EMBO/sd-character-level-ner huggingface dataset:
Figure 4 (A) Scheme of the genetic strategy to delete YAP and TAZ and activate the oncogene SmoM2 in tumor initiating cell (B) Immunostaining for SmoM2 and β4-integrin at different time points upon tamoxifen administration in SmoM2 YAP/TAZ DKO and SmoM2 mice (n=3 animals analysed per genotype and time point (C) Immunostaining for SmoM2 and β4-integrin in rare clones observed at 2 and 8 weeks after tamoxifen administration in Smo2 YAP/TAZ DKO mic (D) Immunohistochemistry for YAP and TAZ in rare dysplasias observed in SmoM2 YAP/TAZ DKO mice 8 weeks after tamoxifen administratio (E) Tunel assay in SmoM2 YAP/TAZ DKO and SmoM2 mice 3.5 days after tamoxifen administration. Arrows indicate tunel-positive cells. Hoechst nuclear staining in blue; Scale bars: 20 μm
The C-terminal membrane anchor determines the localization of Bax and Bak.Figure 4A The influence of the C-terminal membrane anchor (MA) on the differential localization and function of Bax and Bak has been analyzed using the wild-type proteins of Bax (blue) and Bak (green) and MA substitutions of both proteins resulting in the chimeras BaxTBak and BakTBax. B Quantification of HCT116 Bax/Bak DKO cells expressing Bax, BaxTBak, BakTBax or Bak with the expressed protein being present largely cytosolic (white bars), in a mixed distribution between cytosol and mitochondria (grey bars) or largely mitochondrial (black bars). Data represent averages ± SEM from 7 independent experiments with n ≥ 100 cells. C Confocal images of HCT116 Bax/Bak DKO cells expressing Bax, BaxTBak, BakTBax or Bak. The GFP/YFP fluorescence of the expressed protein variants is depicted in the top panels and in green in the merged image on the bottom. The mitochondria were stained by MitoTracker far red depicted in red in the merged images (bottom row). Scale 10 µm. n ≥ 5. D Western blot analysis of Bax, BaxTBak, Bak and BakTBax localization expressed in HCT116 Bax/Bak DKO cells. Cytosol (C) and heavy membrane fraction (HM) of HCT116 Bax/Bak DKO cells are displayed. GAPDH and VDAC serve as fractionation controls. n = 3.
Figure 5Schematic representation of the experimental procedure to obtain the proteomes of wild-type and Fam134s knockout MEFs by mass spectrometry (MS). Principal component analysis (PCA) of analyzed proteomes. Scatter plot showing proteins responsible for driving sample segregation along component 1 and component 2. Proteins driving segregation are significantly enriched (FDR<0.05) for GO cellular component (GOCC) terms associated with endoplasmic reticulum (ER) (blue dots). Unsupervised clustering of ANOVA-significant proteins by GOCC terms. Inset showing the profile plot and the GO-term enrichment (FDR<0.05) of 197 proteins belonging to the highlighted cluster. Physical interaction network of proteins significantly upregulated in Fam134 knockout MEFs. The network was generated using the STRING plugin in the Cytoscape environment with a confidence score set to 0.4. Nodes are protein entities while lines represent physical interactions Red, orange, green: Collagen, stress fiber, ER associated. Insets of (E) showing the Collagen and the ER cluster, respectively. Profile plots showing the Log2 LFQ intensity (G) and representative immunofluorescence (H) of Climp63 (green) in wild-type and Fam134 knockout MEFs. Profile plots showing the Log2 LFQ intensity (I) and representative immunofluorescence (J) for collagen I (red) expression in wild-type and Fam134 knockout MEFs. Scale bar: 10μm. Inset scale bar: 5μm. Nuclei were stained with Hoechst 33342.
Figure 5Phase contrast pictures of ES cells of indicated genotypes after 5 days in neuroectoderm differentiation conditions (N2B27). White arrowheads in the Mbd3∆ panel indicate pockets of ES-like cells. Scale bars represent 100 µm. Expression of indicated genes in the neuroectoderm differentiation time course was measured by RT-qPCR in Control (green) Mta123∆ (magenta) or Mbd3∆ (blue) ES cells. N ≥ 3 biological replicates, error bars indicate SEM. Phase contrast images of embryoid body outgrowths of indicated genotypes after 12 days in differentiation conditions (N2B27 + 10% serum). Scale bars represent 100µm. Expression of indicated genes in embryoid body cultures over time was measured by RT-qPCR Comparison of expression data for wild type, Mbd3∆ or Mta123∆ ES cells in self-renewing (2i) conditions or after 48 hours in differentiation conditions (48h N2B27) with mouse embryonic single cell RNA-seq data Larger shading encloses biological replicates, smaller circles represent individual cells.
Astroglial Stat3 modulates network imbalance in APP/PS1 mice (A) For in vivo two-photon imaging, astrocytes (arrows) and neurons (arrowheads) were labeled with the calcium indicator OGB-1, and astrocytes were co-labeled with sulforhodamine 101 (SR101; arrows).Figure 6For in vivo two-photon imaging, astrocytes (arrows) and neurons (arrowheads) were labeled with the calcium indicator OGB-1, and astrocytes were co-labeled with sulforhodamine 101 (SR101; arrows). Aβ plaques were labeled with the intravital dye methoxy-XO4 (open arrowheads). Scale bar, 50 µm. Calcium imaging of anesthetized animals showed that the hyperactivity of astrocytes in APP/PS1-Stat3KO mice was reduced to levels comparable to WT-Stat3WT mice, but significantly increased in APP/PS1-Stat3WT mice (One-Way ANOVA followed by Bonferroni's multiple comparison test; n = 6 mice (3 female and 3 male) for each group; age, 8 months). Similarly, neuronal activity was also reduced to levels comparable to WT-Stat3WT mice in APP/PS1-Stat3KO mice, but significantly increased in APP/PS1-Stat3WT mice (One-Way ANOVA followed by Bonferroni's multiple comparison test; same mice as in B). The cumulative distributions of neuronal calcium transients in APP/PS1-Stat3KO mice were not different from those of WT-Stat3WT mice (p = 0.31, Kolmogorov-Smirnov test), but significantly different from those of APP/PS1-Stat3WT mice (p = 0.001, Kolmogorov-Smirnov test). To investigate a reciprocal scenario in which astroglial hyperactivity reduces Stat3 activation, we implanted osmotic minipumps into APP/PS1 mice and treated them with the network-normalizing P2Y1R inhibitor MRS2179 or vehicle for 6 weeks. Two-photon imaging of these mice confirmed that astrocytic hyperactivity were reduced by MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test). Two-photon imaging of these mice confirmed that propagating calcium waves were reduced by MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test). MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test). This network normalization induced a reduction of activated Stat3 (pStat3) in astrocytes assessed in fixed brain sections of the same mice (Mann-Whitney test). Scale bars, 50 µm.
Viral genomes from RNA-seq data and titration of coverage(a) The coverage plot across the SARS-CoV-2 genomes (viral coordinate on bottom, colored by sample) from a representative set of clinical positive samples.Figure 3:coverage(a) The coverage plot across the SARS-CoV-2 genomes (viral coordinate on bottom, colored by sample) from a representative set of clinical positive samples. Sample names with the suffixes CN and N are clinical negative (buffer), P are qRT-PCR positive, and CP are Vero 6 cells with virus. (b) Downsampling (right annotation) of the samples and mapping to the SARS-CoV-2 genome to gauge the percent coverage (y-axis) as a function of the viral quantification by qRT-PCR (Ct thresholds, low <20, medium 20-30, and high>30). (c) Average coverage statistics for the low, medium, and high Ct samples, as well as the mean coverage for each of these samples. (d) The cycle threshold (x-axis) vs. the coverage of the genome (y-axis and color depth) for the total RNA-seq.
LL37 and IRGM gene expression is upregulated following NOD2 activation (A‐D).Figure 3LL37 and IRGM gene expression is upregulated following NOD2 activation (A‐D). Alveolar macrophages were incubated in the presence of 10 μg/mL of MDP or 100 ng/mL of LPS for 24 h. Upregulation of (A) LL37 and (D) IRGM gene expression was assessed after specific ligand recognition by quantitative PCR using the Taqman system and the ΔΔCT method for relative quantification. The fold change in gene expression relative to unstimulated cells is depicted. Data are representative of two independent experiments (n = 7-12). Bold lines indicate median values. (B, E) LL37 and IRGM protein levels were measured in cytosolic fractions by western blot analysis. (C, F) Data are expressed as the protein fold increases relative to unstimulated cells and normalized to tubulin calculated by densitometry and are shown as means ± SEM. One representative experiment out of three experiments is presented.
Figure 5HeLa and CCNF K/O cells were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. Ponceau S staining was used as a loading control. U-2-OS cells transfected with non-targeting siRNA (siNC) or with an siRNA targeting Cyc F were lysed using SDS. Whole cell lysates were analyzed by immunoblotting 48h after transfection. H3 was used as a loading control. Heatmap showing gene set variance analysis (GSVA) score for gene sets related to E2F targets (PID_E2F_PATHWAY, ISHIDA_E2F_TARGETS and HALLMARK_E2F_TARGETS). HEK293T cells transfected with empty vector (EV), FLAG-cyclin F WT, FLAG-cyclin F (∆F) and FLAG-cyclin F M309A were treated with MLN4924 for 4 hours and harvested for immunoprecipitation using FLAG antibodies. Immunoprecipitates were immunoblotted as indicated. HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 in the presence of FLAG-cyclin F WT, FLAG-cyclin F (∆F) and FLAG-cyclin F M309A were indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted. HeLa and CCNF K/O cells treated with 10 μM Chk1i (LY2603618) for the indicated time points (h=hours) were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. H2B served as a loading control. HEK293 cells transfected with empty vector (EV) or HA-E2F1 were treated with Chk1i for 20 hours and harvested for immunoprecipitation using HA antibodies. Immunoprecipitates were immunoblotted as indicated. Quantification of immunoprecipitation results of Figure 5G from three independent experiments. Data from 3 independent experiments were plotted with Mean % ± SD. *< 0.05 p-value and calculated by two-tailed paired t-test. HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 were treated with Chk1i for 20 hours and siRNA of cyclin F were indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.
Figure 6A. Stable expression of Myc-tagged wild-type, PIP-box mutant, and catalytic inactive HUWE1 variants in HUWE1-knockout 293T cells. The Western blot shows that expression of the Myc-tagged variants stably transfected in the knockout cell lines, is similar to the endogenous HUWE1 expression. B. Myc-tag immunoprecipitation using extracts of the corrected cell lines described above. As expected, PCNA co-precipitates with wild-type, but not PIP-box mutant HUWE1 (FF). C. iPOND experiment using HUWE1-knockout 293T cells, corrected with wild-type or PIP-mutant HUWE1. The PIP-mutant shows reduced crosslinking to nascent DNA, indicated defective recruitment to replication forks. D. Clonogenic assay showing that stable expression of wild-type HUWE1, but not of the PIP box mutant or the catalytic mutant, corrects the HU sensitivity of the HUWE1-knockout 293T cells. Shown is the average of 3 independent experiments -/+SD. If not indicated otherwise, the p-values shown specify the statistical significance relative to 293T (for the 0.5mM HU condition). P-values are: 0.0003 (293T vs HUWE1-/-; 0.157 (293T vs HUWE1-/- +WT); 0.0045 (293T vs HUWE1-/- +FF); 0.0006 (293T vs HUWE1-/- +C4341A; 0.047 (HUWE1-/- +WT vs HUWE1-/- +FF). E. The S-phase arrest phenotype is also rescued by wild-type, but not PIP-mutant or catalytic mutant HUWE1. S-phase arrested cells were quantified using the BrdU/PI bi-dimensional flow cytometry assay. Shown is the average of 3 independent experiments -/+SD. The p-values shown specify the statistical significance relative to 293T (0.018, 0.957, 0.015, and 0.024 respectively). A quantification of the same data, showing the percentage of cells arrested in S-phase, is shown in Fig EV3A. F. Normal replication tract length, quantified using the DNA fiber assay, is restored by expressing wild-type, but not PIP-mutant HUWE1, in the HUWE1-knockout 293T cells. The "n" numbers of fibers analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. If not indicated otherwise, the p-values shown specify the statistical significance relative to 293T. P-values are: 1.47x10-11 (293T vs HUWE-/- samples), 0.094 (293T vs HUWE-/- +WT samples), 1.40x10-7 (293T vs HUWE-/- +FF samples), 1.22x10-7 (HUWE-/- vs HUWE-/- +WT samples), 0.679 (HUWE-/- vs HUWE-/- +FF samples). G. Alkaline Comet Assay showing that wild-type, but not the FF mutant can correct the breakage phenotype of HUWE1-knockout 293T cells. The "n" numbers of comet tails analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. The p-values indicated are: 4.18x10-43 (293T vs HUWE-/- samples), 6.89x10-41 (HUWE1-/- +WT vs FF samples), and 5.97x10-50 (HUWE1-/- +WT vs C4341A samples).
Figure 1Scheme of SIRF assay. Nascent DNA was pulse-labelled with EdU. Upon fork stalling, proteins located at stalled forks are in close proximity to EdU. EdU was then biotinylated using click reaction, followed by incubating with biotin and target protein antibodies. PLA amplification was used to visualize the localization of target proteins at EdU-labeled stalled forks. B, C. Detection of CST at stalled and active forks with SIRF assay in U2OS (B) and HCT116 (C) cells. To detect CST at stalled forks, cells were pulse-labeled with EdU for 8 min, followed by 4 mM HU treatment for 3 h. To detect CST at active forks, SIRF was performed without HU treatment. Representative SIRF images of CTC1, STN1 and PCNA on normal or stalled forks are shown. Scale bars: 10 µm. Relative SIRF fluorescence intensity was calculated by normalizing to untreated samples and then to their respective EdU-SIRF signals. Mean value in each sample is labeled in the graph. N: the number of cells analyzed in each sample.
a) Immunoblot analysis of AIM2, ASC and LC3-II in lysates of differentiated THP-1 cells left untransfected or transfected with poly(dA:dT) (1.figf4(a) Immunoblot analysis of AIM2, ASC and LC3-II in lysates of differentiated THP-1 cells left untransfected or transfected with poly(dA:dT) (1.5 μg/ml) and left untreated or treated with 3-MA, rapamycin or amino-acid starvation, then separated into an inflammasome-enriched fraction (top) or an autophagosome-enriched fraction (middle). Bottom, immunoblot analysis of total cell lysates. (b) Immunoblot analysis of unprocessed and processed caspase-1 and IL-1β in lysates and supernatants of differentiated THP-1 cells treated as in a. (c) Immunoblot analysis of LC3-I and LC3-II (top) in lysates of mouse BMDMs primed with LPS (20 ng/ml) and left untreated or treated with alum, then left unexposed or exposed for 5 h to live (Live TB) or dead (Dead TB) M. tuberculosis (numbers below lanes as in Fig. 1d). Below, immunoblot analysis of IL-1β in supernatants of LPS-primed mouse BMDMs infected overnight with live M. tuberculosis in the presence or absence of 3-MA (2 mM); treatment with LPS and ATP serves as a positive control. Data are representative of at least two experiments.
Figure 6SUNCR1 protein expression in the gastrocnemius from sedentary or post-running mice (n=4). The mRNA level of SUNCR1 in gastrocnemius and soleus (n=7-8). [Ca2+]i in C2C12 cells treated with 0 or 2 mM SUC (n=18-20). NFAT protein expression in nucleus and cytoplasm of gastrocnemius 0.5 h or 3 h after i. p. injection of 15 mg/kg succinate in C57BL/6J mice (n=4). SUNCR1 protein expression in C2C12 cells transfected with vector or siSUNCR1 (n=3). [Ca2+]i, in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC (n=5-6). enzymes activity (n=9-10) of (J) HK, (K) LDH, and (L) SDH in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC (n=5-6). Representative images and quantification of MyHC-I and IIb immunofluorescent staining (green) in C2C12 cells (n=3). Scale bar in (M) represents 50 μm.
Fig.Figure 1A. Schematic of Ago2 primary sequence with domains colored as in figures throughout manuscript. B. Schematic of crystalized miRNA and target sequences. Vertical lines indicate base pairs observed in structure. miRNA colored red; target RNA colored dark blue (here and throughout manuscript). C. Front and top views of Ago2 bound to seed plus supplementary-paired target RNA. D. miRNA-target duplex with miRNA nucleotides numbered from 5' end (Ago2 removed for clarity). Dashed line indicates disordered nucleotides in bridge region. E. Close-up of seed-paired region of miRNA-target duplex. Fo-Fc electron density map calculated after omitting nucleotides around the seed region (miRNA and target at positions 1-9), contoured at 3.0 sigma, shown as blue mesh. F. Close-up of supplementary-paired region of miRNA-target duplex. Fo-Fc electron density map calculated after omitting nucleotides in the supplementary region (miRNA and target at positions 12-16), contoured at 2.0 sigma, shown as blue mesh.
Figure 2A-C. mtDNA replication in wt (A) and mdi1 ovarioles (B, C) as illustrated by EdU incorporation. Arrows point to mitochondrial DNA, arrowheads to nuclei. In wt ovaries (A), mtDNA replication starts at germarium stage 2B and lasts into egg-chamber stage 2 (S2). In most mdi1 ovarioles (B), mtDNA replication is undetectable at these stages, whereas in some (C) it appears delayed until germarium stage 3. Bars, 10 m. D. Hatching rates and mtDNA content of eggs laid by females of different mutant genotypes relative to eggs laid by wt controls. Each data point represents the mean of 3 independent replicates. Expression of mdi or hAKAP1 in mdi1 significantly restored the mtDNA level and hatching rate. N= 3 x >100 eggs / genotype for hatching rate. The relative mtDNA level was determined as the average of 3 biological repeats. P values of comparing mdi1; nos> mdi to mdi1: hatching rate, P= 1.1995E-05; mtDNA, P= 0.0078. P values of comparing mdi1; nos> hAKAP1 to mdi1: hatching rate, P= 1.6998E-05; mtDNA level, P= 0.0009.
Sept2 interacts with Drp1.Figure 4(A) Western blot analysis of endogenous Sept2 immunoprecipitation from HeLa cells showing that endogenous Drp1 specifically co-immunoprecipitates with Sept2. The asterisks mark the IgG heavy chains. (B) Elyra SIM image of U2OS cells stained for Sept2 (green), mitochondria (GFP targeted to the mitochondrial outer membrane (OM-GFP), shown in red), Drp1 (blue). Yellow arrows point at tetraspeck beads used for image alignment. Insets represent sevenfold enlargements. White arrows in the insets point to sites where Drp1 and Sept2 colocalize on mitochondria. Scale bar: 10 µm, insets are enlarged fourfold. (C) GST pulldown of recombinant Sept2 with GST-Drp1 or GST alone showing that Sept2 is able to interact directly with Drp1. (D) Sept2 immunoprecipitation from mock or CCCP-treated HeLa cells probed for Drp1 and Sept2. (E) Quantification of two independent experiments showing stimulated Drp1 co-immunoprecipitation with Sept2 in FCCP-treated cells. (F) Sept2 immunoprecipitation from mock or Mdivi-1-treated HeLa cells probed for Drp1 and Sept2. (G) Quantification of two independent experiments showing decreased Drp1 co-immunoprecipitation with Sept2 upon Mdivi-1 treatment. (H) Snapshots of a live cell imaging experiment in U2OS cells showing Sept2 (green, Sept2-YFP), Drp1 (red, Drp1-mCherry) and mitochondria (blue, mitotracker DeepRed) during mitochondrial fission. Merged and single channels show the dynamics of Sept2 and Drp1 during mitochondrial fission (yellow arrowheads). Scale bar: 2 µm.
Fig.Figure 5A-F) The indicated yeast strains were transformed with expression plasmids for FYVE2-GFP and their vacuolar membranes were labeled with FM4-64. FRAP was performed using a spinning disk microscope equipped with an emission beam splitter and two synchronized cameras, allowing to simultaneously analyze FM4-64 and GFP fluorescence. The histograms show the kinetics of signal recovery for (A) wildtype (WT), (B) vam3tsf, (C) ypt7T22N, (D) vma16F190Y, (E) vps33P184L, and (F) nyv1∆ cells. Scale bar: 2 µm. Arrows indicate the bleaching area. Means and s.d. are shown for 20 stained vacuoles from 3 independent experiments.
Transfer of α-syn fibrils between neurons occurs through TNT-like protrusions and it can be regulated by the Wnt/Ca2+ pathway.Figure 8Representative confocal images of 1 DIV co-cultured donor (D, loaded with Alexa-568 α-syn fibrils) and acceptor (A, labeled with CTG) neurons. After 20 h post-plating, neurons were treated with 200 ng/mL Wnt5a for 4 h. The insets (right panels) show a magnification of the area depicted in the expanded field (left). Yellow arrows point to α-syn puncta inside acceptor neurons. Percentage of acceptor neurons containing α-syn puncta for each of the indicated treatments. Time lapses of neurons loaded with Alexa-568 α-syn fibrils and cultured in control conditions or treated with 200 ng/mL Wnt5a. Neuron-to-neuron transfer of α-syn puncta (yellow arrows) through a TNT-like structure (yellow arrowheads) is shown upon Wnt5a treatment. Representative confocal images of 1 DIV co-cultured donor (D, loaded with Alexa-568 α-syn fibrils) and acceptor (A, labeled with CTG) neurons. Neurons were obtained from WT or βCaMKII K.O. mice. Yellow arrows point to α-syn puncta inside acceptor neurons in both conditions.
Targeting STAMP2 in preclinical models of PCa results in tumor regression.Figure 10A LNCaP cells were implanted subcutaneously into nu/nu mice. Once tumors reached 5 mm in size, mice (n = 5 per group) were given nanoliposome-encapsulated control siRNA or STAMP2 siRNA as described in Materials and Methods. Tumor volumes were measured at the indicated time points. *P = 0.001. B Same experiment as in (A), but VCaP cells were used instead. *P = 0.001. C A model for STAMP2 effects on growth and survival of PCa cells. Once activated, AR increases STAMP2 expression. STAMP2 catalyzes reduction of Fe3+ to Fe2+, also depleting NADPH levels, which increases ROS. Increased ROS then activate ATF4 expression which in turn activates target genes that adapt PCa cells to oxidative stress resulting in cell proliferation and survival.
Precursor API is trapped in a prevacuolar compartment in vps18 cells.Figure 2Precursor API is trapped in a prevacuolar compartment in vps18 cells. (A) Yeast strain JSR18Δ1 with plasmid pJSR9 (vps18 ts) was grown at the permissive temperature of 26°C. Before labeling, the cells were incubated at either 26° or 38°C for 10 min. Pulse labeling was for 5 min, followed by the indicated chase times. (B) vps18 spheroplasts were shifted to 38°C for 5 min, pulse labeled for 5 min, and then chased for either 0 or 60 min. At each time point the spheroplasts were collected by centrifugation and resuspended in PS0 buffer. The Total (T) fraction either received no treatment, was treated with 50 μg/ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100. The supernatant (S0) and pellet (P0) fractions were collected after centrifugation at 10,000 g for 5 min.