844; MCOLN1, t(4) = 3.246; GLA, t(4) = 3.174; ACADM (acyl-CoA dehydrogenase), t(4) = 4.405; COX6A1 (cytochrome oxidase 6A1), t(4) = 5.584; and the TFEB gene itself, t(4) = 0.593. Significant expression reductions were noted in SBMA NPC lines for all tested TFEB target genes. *P 0.05, **P 0.01. Data in d,e are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.
A. Bio-layer interferometry analysis of netrin-2L/receptor interactions. Other members of the netrin family were also tested: netrin-1, -4 and -G2. (red: netrin-2L; bleu: netrin-1; green netrin-4)
I Effect of high glucose on the expression of podocyte-related markers in primary cultured podocytes isolated from wild-type or KDM6A-KO mice. The primary podocytes isolated from wild-type or KDM6A-KO mice were cultured in normal or high glucose (30 mM) for 48 hours. Protein lysates from the cultured podocytes were subjected to Western blot analysis with the indicated antibodies. *P < 0.05 versus wild-type podocytes in normal glucose, #P < 0.05 versus wild-type podocytes in high glucose (Parametric ANOVA and a Bonferroni post hoc test; n = 3).
(F) Mrp17 targeting and translocation model that requires Tom20-binding motifs and positive charges for targeting (STEP 1) and relies on ubiquitous positive charges for translocation (STEP 2).
C. Boxplots showing the mean mRNA expression of ENAH gene (hMENA) for 22 patients in the 10 groups of cell types (Peng et al., 2019) (total number of cells: 38487, mean of cells for patient: 1603). Shown in each boxplot are the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line). Expressions from each cell-type group were compared to all other groups by using Mann-Whitney U-test (two-sided) and P-values were adjusted for multiple testing using the Benjamini-Hochberg method. Stromal cell-type groups with significantly up-regulated ENAH expression respect to other stromal groups are: Fibroblast, ***q=0.0002; Stellate, ***q= 1.5e-07. Non-stromal cell-type groups with significantly up-regulated ENAH expression with respect to other non-stromal groups are: Ductal 1, **q=0.0013.
(B) Survival curves of recipient mice are shown (n = 9-10 mice per group). Log-rank test was used to generate p values, **** p <0.0001.
(C) The top panels show representative images of GFP-transfected acceptor cells after 24 h co-culture with donor cells containing α-synucleinfibrilsATTO-550. Immunofluorescence was performed with endo-lysosomal organelle markers (top panel: EEA1, middle panel: Vamp3 and Lamp1: bottom panel) and revealed that α-synucleinfibrils co-localized with lysosomal vesicles (see arrow); in red: α-synucleinfibrils, in green: organelle markers and in blue: nuclei. Scale bar represents 10 μm, arrows pinpoint assemblies co-localizing with organelle markers. The bar graph at the bottom shows the quantification of the percentage of co-localization of α-synucleinfibrils with organelle markers, revealing that α-synuclein puncta co-localized mostly with lysosomal vesicles. Data show mean ± s.e.m. from three independent experiments.
B Lipofectamine-coated latex beads damage lysosomes and induce their K48-ubiquitination. Cells expressing mCherry-Gal3 were incubated with Lipofectamine-coated latex beads for 3 h and stained with K48 chain-specific antibodies.
G LNCaP cells stably expressing wild-type STAMP2 were treated with or without DPI (1 μM) for 4 h, harvested, and used in the NADPH/NADP+ assay. *P = 0.018.
(A) Representative frame of a 16 hour time-lapse microscopy experiment; GFP+ MM cells (green) were co-cultured with mouse bone marrow derived macrophages (gray) in the presence of 200 nM BoxA. Arrows indicate GFP+ MM cells engulfed by macrophages. This experiment was repeated twice.
(F) RNAi of all tested genes in the TOR-PI3K pathways had a statistically significant effect on LTG fluorescence levels. Known negative regulators of autophagy are shown with gray bars; positive regulators are shown with white bars. (Pten, P = 0.007; Tsc1, P = 0.027; Tsc2, P = 0.025; RheB, P = 0.005; Tor, P = 0.016; and S6k, P = 0.012). dsRNA corresponding to a human gene (Hs) was used as a negative control in E and F. Results represent the mean value ± SD from at least three independent experiments.
% of ventricle wall thickness (%WTC) in 24 month-old mice treated or not with navitoclax. Data are mean±S.E.M of n=6 mice per treatment group.
D. Quantification of area under curve for insulin tolerance test in C. Data represent mean ± SEM, Welch's t-test.
(E) GST pull-down assay of in-vitro translated and radiolabelled myc-tagged cGAS, RIG-I, and TLR3 with GST or GST-tagged IRGM.
Representative immunoblots of ISR component eIF2α and quantification of phospho-eIF2α (p-eIF2aSer51) relative protein expression in quadriceps skeletal muscle (WT n=3, TG n=4).
Alteration of the glomerular ultrastructure in TfebiEC-/- mice at p17 (mice n=5). Representative transmission electron micrographs of renal tissue. Magnification: i) x6000, ii) x20000), iii-iv) x10000, v-vi) x25000; scale bars: 1µm). Asterisks indicate the accumulation of extracellular matrix in mesangium; white and black triangles indicate the fusion of podocyte foot processes and the lack of endothelial fenestrae, respectively.
CD31 and CD34 double positive endothelial cells were isolated from wild-type or EVL-/- P5 retinas by FACS and analyzed by RNA-sequencing. Two independent experiments; in total 18 retinas from WT mice and 18 retinas from EVL-/- mice from three different litters, each.
(A) Processing of L-PGAM5 to S-PGAM5 in depolarized mitochondria of wild type (WT), Slp2-/-, Yme11-/-, and Parl-/- MEFs. PGAM5 processing was monitored by immunoblotting at the indicated time points after inhibition of cytosolic protein synthesis with cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown. L-PGAM5 levels at t=0 was set to 100%. Two-way ANOVA analysis (n=3; *, p<0.05, **, p<0.01. ****, p<0.0001). Arrowheads denote intermediate PGAM5 cleavage products.
(C). Tsix RNA levels in female mESCs infected with shRNA directed against Luciferase (Control, black) and KAP1 (Kap1 KD, grey), during differentiation. Mean ±standard deviation from four independent experiments are shown. Statistical significance was determined using 2-way ANOVA. (**p ≤ 0.01). Values have first been normalised to a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha.
EVs were purified by SEC from supernatants of Jurkat cells. (C,D) Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with 2 x 109 particles. Captured EVs were detected with either APC-labeled anti-CD81, anti-CD63 or anti-CD3E. (C) Median APC fluorescence values for the different bead populations is shown as the ratio to the median APC fluorescence of control beads (log10 scale). shown. (D) Heat-map representation of the median APC fluorescence values for the different bead populations detected with anti-CD63 or anti-CD3E antibodies relative to the values detected with anti-CD81.
D and E) Images of lobules II and X taken from C1.4 at 70 d. GFP is green and Calbindin staining is red. In lobule II, the number of PCs is clearly reduced and of the five remaining PCs, four are wild-type (GFP-positive). In lobule X (where no degeneration occurs during the normal npc1−/− life span), PC density is normal and the majority of PCs (23 of 32; 72%) are mutant, as expected based on npc1−/− contribution to this mouse (67%).
Foci -deficient cells display poor traction forces in their synapse. WT or WASP-/- T cells were incubated on polyacrylamide substrates covalently functionalized with anti-CD3 and ICAM1, and traction force measurements were carried out as described in 'Methods'. The images in the right show traction force maps without (left panels) or with (right panels) force vectors. p value, **P<0.005 as determined by using Mann-Whitney two-tailed test between populations of cells within the same experiment, n = 9 and 11.
E) Schematic representations of xanthine and the methylxanthines used in this study.
Kaplan-Meier curves for overall survival (OS) in lung adenocarcinoma TCGA_LUAD (RASSF1 mRNA high/low cutoff FKPM 5.85) and squamous cell carcinoma patients TCGA_LUSC (RASSF1 mRNA high/low cutoff FKPM 6.52). Significance derived from log-rank test.
E. Co-IP experiments examining protein interactions between ATG8a with VISP1 or VISP1mUIM. N. benthamiana leaves were agroinfiltrated and treated with 2 mM 3-MA at 48 hpi, and then for IP with anti-Flag beads 12 h later.
(j) Immunoblot of beclin 1 in WT and CftrF508del mice. Representative of seven mice for each group.
G Western blot analysis of protein extracts from artificial muscles (PF) and host TA (Ctrl) reveals remarkable expression of muscle- and vessel-specific proteins (n = 5).
E Expression of CD69, an activation marker, following CRISPR editing with indicated gRNA combinations or the non-targeting (negative) control (NTC) - data shown for ATTO-550 positive (RNP-containing) and negative cells (n=6; paired t-test, one-tailed). Inset flow cytometry plot depicting representative gating of ATTO-550 positive and negative cells.
D Immunostaining of FOXP3 in Traf6fl/flCD4Cre+ and wild type (Traf6fl/flCD4Cre-) derived suspensions of lymph node and spleen cells. (blue: DAPI, green: FOXP3, Scale bars: 50 μm).
(G) Peroxisomal membrane protein (PMP) 34-FusionRed also co-localized with MITOL-GFP in Parkin-expressing HeLa cells after 3 hours of CCCP treatment. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. (H) Correlation statistics for the localization of MITOL-GFP and PMP34-FusionRed. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.
Endogenous levels of free cholesterol revealed by filipin staining of (C) PS-DKO cells under the indicated treatments. Graphs show the filipin fluorescence intensity as a percentage over VEH.
(F) HeLa CCL2 cells were co-transfected with HA-Ect2 and either wild type Flag-β-catenin (+WT), Flag-β-catenin S60A mutant (+S60A), or Flag-β-catenin S60D mutant (+S60D) and then treated with 200 ng/mL nocodazole for 18 h and released by incubation in fresh medium for 2 h (telophase). The resulting cell lysates were used for immunoprecipitation analysis with anti-HA antibody. Immunoprecipitated beads were separated by 10 % SDS-PAGE followed by immunoblot analysis with the indicated antibodies.
(A) qPCR levels of collagen in PSCs, normalized to RPLP0 and relative to control
O, P FISH of β-catenin in mandible slices infected with RUNX2 shRNA lentivirus (knockdown) or scrambled shRNA lentivirus (scramsh) via RNAscope. Data information: Scale bars = 50 µm.
(E) Whole Exome Sequencing (WES) and targeted Sanger sequencing identified compound heterozygous variants in OTULIN at position c.258 G>A, p.M86I on the paternal allele and at position c.500 G>C, p.W167S on the maternal allele.
(B) Non-targeted metabolome reveals 71 singinifcatly altered metabolites in young wild-type versus young +/atpcl mutants, while 439 metabolites are altered in the midlife groups out of 1,658 metabolite compounds. N=6 per group.