EMBO/sd-character-level-ner huggingface dataset:
Figure 3Figure 3. Synthetic lethality assay. The Prp5 domain structure, and the mutations in some of the domains (indicated) are schematized at the top. Each of these PRP5 genes (wild-type or mutants, plasmid-borne) was used to replace the chromosomal PRP5. The numbers in parentheses are the relative ATPase activities of the wild-type (SAT) Prp5 and some of the Prp5 mutants tested before (Xu Query, 2007). The U2 status, either containing Ψ42 and Ψ44 (WT-U2 and snR81Δ pus1Δ with pΨ42 or Ψ44) or lacking Ψ42 and Ψ44 (snR81Δ pus1Δ), is indicated (on the right). Upon transformation with the plasmids carrying PRP5 genes (wild-type or any of the mutants), cells were grown on the regular medium (-FOA) or on medium containing 5-FOA (+FOA).
a) Sequence alignment of N-CRD and C-CRD of GAL8.f4(a) Sequence alignment of N-CRD and C-CRD of GAL8. The residues important for interaction with the Galbi peptide and NAD are marked by yellow and magenta circles, respectively. (b) Detailed view of the peptide-binding site of C-CRD superposed with N-CRD. The residues for the Galbi peptide (cyan) interaction are labelled and the side chains of the corresponding N-CRD residues (magenta) are shown. (c) A detailed view of the NAD-binding site of N-CRD superposed with C-CRD. The residues for NAD (purple) interaction are labelled and the side chains of corresponding C-CRD residues (yellow) are shown.
Figure 5A. Overexpression of RNase H1 in Rnaseh2b-/- cells restores RNase H activity against RNA:DNA hybrids to 81 ± 10% of wildtype levels, while overexpression of RNase H2B restores cellular enzyme activity for cleavage of both RNA:DNA and DRD:DNA substrates (RER). Rnaseh2b-/- MEFs were complemented with Rnaseh1 (+H1), Rnaseh2b (+H2B) or EGFP by retroviral infection. Mean of n=3 independent experiments ± SEM. B, C. DNA damage is reduced to wildtype levels by complementation with Rnaseh2b but not Rnaseh1, measured by 53BP1 foci formation in detergent-extracted fixed cells. (B) Representative images (scale bar, 10 µm). (C) At least 150 cells were counted for each cell line in three independent experiments. Mean ± SEM, **** = p<0.0001 two-tailed t-test. B, C. DNA damage is reduced to wildtype levels by complementation with Rnaseh2b but not Rnaseh1, measured by 53BP1 foci formation in detergent-extracted fixed cells. (B) Representative images (scale bar, 10 µm). (C) At least 150 cells were counted for each cell line in three independent experiments. Mean ± SEM, **** = p<0.0001 two-tailed t-test. D-F. CCL5 (D) and CXCL10 production (E), as well as ISG induction (F) in Rnaseh2b-/- MEFs are reduced close to wildtype levels (Rnaseh2b+/+), by complementation with Rnaseh2b but not Rnaseh1. Mean of n=6 independent experiments ± SEM for complemented cells; n=3 independent experiments for Rnaseh2b-/- parental and Rnaseh2b+/+ controls cells. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001 two-tailed t-test indicates significantly reduced expression compared to Rnaseh2b-/- parental cells. D-F. CCL5 (D) and CXCL10 production (E), as well as ISG induction (F) in Rnaseh2b-/- MEFs are reduced close to wildtype levels (Rnaseh2b+/+), by complementation with Rnaseh2b but not Rnaseh1. Mean of n=6 independent experiments ± SEM for complemented cells; n=3 independent experiments for Rnaseh2b-/- parental and Rnaseh2b+/+ controls cells. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001 two-tailed t-test indicates significantly reduced expression compared to Rnaseh2b-/- parental cells.
Figure 3(A) Comparative expression by qPCR of Dux, two downstream targets of DUX (Zscan4 and Tdpoz4), and three targets of DPPA2 and DPPA4 independent of DUX (Mael, Tdrd1, Prex2) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DPPA2, DPPA4 or GFP as a control Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test. (B) Average coverage normalized for sequencing depth of the ChIP-seq signal of DPPA2, DPPA4 or DPPA4 truncated of the SAP domain overexpressed in mESCs in a window of 500bp around the Dux gene (n=2). Total input (TI) is shown in gray. Peaks over the Dux gene were called in DPPA2, DPPA4 and DPPA4ΔSAP ChIP-seq. (C) Heatmap showing the distribution of DUX, DPPA2, DPPA4 and DPPA4ΔSAP coverage in a ± 5kb window around the TSS of DUX-dependent, DPPA2 and DPPA4-dependent and random genes in mESCs. (D) Boxplot/Jitterplot representing the percentage of GC nucleotide contents in the peaks from DPPA2, DPPA4 and DUX ChIP, and in a random shuffle of peaks.
a) ATG4B depletion increases steady-state LC3-II levels.f7a) ATG4B depletion increases steady-state LC3-II levels. Immunoblots for indicated proteins in NIH/3T3 cells transfected with scrambled siRNAs (scr) or siRNAs against ATG4B (siATG4B) in presence/absence of EGF. Bars represent mean±s.e.m. *P0.05, ***P0.001 compared with scr; Student's t-test, n=4. (b) siATG4B cells exhibit increased autophagic vesicles. Representative electron micrographs depicting autophagic vesicles (indicated by black arrows) in scr and siATG4B NIH/3T3 cells. (c) ATG4B depletion does not increase autophagic flux in serum-fed cells. Immunoblots for indicated proteins in lysates from scr or siATG4B NIH/3T3 cells in presence/absence of lysosomal inhibitors, ammonium chloride and leupeptin (Inh) for 2 h. Scale bars, 1 μm. Bars represent mean±s.e.m., n=3. (d) siATG4B cells display increased nuclear LC3-II. Images for LC3-II (red) in scr or siATG4B NIH/3T3 cells in presence/absence of EGF (10 min). Scale bar, 5 μm. Bars represent mean±s.e.m. ***P0.001 compared with scr; Student's t-test, 60 cells from n=2. (e) ATG4B deficiency increases P-ERK/LC3-II colocalization. Immunofluorescence (IF) for P-ERK (green)/LC3 (red) colocalization in scr or siATG4B NIH/3T3 cells in presence/absence of EGF (10 min). Scale bars, 10 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student's t-test, 60 cells from n=2. (f) ATG4B-deficient cells display increased nuclear P-ERK/LC3 colocalization. IF depicting P-ERK (green)/LC3 (red) colocalization in nuclei of scr or siATG4B NIH/3T3 cells in presence/absence of EGF. Scale bars, 5 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student's t-test, 60 cells from n=2. (g) ATG4B deficiency augments EGF-induced MEK and ERK phosphorylation. Immunoblots for P- and total MEK and ERK, LC3 and GAPDH in total lysates from scr or siATG4B NIH/3T3 cells in presence/absence of EGF. Bars represent mean±s.e.m., n=3. h) ATG4B deficiency increases nuclear P-ERK levels. Native (top)/inverted images (bottom) showing nuclear P-ERK content. P-ERK fluorescence in untreated (Con)/EGF-treated scr or siATG4B NIH/3T3 cells is shown. Scale bars, 5 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student's t-test, 60 cells from n=2. Nuclei are blue (DAPI).
Heat shock suppresses AMPK and interferes with cellular responses to metabolic stressors.Figure 5A Reporter cells were heat-shocked at 43°C for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting. B Immediately following heat shock at 43°C for 40 min, AMPK complexes were immunoprecipitated from HEK293 cells and in vitro kinase assays were performed as described in Figure 3D using recombinant human ACC1 proteins as substrates. C Following heat shock at 43°C for 40 min, both detergent-soluble and detergent-insoluble fractions of cellular proteins were extracted from HEK293 cells as described in Materials and Methods for immunoblotting. D, E Immediately following heat shock at 43°C for 40 min, HEK293 cells were subjected to nutrient deprivations for 4 h. AMPKα Thr172 and ACC Ser79 phosphorylation was measured by immunoblotting. F-H HSF1WT or HSF1S121A plasmids were transfected into HSF1-deficient HEK293T cells. Transfected cells were heat-shocked at 43°C for 40 min. HSP mRNAs were quantitated by qRT-PCR following overnight recovery. HSF1-DNA binding was measured immediately after heat shock (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001). I Schematic depiction of the opposite impacts of metabolic and proteotoxic stress on AMPK and HSF1. While metabolic stress activates AMPK to suppress HSF1, proteotoxic stress suppresses AMPK to enhance HSF1 activation. Dashed arrow signifies HSF1 activation independent of AMPK.
Figure 5(A) Lysates from THP1 cells infected with HSV-1 KOS (MOI 10) were subjected to immunoprecipitation using an anti-STING antibody, and the presence of TBK1, STING, and ICP27 in the precipitates was detected by Western blotting. (B, C) THP1 cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h. The cells were fixed and stained with DAPI and anti-ICP27 together with anti-pTBK1 (B). The stainings were visualized by confocal microscopy. Arrowheads, colocalizations between ICP27 and pTBK1. Scale bar 10 μm. (B, C) THP1 cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h. The cells were fixed and stained with DAPI and anti-ICP27 together with anti-STING (C). The stainings were visualized by confocal microscopy. Arrowheads, colocalizations between ICP27 and STING. Scale bar 10 μm. (D) TBK1 was immunoprecipitated from whole cell lysates from HEK293T cells transfected with ICP27 and each of the adaptor proteins TRIF, MAVS, and STING. The precipitates were immunoblotted for TBK1, ICP27, and STING. (E) THP1 control and STING KO cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h and total lysates were generated. TBK1 was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27, phospho-TBK1 (pTBK1), and TBK1. (F) THP1 control and TBK1 KO cells were infected with HSV-1 KOS (MOI 10) for 8 h and total lysates were generated. STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING. (G) THP1 cells were infected with HSV-1 KOS (MOI 10) for 8 h in the presence or absence of the TBK1 inhibitor BX795 (BX, 200 nM). Total lysates were generated and STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING. (H) THP1 cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h and total lysates were generated. Phospho-TBK1 was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and total TBK1. (I) TBK1 was immunoprecipitated from whole cell lysates from HEK293T cells transfected with ICP27 and STING (either WT or S366A). The precipitates were immunoblotted for TBK1, ICP27, and STING.
Figure 4(A) Analysis of telomeric aberrations by Telo-FISH in doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. Two independent experiments were performed. Counted chromatids: WT/empty: n=1824; WT/PARN: n= 1744; KO/empty: n=1452; KO/PARN: n=1920. Averages are shown and χ2-tests were applied to compare KO/empty with either WT/empty or KO/PARN. (B) TRF analysis of telomere length (kb) in KO/empty and KO/PARN cells treated with 10 ng/ml Dox for 72h. A control Western blot with PARN and Actin is shown below for the corresponding samples.
Figure 4(A) Immunoblot showing p-C/EBP β, C/EBP β, AEP, APP, and Tau expression and processing in mouse colon of 3xTg mice with neither DSS treatment nor single side vagotomy, with both DSS treatment and single side vagotomy, or with DSS treatment but no single side vagotomy. (B) Immunoblot showing p-C/EBP β, C/EBP β, AEP, APP, and Tau expression and proteolytic processing in mouse brain. (C) AEP activity assay in colon lysates from pre-vagotomy DSS-treated, DSS-treated and vehicle-treated 3xTg mice. DSS treatment escalated AEP activity in colon of 3xTg mice, which was reversed by vagotomy. Data represent the mean ± SEM; representative data of three samples; ***P=0.0002 (***P< 0.001), **P=0.0013 (**P < 0.01) compared with control, one-way ANOVA. (D) AEP activity assay with brain lysates from different brain hemispheres of single side pre-vagotomy DSS-treated 3xTg mice. Data represent the mean ± SEM; representative data of three samples; *P =0.0162 (*P < 0.05) compared with control, one-way ANOVA. (E) Gastrointestinal permeability barrier defect as determined by FITC-dextran translocation in pre-vagotomy DSS-treated, DSS-treated and vehicle-treated 3xTg mice. Data represent the mean ± SEM; representative data of three samples; *P=0.0126 (*P < 0.001), ***P=0.0002 (***P < 0.001) compared with control, one-way ANOVA. (F) Pro-inflammatory cytokines IL-6, IL1-β, and TNFα concentrations in colon lysates of vehicle-treated 3xTg mice, DSS-treated single side vagotomy 3xTg and DSS-treated 3xTg, respectively. Representative data of three samples; data are shown as mean ± SEM. *P=0.0372 (*P < 0.05), **P=0.0050 (**P < 0.01), ***P=0.0008 (***P <0.001), ****P<0.0001 compared with control, two-way ANOVA. (G) Immunofluorescent staining of AEP (red) and C/EBP β (green), cleaved Tau N368 (red) and AT8 (green), cleaved APP C856 (red) and Aβ (green), T22 (red) and AT8 (green) and Iba-1(red) of the cerebral cortex in different brain hemispheres of pre-vagotomy DSS-treated 3xTg mice. Scale bar: 20 μm. White arrows indicate that C/EBP β and AEP, Tau N368 and AT8, as well as T22 and AT8 were co-localized in mouse brain, respectively. (H) Representative electron microscopy of the synaptic structures in hippocampus. Red stars indicate the synapses. (Scale bar: 1 μm). (I) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining. (Scale bar: 5 μm). (J) Quantitative analysis of the synaptic densities in DSS-treated mice. Representative data of five samples, data are shown as mean ± SEM. ****P < 0.0001, unpaired t tests. (K) Quantitative analysis of the spine density. Representative data of five samples, data are shown as mean ± SEM. ***P =0.0001 (***P < 0.001), unpair t tests.
Figure 1Left: Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured at low/high density Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells. Left: Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured with/without serum Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells. Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured with/without Aphidicolin (Aphi) (C). Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells. Immunoblot estimation of cGAS in nuclear/cytosolic subcellular fraction of BMDMos cultured under indicated conditions. Lamin B and α-Tubulin are nuclear and cytosolic markers respectively. Immunoblot estimation of cGAS in nuclear/cytosolic subcellular fraction of BMDMos cultured under indicated conditions. Lamin B and α-Tubulin are nuclear and cytosolic markers respectively. Immunoblot estimation of cGAS in nuclear/cytosolic subcellular fraction of BMDMos cultured under indicated conditions. Lamin B and α-Tubulin are nuclear and cytosolic markers respectively. Flow cytometric analysis of cell cycle of BMDMos Flow cytometric analysis of cell cycle of BMDMos Flow cytometric analysis of cell cycle of BMDMos
Figure 5A. Schematic representation of the experimental procedure. B. Tumor volume quantification measured by MRI analysis of Tie2-GFP (n=6) or Tie2-IFNα mice (n=9) at the indicated time points post-transplant, one additional Tie2-IFNα mouse showed a tumor volume at day 21 of more than 820 mm3 and was statistically rejected by the Grubbs' test and not further analyzed; mean values are shown; error bars=S.E.M.; p=0.051, by Mann-Whitney test. C. Images (top panels) and corresponding contrast-enhanced MRI (middle and bottom panels) of the liver from representative Tie2-GFP mice or Tie2-IFNα mice that were intrahepatically injected with 5x103 CT26. White dashed lines identify macroscopic lesions. Red and green dashed lines identify hepatic or extrahepatic CRC metastases respectively from representative MRI z-stacks. Tumors detected by MRI analysis appeared as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences, respectively. Scale bars=5mm. D. Percentage of mice with peritoneal carcinomatosis (defined by the presence of multiple intra-peritoneal lesions) determined by MRI analysis from the same mice described in B. Note that while intrahepatic number of lesions remained similar between time points, extrahepatic tumor spreading was significantly reduced in Tie2-IFNα mice. P-values were calculated by Fisher's exact test. E. Confocal immunofluorescence images of representative liver sections from Tie2-GFP mice at distal (upper panels) or proximal (bottom panels) areas to CRC liver metastasis, 30 days post HSPC transplant. Note that TEMs were identified as GFP+ cells and by the concomitant expression of different levels of the myeloid cell marker CD11b+ as highlighted in the inset (merge panels, white arrows); the dashed line identifies the metastasis margin; ✱=CRC metastatic area; scale bars=100μm; insets depict the corresponding areas identified by dashed squares in the merge panels, magnified 1.5-folds. F, G. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible genes Irf7 and Oas1 within liver of Tie2-GFP (n=4) or Tie2-IFNα (n=4) mice euthanized 30 days post-transplant. The basal expression of Irf7 estimated in liver of control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values observed. Mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test. H. Kaplan-Meier survival curves of the indicated groups of mice. Tie2-GFP (n=3); Tie2-IFNα (n=5); p=0.007 by log-rank/Mantel-Cox test.
expression induces senescence-specific gene expression changes.Figure 4Immunofluorescence (top), western blot (middle) and RT-qPCR analyses (bottom; mean fold-change ±S.D. from two biological replicates) confirm HMGB1 knockdown in IMR90. PCA analysis plot (top) of control (black) and HMGB1-knockdown replicates (blue). Scatter plot (bottom) showing significantly up- (>0.6 log2-fold change; orange) or downregulated genes (<-0.6 log2-fold change; green) upon HMGB1 knockdown.   Bar plots showing GO terms associated with the up/downregulated genes from panel B and their enrichment P-values (-log; right).   Gene set enrichment analysis of HMGB1-KD data. Normalized enrichment scores (NES) and associated P-values for each gene set are shown. Representative images of IMR90 overexpressing HMGB1-GFP, immunostained for p21 and CTCF, and counterstained with DAPI. IMR90 transfected with empty vectors provide a control. Bar: 5 μm. Bar plots showing RT-qPCR data (mean mRNA fold-change ±S.D., from two biological replicates) for selected genes in HMGB1-overexpressing compared to control IMR90. The mean fold-change for each mRNA from HMGB1-knockdown RNA-seq data is also shown for comparison (magenta bars). *: P<0.01; unpaired two-tailed Student's t-test.
Figure 5A) Bar graph of TUNEL-positive cells in the retina of mice treated with 5 mg/kg anti-VEGF-A, anti-ANG-2, and 3, 5, and 10 mg/kg anti-VEGF-A/ANG-2. Error bars show SEM with n = 6 animals per group and * denote all significant changes after one sided ANOVA (**, P = 0.0041) and Tukey's multiple t-test. IgG control is significantly different from anti-VEGF-A/ANG-2 mid (**, P = 0.0077) and -high (**, P = 0.0028). B) IgG control, C) anti-VEGF-A, D) anti-ANG-2, E-G) anti-VEGF-A/ANG-2 low, mid, high: representative micrographs of whole-mount retinae preparations display the reduction of TUNEL-positive photoreceptor cells (green) clustered around focal neovascular lesions in anti-VEGF-A/ANG-2 (E-G) and, to a lesser extent, anti-VEGF-A- (C) and anti-ANG-2 (D)-treated groups compared with IgG-treated controls (B), scale bar 200 μM. H) ERG was used to assess retinal function in response to large-field flash stimuli under both scotopic and photopic conditions. Representative photopic (light-adapted) and scotopic (dark-adapted) flash response series show increased amplitude and reduced latency after treatment with bispecific VEGF-A/ANG-2 compared with IgG control (both 3 mg/kg). ERG shows reduced depression of B-wave responses in JR5558mice treated with anti-VEGF-A/ANG-2 compared to IgG control.I) Maximum photopic ERG amplitude was increased using the rodent crossreactive bispecific VEGF-A/ANG-2 compared to single anti-VEGF-A or anti-ANG-2 treatments (all at 3 mg/kg). There was a significant increase in amplitude at stimuli >2 log (cd*s/m2) between the bispecific VEGF-A/ANG-2 and IgG control group under scoptopic and phototopic conditions. *Denotes significance after ANOVA and Dunnett's multiple t-test compared to IgG control compared for each stimuli separately, error bars show SEM with n = 8 animals per group. Under phototopic conditions anti-VEGF-A/ANG-2 reached significance at 2.58 and 2.30 cd*s/m2 with P = 0.0498 and *P = 0.0479, respectively. Under scotopic conditions all stimuli reached significance for anti-VEGF-A/ANG-2 with P-values of -2.3 cd*s/m2 (P = 0.0498); -1.93 cd*s/m2 (P = 0.0035); -1.33 -1.03 cd*s/m2 (P 0.0001); -0.73 cd*s/m2 (P = 0.0002); -0.43 cd*s/m2, (P = 0.0004); -0.13 cd*s/m2, (P = 0.0024) and 0.18 cd*s/m2 (P = 0.0031). In addition anti-ANG-2 treatment reached significance at -0.13 cd*s/m2 (P = 0.011) and 0.18 cd*s/m2 (P = 0.005), respectively.
Figure 3A A drop in PKAc1-Ty-iKD expression is detected as early as 24 hours upon ATc treatment with almost no protein detectable after 36 hours. B Conditional down-regulation of PKAc1-Ty-iKD leads to the absence of plaque formation.Scale bars = 1 mm (B) C, D Down-regulation of PKAc1-Ty-iKD upon ATc treatment leads to increased parasite dispersion after 40 hours and premature egress (data is from three independent biological replicates).Scale bars = 20 μm (C) C, D Down-regulation of PKAc1-Ty-iKD upon ATc treatment leads to increased parasite dispersion after 40 hours and premature egress (data is from three independent biological replicates).Data information: In D, data are presented as mean ± SD. E Prematurely egressed PKAc1-Ty-iKD parasites after 24 hours of ATc treatment invade and exit fully lysing the monolayer of HFF cells while the non-treated parasites invade and initiate a new lytic cycle. Scale bars = 2.5 mm (E)