EMBO/sd-character-level-ner huggingface dataset:
Centriole loss in NSCs is accompanied with changes in neurite growth cone morphologyA.Figure 3A. Correlative plot of changes in protein abundance between control neurons and neurons treated with Centrinone-B (day7/day3). Pearson correlation R=0.5716, p<0.001. Specific, significantly changing growth cone proteins highlighted in red. B. Protein abundance profile over time for axon-related proteins Trim46 and Tau in control neurons and neurons treated with Centrinone-B (+C). C. Protein abundance profile over time for growth cone-related proteins Basp1, Gap43 and Marcks in control neurons and neurons treated with Centrinone-B (+C). D. Representative images of fan-like growth cones at day 5 of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions. Growth cone visualized by immunostaining for phalloidin. Scale bar = 5µm. E. Quantifications of the average area (µm²) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. n = 25-83 growth cones in 3 independent experiments. F Data information: Data represent mean ± SEM. One-way ANOVA including Tukey's post-hoc analysis *** p<0.001, ** p<0.005, * p < 0.05, ns p≥0.05 F. Representative images of different growth cone morphological categories: fan-like, torpedo-like and bulb-like. Scale bar = 5µm. G. Quantifications of the ratios of different subtypes (fan-like, torpedo-like, bulb-like) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. H. Quantifications of the average area (µm²) of different subtypes (fan-like, torpedo-like, bulb-like) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. n = 5-55 growth cones in 3 independent experiments. Da Data information: Chi-square-test including post-hoc analysis with Bonferroni correction (G), One-way ANOVA including Sidak's post-hoc analysis (H). *** p<0.001, ** p<0.005, * p < 0.05, ns p≥0.05
Figure 5The levels of PIF4-Myc are negatively correlated with the levels of KIN10-GFP. Total proteins were extracted from mesophyll protoplasts transfected with different amounts of KIN10-GFP and PIF4-Myc and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. Ponceau S staining is shown for equal loading. KIN10 reduces the stability of PIF4 through 26S proteasome-mediated degradation. Transfected mesophyll protoplasts were pre-treated with or without 20 µM MG132 for 4 hours and then incubated in the presence of 100 µM cycloheximide (CHX) for the indicated time. Total protein was extracted from the protoplasts, and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. Ponceau S staining is shown for equal protein loading. Representative western blots are shown in (B). KIN10 reduces the stability of PIF4 through 26S proteasome-mediated degradation. Transfected mesophyll protoplasts were pre-treated with or without 20 µM MG132 for 4 hours and then incubated in the presence of 100 µM cycloheximide (CHX) for the indicated time. Total protein was extracted from the protoplasts, and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. Quantification of relative PIF4-Myc levels is shown in (C). PIF4-Myc levels are presented as values relative to those at 0 h. In (C), different letters above each point indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n = 3). Error bars indicate s.d. (n = 3). Western blotting showing endogenous PIF4 protein levels in the seedlings grown Total protein extracted from the seedlings was analyzed by immunoblotting with anti-PIF4 antibody. Ponceau S staining shows equal protein loading. Western blotting showing endogenous PIF4 protein levels in the seedlings grown Total protein extracted from the seedlings was analyzed by immunoblotting with anti-PIF4 antibody. Ponceau S staining shows equal protein loading.
Figure 5(A) Western blot analysis of the protein levels of FTO, HIF1A, UCP1 and PPARGC1A in differentiated beige adipocytes from Ftoflox/flox mice infected with retroviruses MSCVhygro expressing Vec, Cre or together with lentiviruses pCDHpuro expressing wild-type (WT)- or mutant (MUT)-FTO. (B) HPLC-QgQ-MS/MS quantification of the m6A/A in mRNA of siCtrl or siFto 3T3-L1 cells. FTO knockdown efficiency was measured by Western blot. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001. (C) Distribution of m6A peaks in Hif1a mRNA transcript in siCtrl and siFto 3T3-L1 cells. Blue boxes represent exons, and blue lines represent introns. The m6A peaks were in the dashed red box. (D) Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis of m6A levels of Hif1a mRNA in siCtrl and siFto 3T3-L1 cells. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001. (E) Western blot analysis of FTO in input and HA-RIP groups. (F) RIP-qPCR analysis of the association of Hif1a with HA in 3T3-L1 cells overexpressing HA-tagged Vec or FTO. Enrichment of Hif1a with HA was measured by qPCR and normalized to input. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001. (G) Synonymous mutation sites in Hif1a. (H) Western blot analysis of the protein levels of HIF1A, UCP1, PPARGC1A, PPARG and GFP in 3T3-L1 cells infected with lentiviruses pCDHpuro expressing Vec, WT- or MUT-HIF1A.
The recovery of histone acetylation levels at later time points after UV is dependent on NER.Figure 3(A) Representative western blots of WCE from NER-proficient HeLa cells and NER-deficient XP4PA (XP-C) and XP2OS (XP-A) cells obtained at indicated time points after UV irradiation (16 J/m2) and stained with the indicated antibodies (B) Quantification of histone acetylation levels of NER-proficient cells (HeLa) and NER-deficient (XP-A and XP-C) patient cells at the indicated time points after UV-irradiation (16 J/m2). Histone acetylation levels are normalized against histone H4 levels. Average of at least 5 independent experiments and error bars represent SEM. Significant differences (T-test) are indicated with * (p<0.1), ** (p<0.05) and *** (p<0.01)
Figure 3(A) Steady state levels of proteolytic substrates of YME1L. Whole cell extracts of Yme1l-/-, Parl-/-, and Slp2-/- MEFs were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. (B) PGAM5 processing depends on proteolytically active PARL. PARL and catalytic inactive PARLS277A harboring C-terminal FLAG-epitopes were expressed under the control of a tetracycline (tet)-inducible promoter in wild type (WT) and PARL-/- FITR293T cells as indicated. Processing of L-PGAM5 to S-PGAM5 was monitored by immunoblotting. (C) Accelerated processing of L-PGAM5 in Slp2-/- cells. Processing of L-PGAM5 was analyzed by immunoblotting in wild type (WT), Yme1l-/-, Parl-/- and Slp2-/-MEFs after inhibition of cytosolic protein synthesis by cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown in the lower panel. Two-way ANOVA analysis (n=3; ****, p<0.0001). (D) The accelerated processing of L-PGAM5 in Slp2-/- cells is mediated by PARL. PARL was depleted from Slp2-/- cells by RNAi prior to CHX treatment. A quantification of L-PGAM5 levels at different time points is shown. Two-way ANOVA analysis (n=3; **, p<0.01. ****, p<0.0001). (E) PGAM5 associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells expressing PARL-FLAG (PARL-WT) or PARLS277A-FLAG (PARL-S277A) were solubilized in digitonin were analyzed by BN-PAGE and immunoblotting using FLAG- and PGAM5-specific antibodies.
Figure 1A Double-plotted representative actograms of the indicated genotypes. Flies are monitored in LD for 4 days and then DD for 7 days. Dicer2 (dcr2) is co-expressed to enhance the effects of RNAi. White boxes indicate the light phase and black boxes indicate the dark phase. Gray shades indicate DD. G4, GAL4; U, UAS. B-D The period of DD locomotor rhythms of flies with Nipped-A knocked down and controls. Data information: (B-H) Error bars represent standard error of the mean (SEM). Digits on the bars are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using One-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###P<0.001, * compared with the GAL4 controls; # compared with the UAS controls. White bar indicates UAS or GAL4 controls. Gray bar indicates flies with Nipped-A knocked down. G4, GAL4; U, UAS. E, F Using temperature-sensitive GAL80 system to control Nipped-A RNAi expression. (E) The period of DD locomotor rhythms of flies raised at 18ºC and tested at 29ºC. (F) The period of DD locomotor rhythms of flies raised at 29ºC and tested at 18ºC. Data information: (B-H) Error bars represent standard error of the mean (SEM). Digits on the bars are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using One-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###P<0.001, * compared with the GAL4 controls; # compared with the UAS controls. White bar indicates UAS or GAL4 controls. Gray bar indicates flies with Nipped-A knocked down. G4, GAL4; U, UAS. G, H The period of DD locomotor rhythms of flies treated with RU486 to activate the pdfG4-geneswitch (pdf-GS) driver (G) or vehicle control (H). Data information: (B-H) Error bars represent standard error of the mean (SEM). Digits on the bars are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using One-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###P<0.001, * compared with the GAL4 controls; # compared with the UAS controls. White bar indicates UAS or GAL4 controls. Gray bar indicates flies with Nipped-A knocked down. G4, GAL4; U, UAS.
Newly identified NMD factors.Figure 2RNAi was induced with an empty vector as a negative control (panel I), whereas a smg-2 clone (panel II) was used as a positive control. Panels i and ii show brightfield images of the PTCxi strain treated with the negative and positive controls, respectively. Depletion of five genes (panels III to VII) resulted in increased GFP expression. Panels iii to vii show brightfield images of the phenotypes of the affected worms. The scale bars correspond to 100 μm. Depletion of the novel NMD genes in C. elegans leads to upregulation of the PTCx NMD reporter mRNA, which was monitored by quantitative RT-PCR relative to the expression of ama-1 reference gene. The values shown are the average fold-change (mean ± SEM) from at least three independent experiments relative to empty vector-depleted worms. Statistical analysis was performed using the Mann-Whitney U-test for non-parametric distributions. *P < 0.05.
Figure 9Averaged protein stability was calculated from the data for total mutant protein levels and mutant C. elegans heat shock paralysis (Fig S2, 3 and 8). Averaged mutant Munc18-1 function was obtained from the data for MEA plate assay, antibody uptake, and synaptopHluorin release (Fig S4 and Fig 5-7). See Materials and Methods for details.
a) Time-lapse microscopy.f2(a) Time-lapse microscopy. HeLa H2B-mCherry-α-tubulin-EGFP cells were cultured in the presence and absence of etoposide and cisplatin, respectively. Treated cells frequently developed multiple nuclei, including micronuclei (upper panel), and demonstrated evidence of multipolar spindles and misaligned chromosomes (lower panel). Cells were also much larger than untreated cells (see also Supplementary Fig. S3). Scale bar, 10 μm. (b) Statistical analysis of the data shown above. At least 100 cells were counted for each drug exposure time according to the classification used in Fig. 1. Values are means±s.d. (n=3).
Figure 6A. HeLa-Flag-Bclaf1 and Bclaf1 WT HeLa cells treated with or without TNF (10 ng/ml) for 2 hours were separated into the cytoplasmic and the nuclear fraction. Endogenous Flag-Bclaf1 complex in each fraction was immunoprecipitated with M2 beads and analyzed by western blotting. B. HeLa-Flag-Bclaf1 cells transfected with siCtrl or sip65 were treated with TNF (10 ng/ml) for 2 hours. The nuclear fraction was isolated and subjected to immunoprecipitation with an anti-Flag or control IgG antibody, followed by western blotting analysis. C. 293T cells transfected with the indicated plasmids were immunoprecipitated with the anti-Flag M2 beads followed by western blotting analysis. A diagram of full-length Bclaf1 and its deletion mutants are presented. 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.
KZFPs IFs and interactome A.Figure 1IF by confocal microscopy performed on KRAB-HA overexpressing cells. Staining was performed with anti-HA (Alexa-488, green), anti-HSPA5 (Alexa-647, red) and DNA was stained with Dapi (blue). Force-directed network representing detected interactions. Each bait KZFP is represented by a blue circle ("node") and linked to green nodes that represent its preys. Specific preys, such as KAP1 is represented in red and its associated proteins SIRT1, SMARCAD1 and HP1α, HP1γ in orange. All interactions represented are below the false discovery rate of 1%. The topology of the network is established by a force-directed process that follows certain rules: all nodes repel each other and are attracted to the center by artificial "gravity" and nodes with links attract each other. Weighted links between any nodes are based on the average fold change over controls. Zoom-in on KAP1 core of the interactome. D&E. Zoom-in on KZFPs enriched with unique interactors. F. Zoom-in on the subset of KZFPs not connected to the main interactome.
Figure 1FACS analysis of the E3: T-GR line and scheme for the evaluation of reversion efficiency. Reversion of E3 EpiSCs from GFP-positive (left) and -negative EpiSCs (right). Macroscopic view of AP-stained dishes on day 5 (top). Quantification of reversion efficiency based on the number of AP-positive colony (middle) and GFP-positive cell (bottom). Top left: Scheme for the chimera contribution assay. Bottom left: Representative brightfield/mCherry images from chimeras at E7.5. Scale bar: 100um. Right: Percentage of mCherry-positive cells in chimeric embryos measured by FACS. Blue circles and red circles represent the percentage of mCherry-positive cells derived from control and T-induced PSCs (GOF-positive E3 EpiSCs [E3G], reverted E3 EpiSCs [rE3], and 2iLIF ESCs), respectively. Western blot of the indicated proteins in T-inducible PSCs stimulated as indicated for 24 hr. GAPDH was used as a loading control.
Figure 7A. Both phosphorylated pRb and pRb∆K7 do not co-precipitate with E2F1. Protein lysates from Rb∆K7 or control wildtype thymocytes were immunoprecipitated with antibodies for pRb, for pSer243/Thr246-pRb or for pSer605-pRb, followed by immunoblotting for pRb or E2F1. Anti-pRb antibody efficiently co-immunoprecipitated E2F1 in control and Rb∆K7 cells (lanes 2-3, 11), whereas the two phospho-pRb antibodies failed to co-immunoprecipitate E2F1 in either cell (lanes 7-10). B. Left, top, effect of retrovirus-mediated transduction of under-phosphorylated Rb∆K11 (rRb∆K11) on Rb-deficient mammary tumor cell proliferation as determined by MTT assay (n=4-5 technical replicates). Left, bottom, quantification of SA-βGAL in rRb∆K11-transduced cells. Bars represent mean ±SD; P value calculated by two-tailed unpaired student's t-test. Right, representative images of SA-βGAL staining showing robust senescence (arrows) in rRb∆K11-transduced cells. Original magnification: 200x. C. Schematic diagram illustrating how unphosphorylatable Rb∆K11 allele or control wildtype Rb DNA was introduced in vivo (top) or ex vivo (bottom) into retinal progenitors. i.v.c., in vitro culture. D. Suppression of retinal progenitor division in vivo (grey bars) and ex vivo (black bars) by Rb∆K11 and p16 but not by wildtype pRb. Shown are quantification of BrdU+;GFP+ cells relative to all GFP+ cells in samples transfected with the indicated vectors (bars represent mean ±SD; ** denotes p<0.01; *** p<0.001 by one-way ANOVA and Tukey's multiple comparison analysis, n=3 biological replicates). E. Representative images illustrating the effect of indicated plasmids. NBL: neuroblastic layer, GCL: ganglion cell layer; scale bar: 10 μm. Arrows point to yellow cells that are Green+ and BrdU+. F. Vitamin C reduces cellular senescence induced by retroviral-mediated over-expression of Rb∆K11. Top - images of SA-βGAL staining in human diploid cells, IMR90, transduced with control or rRb∆K11 virus with or without vitamin C added at the time of infection. Arrows point to SA-βGAL-positive cells. Bottom left, quantification of SA-βGAL staining (bars represent mean ±SEM; n=4 biological replicates). P values by one-way ANOVA, Tukey's multiple comparisons test. Bottom right, dot-blot analysis with antibody for 5hmC on DNA extracted from IMR90 cells treated or not with vitamin C. Original magnification: 200x. G. A schematic model for the induction of DDR, senescence and diabetes in Rb∆K7 mice and the effect of vitamin C diet. Normal pancreatic β-cells can re-enter the cell cycle in response to mitogenic signals. In contrast, Rb∆K7 β-cells display a repressive chromatin and stable differentiation state, which, in response to mitogenic signals, elicits a DDR, cellular senescence and SASP, leading to gradual islet degeneration. We propose that the epigenetic modifier, vitamin C, prevents the establishment of a stable repressive state during differentiation of Rb∆K7 β-cells, thus reduces DDR and senescence in response to mitogens, facilitating cell cycle re-entry/regeneration and alleviating diabetes in Rb∆K7 mice. SC denotes senescent cell.
Figure 5A. Immunofluorescence imaging of endogenous ABL1 in the panel of NSCLC cell lines; scale bars=20 μm. B and C Quantification of nuclear versus cytoplasmic localization of (B) endogenous ABL1 in NSCLC cell lines and (C) tetracycline-inducible expression of ABL1-WT, ABL1-R351W and ABL1-G340L in Beas-2B cells. Error bars represent ±S.D., n=6-10 images, *P<0.005 by two-tailed unpaired Student's t-test (Beas-2B vs H1915 P=6.6×10-5, vs H2110 P=0.0001; WT vs R351W P=0.003, vs G340L P=0.002).
Figure 1A, B In-gel kinase assay of OST1 activity in ABA receptor mutants under cold stress. Ten-day-old wild type, ost1-3, pyr1 pyl1,4, pyr1 pyl1,2,4, pyr1 pyl4,5,8 and pyr1 pyl1,4,5,8 mutants were treated at 4°C for 2 h. Total protein extracts were prepared and separated on SDS-PAGE gel containing 0.1 mg/mL GST-∆ABF2 as a substrate, and incubated with 60 μCi of [γ-32P]ATP. Representative pictures are shown in (A) and relative kinase activity is shown in (B). Data information: In (A , Top, gel autoradiograph; bottom, Coomassie Brilliant Blue (CBB) staining of RuBisCO large subunit was used as a loading control. The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In (B , each bar represents the mean ± SEM of three biological repeats. *P < 0.05 (two-tailed t-test). C, D In-gel kinase assay of OST1 activity in ABI1-OE14 and abi1 abi2 hab1 mutants under cold stress. Representative pictures are shown in (C) and relative kinase activity is shown in (D). Data information: In C), Top, gel autoradiograph; bottom, Coomassie Brilliant Blue (CBB) staining of RuBisCO large subunit was used as a loading control. The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In D), each bar represents the mean ± SEM of three biological repeats. *P < 0.05 (two-tailed t-test).
Cultured slices expressing hTauAT show a progressive increase in epileptiform activity and mossy fiber sprouting which can be prevented by CEF application.Figure 7A. Example of electrophysiological fEPSP recording in stratum pyramidale of area CA3 in a control slice (DIV30) during wash-in of 50 µm picrotoxin. This leads to blockage of GABAergic transmission followed by epileptiform discharges (burst) occurring approximately after 3 min. The zoom-in reveals that such an epileptiform discharge (burst) consists of repetitive firings.B. Similar example trace as in (A) recorded in a hTauAT expressing slice (red) demonstrating an increase in burst frequency as well as in firings per burst due to hTauAT expression.C. Quantification of bursts per minute in DIV 10 slices from control littermates and hTauAT slices demonstrating a ~2-fold increase in hTauAT slices (n=8 slices per group).D. Firings per burst are increased ~1.5-fold already at the early time point DIV10 due to hTauAT expression.E. Analysis of burst frequency in slices at DIV 30 demonstrates the progression of the pathological effect beyond that observed at DIV 10. Burst frequency is enhanced in hTauAT expressing slices (n = 10 slices). If Ceftriaxone (CEF) is applied to hTauAT slices the pathological burst frequency enhancement is partly prevented (n = 5 slices).F. Firings per burst are strongly enhanced due to hTauAT expression in slices at DIV 30. Ceftriaxone prevents this pathological phenotype if applied during the entire period of cultivation (n = 6 slices). G. Example of a control littermate organotypic slice culture at DIV 24 stained against NeuN (red) and pan-Tau (K9JA antibody, green). Axonal projections (green) of granule cells (red) in the dentate gyrus are depicted.H. Hippocampal slice culture expressing hTauAT stained against NeuN (red) and pan-Tau (green) showing mossy fiber sprouting in the hilar region, CA3 and dentate gyrus (white arrows).
miR-155 expression is induced after mycobacterial infection.Figure 1(A) miR-155 expression levels were examined in the lungs of normal uninfected or H37Rv infected BALB/c mice 6 weeks postinfection (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. (C and D) RAW264.7 cells were infected with BCG at an MOI of 5 for the indicated time points (C) or at indicated MOIs for 24 h (D). The expression levels of miR-155 were examined by real-time PCR. (E and F) RAW264.7 cells were infected with H37Ra at an MOI of 5 for the indicated time (E) or at indicated MOI for 24 h (F). The expression levels of miR-155 were examined by real-time PCR. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.