EMBO/sd-character-level-ner huggingface dataset:
Neurons derived from Mecp2 null NPCs are morphologically more immature than wt controls and establish less active networks A,B: Wt or Mecp2 null NPCs plated on the grid of 64 MEA electrodes.Figure 2A,B: Wt or Mecp2 null NPCs plated on the grid of 64 MEA electrodes. Scale bars: 50 µm. C-G: Line graphs represent the changes through time of 5 parameters describing neuronal activity. Difference between genotypes and through time were analysed using Two-way ANOVA for repeated measures and Bonferroni post-hoc test; * refers to differences between genotypes (*: p-value<0.05; **: p-value<0.01; ****: p-value<0.0001); # refers to differences between the two time points (#: p-value<0.05; ##: p-value<0.01). Sample size: n=8 wells deriving from three independent preparations. Values are represented as mean ± SEM. H-K: Raster plots (H and I) and their quantitation under basal conditions and upon 100µM 4-AP exposure. The magnitude of the response after stimulation is expressed either as fold change (J) or as the averaged maximal values reached after 4-AP exposure (K). Dotted line in J represents untreated controls (set to 1). Values are represented as mean ± SEM. Student's t-test, *: p-value<0.05. Sample size n=8 wells deriving from three independent preparations. L: Wt and null neurons counterstained with an antibody against Tuj1 and DAPI at different time points. The binary mask is representative of a neuron used to study morphology. Scale bars: 30 µm. M-Q: Each graph represents a different morphological parameter analysed at selected time point. Two-way ANOVA indicated both a significant time effect ((F 4,20)=282,5 and p<0,0001 for the number of intersections; (F 4,26)=104,5 and p<0,0001 for the total dendritic length; (F 4,20)=58,51 and p<0,0001 for the maximal dendritic length; (F 3,22)=38,32 and p<0,0001 for polarization) and genotype effect ((F 1,20)=25,1 and p<0,0001 for the number of intersections; (F 4,26)=16,0 and p=0.005 for the total dendritic length; (F 1,20)=17,24 and p=0,005 for the maximal dendritic length; (F 1,22)=18,76 and p<0.003 for polarization). Bonferroni multiple comparison test was used to assess difference between wt and null neurons at each analysed time point. The dotted line in panel P represents the cut-off above which a culture is defined polarized (Horton et al, 2006). Values are represented as mean ± SEM; *: p-value<0.05; **: p-value<0.01; ****: p-value<0.0001. Sample size: n≥3 wells deriving from three independent preparations; for each well we measured at least 15 cells.
Figure 1A. Experimental scheme of Cripto expression analysis in macrophage (MP) subpopulations from wild-type skeletal muscles at different time points after Cardiotoxin (CTX) injection. B. Representative flow cytometry dot plots of CD11b+ and CD11b+/Cripto+ cells at day 2, 3 and 5 after injury. PE (Phycoerythrin/free channel). Data are mean±SEM (n=5 biological replicates; P≤0.00008, Student's t-test). C. Representative flow cytometry dot plots of CD11b+/F4/80+/Ly6CHigh/Low and CD11b+/Cripto+/F4/80+/Ly6CHigh/Low cells at day 2, 3 and 5 after injury. D. Quantification of F4/80+/Ly6CHigh/Low cells in the CD11b+ and CD11b+/Cripto+ cell population from injured muscles at the indicated time points. Data represent mean±SEM (n=5 biological replicates; ***P<0.001, Student's t-test).
Enhanced LC3‐II levels in the liver and kidney of bi‐1‐deficient mice.Figure 7(A) The levels of LC3, Atg5/Atg12 complex, Beclin‐1, Bcl‐2, Bcl‐XL, IRE1α and Hsp90 were monitored in liver protein extracts of bi‐1+/+ and bi‐1−/− at 6‐month‐old mice. Each well represents independent mice. (B) A similar analysis was performed for indicated markers in kidney protein extracts as described in liver. (C) bi‐1+/+ and bi‐1−/− mice were injected with 50 ng/ml of tunicamycin by intraperitoneal injection (N=3) and then LC3 levels were monitored in liver protein extracts by western blot analysis. Right panel: quantification of relative LC3‐II levels, **P0.005.
Figure 2Figure 2a. Experimental outline. AS CDK12 HCT116 cells were arrested by serum starvation for 72 h and released into the serum-containing medium with or without 3-MB-PP1. DNA content was analyzed by flow cytometry at indicated time points after the release.  Figure 2b. CDK12 kinase activity is needed for G1/S progression in cells arrested by serum starvation . Flow cytometry profiles of control (-3-MB-PP1) or inhibitor (+3-MB-PP1) treated cells from the experiment depicted in Fig. 2a. The red arrow points to the onset of the G1/S progression defect in 3-MB-PP1-treated cells. To better visualize the G1/S delay in the presence of the inhibitor the 24 h time point is also shown. n=3 replicates; representative result is shown.Figure 2d. CDK12 protein levels peak in the G0/G1 phase of the cell cycle. Western blots show levels of proteins at indicated time points after the release of serum starved AS CDK12 HCT116 cells. Corresponding cell cycle phases are depicted above time points. A representative Western blot from 3 replicates is shown. Figure 2e. Experimental outline. AS CDK12 HCT116 cells were arrested by serum starvation for 72 h and released into the serum-containing medium. 3-MB-PP1 was either added or not at indicated time points after the release. Propidium iodide- or BrdU-stained DNA content was measured by flow cytometry at 16 h after the release. Note, that for the BrdU staining the 3-MB-PP1 was added only at the time of the release (0 h) and 3, 4, 5 and 6 h after the release. Figure 2f, g. Inhibition of CDK12 in early G1 perturbs normal cell cycle progression. Quantification of cells (%) in cell cycle phases from flow cytometry profiles of propidium iodide (f) and BrdU (g) labeled cells upon addition of 3-MB-PP1 at indicated time points after serum addition in the experiment depicted in Fig. 2e. CTRL in Fig. 2g = control sample without 3-MB-PP1. n=3 replicates, representative result is shown. Figure 2h. Short-term CDK12 inhibition does not activate DNA damage checkpoints. Western blot analyses of phosphorylation of depicted DNA damage response markers upon inhibition of CDK12 for indicated times. CPT corresponds to 5 µM camptothecin. A representative Western blot from 3 replicates is shown. FUS is a loading control.
miR-155-induced autophagy promotes the formation of mycobacterial autophagosomes.Figure 6(A) RAW264.7 cells stably expressing GFP-LC3 were transiently transfected with control or miR-155 mimic and then infected with Texas Red-labeled BCG for 1 h. The co-localization of BCG with LC3 was detected by confocal microscopy. (B) Quantification of the co-localization of BCG with LC3-positive autophagosomes is shown. (C) RAW264.7 cells were transiently transfected with control or miR-155 mimic, and then infected with Texas Red-labeled BCG for 1 h. Endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (Green). The co-localization of BCG with endogenous LC3 was detected by confocal microscopy. (D) Quantification of the co-localization of BCG with LC3-positive autophagosomes is shown. (E) After transient transfection with control or miR-155 mimic, RAW264.7 cells were infected with Texas Red-labeled BCG for 1 h, and then were labeled with a specific fluorescent dye MDC (50 µM) for autophagic vacuoles. The co-localization of BCG with MDC-positive autophagic vacuoles was detected by confocal microscopy. (F) Quantification of the co-localization of BCG with MDC-positive autophagosomes is shown. Cells treated with rapamycin were used as a positive control. Arrows indicate the co-localization of BCG with autophagosomes; scale bar = 5 µm. Data are shown as the mean ± SEM of three independent experiments (n = 100 phagosomes). **, p<0.01; ***, p<0.001.
Depletion of lamin A/C expression causes impaired ciliogenesis in RPE cells.Figure 3A RPE cells were infected with lentiviruses encoding shRNAs specific to lamin A/C (shLamin A/C) or luciferase (shLuc). Exogenous lamin A was re-expressed in the lamin A/C-depleted cells (shLamin A/C-Lamin A). An equal amount of whole cell lysates was analyzed by immunoblotting with antibodies as indicated. B The cells were serum-starved for 48 h and stained for acetylated tubulin (green), γ-tubulin (red) and DNA (blue). The depletion of lamin A/C (shLamin A/C) led to impaired ciliogenesis, as manifested by no cilia (inset 1) or short cilia (inset 2). Scale bars, 20 μm or 2 μm (insets). C The percentage of the cells with cilia in the total counted cells (n ≥ 626) was measured. D The length of cilia was measured (n ≥ 87 in each group). E Cells were stained for acetylated tubulin (green) and γ-tubulin (red) after various intervals of serum starvation. The percentage of cilia-positive cells at each time point (n ≥ 372) was measured.
a–d) IB3-1 cells transfected for 24 h at 37 °C with 1 μg of pGFP–CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM human p62 siRNA or scrambled oligonucleotides.figf6(a-d) IB3-1 cells transfected for 24 h at 37 °C with 1 μg of pGFP-CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM human p62 siRNA or scrambled oligonucleotides. (a) Left: CFTR revealed with anti-GFP primary antibody in IB3-1 cell lysate. Top arrow, complex-glycosylated form (apparent Mr of band C about 170K, revealed as 210K with GFP tag); bottom arrow, core-glycosylated form (apparent Mr of band B about 150K, revealed as 190K with GFP tag). Uncropped images of blots are shown in Supplementary Information, Fig. S10. Right: quantification of bands B (black) and C (white) normalized to β-actin levels. Values are means ± s.d. for three independent experiments. (a-d) IB3-1 cells transfected for 24 h at 37 °C with 1 μg of pGFP-CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM humanp62 siRNA or scrambled oligonucleotides.(b) Cells immunostained with an anti-GFP antibody under non-permeabilizing conditions. Serial confocal sections were collected from top to bottom of cell. (c) Side view (X-Z) of a z-stack of confocal images taken in b. (c) Side view (X-Z) of a z-stack of confocal images taken in b. (a-d) IB3-1 cells transfected for 24 h at 37 °C with 1 μg of pGFP-CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM humanp62 siRNA or scrambled oligonucleotides.(d) Surface biotinylation assays was performed with membrane-impermeable sulpho-NHS-LC-biotin. The cells were fractioned to obtain the plasma membrane fraction, and biotinylated proteins were precipitated with streptavidin beads. Immunoblot of GFP to reveal the C form. E-cadherin and β-actin were used as positive and negative controls, respectively. (e) IB3-1 cells transfected with either HA-tagged human beclin 1 or empty vector. Immunoblot of PIASy, TG2, phospho-p42/44 and PPAR-γ proteins. (f) IB3-1 cells transfected with either 50 nM human p62 siRNA or scrambled oligonucleotides. Immunoblot of p62 and phospho-p42/44. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (g) IB3-1 cells transfected with either 50 nM human beclin 1 siRNA or scrambled oligonucleotides in the presence or absence of cystamine. Immunoblot of phospho-p42/44. (h) IB3-1 cells transfected with either 50 nM humanbeclin 1 siRNA or 50 nM humanATG5 siRNA or scrambled oligonucleotides in the presence or absence of cystamine. TNF-α secretion. Each bar represents the mean ± s.d. for n = 3 experiments. Asterisk, P 0.01; ANOVA.
Figure 4A. ISG activation and cytokine secretion in Rnaseh2b-/- MEFs is markedly impaired by cGAS or STING siRNA depletion. Upper left and lower panels: RT-qPCR of IFIT1, cGAS and STING transcripts, 48 h after siRNA targeting luciferase (control), cGAS or STING. Upper right panel: CCL5 is significantly reduced in culture supernatants 48 h after cGAS or STING depletion. Concentration of CCL5 (ELISA), normalised to luciferase siRNA control levels in each experiment. Mean from three independent experiments using one Rnaseh2b-/- MEF line; error bars, SEM. * = p<0.05, ** = p<0.01, two-tailed t-test for RT-qPCR, one sample t-test for CCL5ELISA. B-D. ISG induction and cytokine secretion is abolished in Rnaseh2b-/- cGAS-/- MEFs. cGAS was targeted by CRISPR/Cas9 genome editing of a Rnaseh2b-/- MEF line to inactivate cGAS/STING signalling. In addition to sequence validation, functional inactivation of cGAS was confirmed in Rnaseh2b-/- cGas-/- CRISPR lines by the absence of CCL5 secretion in response to dsDNA (Appendix Fig S4). CCL5 (B) and CXCL10 (C) production, as well as ISG expression (D) was abrogated in Rnaseh2b-/- cGAS-/- clones, assessed by ELISA and RT-qPCR respectively. Four independent experiments, n=2 Rnaseh2b-/- cGAS-/- clones, n=4 Rnaseh2b-/- cGAS+/+ clones, error bars, SEM of each experiment * = p<0.05, ** = p<0.01, *** = p<0.001, two-tailed t-test. B-D. ISG induction and cytokine secretion is abolished in Rnaseh2b-/- cGAS-/- MEFs. cGAS was targeted by CRISPR/Cas9 genome editing of a Rnaseh2b-/- MEF line to inactivate cGAS/STING signalling. In addition to sequence validation, functional inactivation of cGAS was confirmed in Rnaseh2b-/- cGas-/- CRISPR lines by the absence of CCL5 secretion in response to dsDNA (Appendix Fig S4). CCL5 (B) and CXCL10 (C) production, as well as ISG expression (D) was abrogated in Rnaseh2b-/- cGAS-/- clones, assessed by ELISA and RT-qPCR respectively. Four independent experiments, n=2 Rnaseh2b-/- cGAS-/- clones, n=4 Rnaseh2b-/- cGAS+/+ clones, error bars, SEM of each experiment * = p<0.05, ** = p<0.01, *** = p<0.001, two-tailed t-test. E. ISG induction in Rnaseh2bA174T/A174T mice is STING dependent. RT-qPCR of RNA extracted from hearts from Sting+/+ Rnaseh2bA174T/A174T (n=4) and Sting-/-Rnaseh2bA174T/A174T (n=6) three-month old mice. F. Absence of STING does not significantly decrease basal ISG expression. RT-qPCR of RNA extracted from hearts from Sting+/+ (n=3) and Sting-/- (n=5) three-month old mice. (E, F) Each data point represents the mean of technical triplicates from one mouse. Error bars, SEM. * = p<0.05, Mann-Whitney U test.
FBXO7 interacts with the proteasomal subunit PSMA2 and binds to the proteasome.Figure 3A. Lysates of HEK293T cells, transfected with the indicated plasmids, were subjected to immunoprecipitation (IP) with the FLAG antibody (PSMA2), followed by immunoblotting (IB) with the myc antibody (FBXO7). B. Recombinant PSMA2 together with gluthathione bead-coupled GST-FBXO7 or GST only were incubated in Co-IP buffer. Precipitated proteins were subjected to immunoblotting with the PSMA2 antibody. 2% input was used as loading control for PSMA2. GST-coupled proteins were visualized by Coomassie staining. C. Schematic depicts FBXO7 full length and FBXO7 deletion mutants used in mapping analyses. UbRD = ubiquitin-related domain, FP = FBXO7/PI31 domain, F-box = F-box domain, PRR = proline rich region. D. Indicated FBXO7 expression plasmids were used to transfect HEK293T cells together with the FLAG-PSMA2 plasmid. Lysates were then subjected to IP with the myc antibody followed by IB with the FLAG antibody. E. HEK293T cells were transfected with myc-FBXO7 full length or ΔUbRD and subjected to fractionation using a 10-40% linear glycerol gradient. Fractions were immunoblotted for myc (FBXO7), Rpt6 (26S/30S proteasome) and PSMB5 (20S proteasome). F. Cortical lysates were subjected to fractionation using a 10-40% linear glycerol gradient. Fractions were immunoblotted for FBXO7, Rpt6 (26S/30S proteasome) and PSMA2 (20S proteasome). G. GST or GST-Rad23 bound to glutathione Sepharose were used to precipitate proteasomes from brain lysates followed by IB with the FBXO7, PI31 or PSMA2 antibody. H. HEK293T control lysates and FLAG-FBXO7 cells were subjected to immunoprecipitation, followed by digestion. The resulting peptide mixtures were analyzed by MS; ratio FBXO7 versus control; mean of three independent measurements based on the intensity of the relevant protein. I. STRING analysis of the proteins listed in figure 3H (string-db.org). PSMA2, PSMA6, PSMB1, PSMB3, PSMB4, PSMB6 are members of the gene ontology term (GO) ´"proteasome core complex" (GO:0005839).
altFUS is involved in mitochondrial dysfunction and autophagy processes A, Representative traces of TMRE fluorescence measured by flow cytometry in mock transfected cells and cells overexpressing the bicistronic FUS-R495x or the monocistronic FUS(Ø)-R495x constructs (n=4, minimum of 50 000 live cells per independent replicates).Figure 3A, Representative traces of TMRE fluorescence measured by flow cytometry in mock transfected cells and cells overexpressing the bicistronic FUS-R495x or the monocistronic FUS(Ø)-R495x constructs (n=4, minimum of 50 000 live cells per independent replicates). Mean fluorescence intensity of mock transfected cells treated with a decoupling agent, FCCP, is indicated by a grey dotted line. B, Mean TMRE fluorescence intensity measures in mock transfected cells, cells over-expressing altFUS, FUS, FUS(Ø), FUS-R495x or FUS(Ø)-R495x, or mock transfected cells treated with FCCP across 4 independent experiments (n=4, mean ± SD). Statistical significance is relative to the mock condition unless otherwise indicated (*** = p value < 0.001, **** = p value < 0.0001, n.s. = non-significant, two-way ANOVA with Tukey's multiple comparison correction). C, Representative image by stimulated emission depletion microscopy (STED) of altFUS-Flag (green) localization within mitochondria (Tomm20 marker in red). The white bar across the mitochondria represents the region of interest quantified The white scale bar corresponds to 3μm. D, Relative fluorescence histogram for altFUS-Flag and Tomm20 across the region of interest highlighted by a white line on panel C. E, Scatter plot of the proteins identified by AP-MS indicating their enrichment (Fold-change over control) and their SAINT probability score. Proteins above the 0.8 threshold (grey line) are indicated in black, others in grey. AltFUS is indicated in red (bait) and preys known to regulate the autophagy or the cellular stress response are indicated in green. F, Subcellular localizations of proteins identified by AP-MS G, Enrichment of biological processes in altFUS interacting proteins compared to the Human mitochondrial proteome (Fisher's Exact test with FDR < 0.1 %). The number of proteins identified in each GO term is indicated next to the corresponding bar.
Figure 6A, B PPL-C99FL lacking a stop codon was in vitro translated in RRL in the presence of canine pancreatic microsomes or buffer control in reactions supplemented with ε-TDBA-Lys-tRNA analogue. Reactions were carried out in the presence (A) or absence (B) of 2 μM HisTrx-SGTA. Samples were subjected to UV light-induced photo-cross-linking and processed as described for Fig 5. "x SRP54" and "x HisTrx-SGTA" indicate cross-linking adducts between the translated nascent chain and endogenous SRP54 or recombinant HisTrx-SGTA, respectively, whilst "endo. SGTA" indicates cross-linking adducts with endogenous SGTA. Cross-linking products between stalled PPL-C99FL and SGTA are also indicated with arrowheads, whilst arrows indicate unmodified translation products. C, D chemical cross-linking using DSS reagent was carried out. E DSS-mediated HisTrx-SGTA cross-linking efficiency to PPL-C99FL in the absence or presence of ER-derived microsomes was quantified. For each repeat, intensity of the cross-linked SGTA adduct in the absence of microsomes was set as 100%. Shown is the mean with standard error of mean for n=3 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction.
Figure 3(A) Co-expressed FLAG-CAMDI and HDAC6-HA interacted with each other in SH-SY5Y cells. n=3 independent experiments. (B) Endogenous interaction of CAMDI with HDAC6 in the mousebrain. Co-IP assay was performed with the indicated antibodies using E16 mousebrain. n=3 independent experiments. (C, D) N-terminal region of CAMDI recognizes the second HDAC domain of HDAC6. SH-SY5Y cells were co-transfected with HDAC6-HA and each FLAG-CAMDI fragment (C) (fragment 1, 1-250; 2, 251-500; 3, 501-750; 4, 751-1000; 5, 1001-1451 amino acids) or co-transfected with FLAG-CAMDI and each HDAC6-HA fragment (D) (fragment 1 (DD1), 1-440; 2 (DD2), 441-840; 3, 841-1215 amino acids). Co-IP assay was performed with the indicated antibodies. n=3 independent experiments. **, p<0.01, ***, p<0.001, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM. (E) Schematic illustration of interaction between CAMDI and HDAC6.
PI3KC2β depletion disturbs endosomal trafficking with increase of VEE (APPL1+) and recycling (Rab11a+) endosomes.Figure 5A Quantification of internal transferrin in sh-Control or sh-PI3KC2β cells at different incubation time points (ImageJ software). Results are expressed as fold change of internalized Alexa Fluor®546-transferrin. Data represent mean ± SEM of cell fields randomly selected (dots n=13-20). Data are collected from 4 independent experiments. ***p<0.001 Mann-Whitney test. B Analysis of APPL1, EEA1, LAMP1 and Rab11a staining in sh-Control and sh-PI3KC2β hCMEC/D3 cells. White dotted lines delineate cells; Scale bar, 10µm; nuclei are shown in blue (DAPI). Quantification was performed to determine number of positive vesicles per cell (ImageJ software). Nuclei are shown in blue (DAPI). Data represent mean ± SEM of cell fields randomly selected (n=13-20 for (C) and n=11-20 for (D)). Data are collected from at least 3-4 independent experiments. For violin plots (D), thick lines represent median and thin lines interquartile; ***p<0.001; **p<0.01; *p<0.05, unpaired t-test or Mann-Whitney, as accordance. Quantification was performed to determine average size of positive vesicles per cell (ImageJ software). Nuclei are shown in blue (DAPI). Data represent mean ± SEM of cell fields randomly selected (n=13-20 for (C) and n=11-20 for (D)). Data are collected from at least 3-4 independent experiments. For violin plots (D), thick lines represent median and thin lines interquartile; ***p<0.001; **p<0.01; *p<0.05, unpaired t-test or Mann-Whitney, as accordance.
Svb repressor promotes enterocyte differentiationA Drawing of apical-basal organization of the intestinal epithelium and expression of svb mRNA as revealed by in situ hybridization.Figure 5Drawing of apical-basal organization of the intestinal epithelium and expression of svb mRNA as revealed by in situ hybridization. The inlet shows an enlarged view. esg-Gal4 midguts expressing mCherry and a svb::GFP rescue mini-gene, consisting of svb-cDNA tagged by GFP (green) and driven by E and 7 svb enhancers (see Fig 1). Samples were stained for GFP (green), mCherry (red) and Prospero (white). B' shows closeup views. The drawing at left schematizes the svb locus, with position of E3N and 9CJ2 enhancers. Close-up shows 9CJ2 sequence with binding sites for Pdm1 (orange). The right subpanels are posterior midguts showing expression of wild type 9CJ2 svb enhancer, and expression of 9CJ2-Pdm-mt in which Pdm1 binding sites have been mutated. esgts midguts expressing GFP alone (control), or expressing SvbREP. Samples were stained for GFP (green) and Prospero (red). Closeups correspond to boxed regions, with DAPI shown in purple and GFP-positive cells outlined in yellow. esgts-F/O midguts expressing GFP alone (control), or expressing SvbREP. Samples were stained for GFP (green) and cleaved DCP1 (red). E' shows quantification of the number of GFP-positive cells in esgts guts expressing GFP alone (ctrl), or DIAP, SvbREP, and SvbREP + DIAP. ReDDM lineage tracing in control midguts, or in midguts expressing SvbREP, and quantification of the percentage of progenitors (GFP-positive, RFP-positive) versus differentiated cells (GFP-negative, RFP-positive). esgts midguts expressing GFP alone (control), or expressing SvbREP. Samples were stained for GFP (green) and Pdm1 (red); arrows show enlarged GFP-positive cells which are also positive for Pdm1.