EMBO/sd-character-level-ner huggingface dataset:
Figure 2Two-week old AG129 mice (n=2-3 /group/time point) were infected with EV-A71 S41. (A) At various time points post-infection, mice were euthanized and the relative expression of PRPH was detected by Western blot in tissue homogenates (brainstem, spinal cord and limb muscle). Actin and GAPDH serve as loading controls, and protein expression was compared to age-matched non-infected mice. Two-week old AG129 mice (n=2-3 /group/time point) were infected with EV-A71 S41. (B) Immunostaining of PRPH and EV-A71 particles on brainstem and spinal cord sections harvested at 6 d.p.i. Two-week old AG129 mice (n=2-3 /group/time point) were infected with EV-A71 S41. (C) Immunostaining of NMJs (nAChR), PRPH, and EV-A71 particles (VP0) in limb muscle sections harvested at 6 d.p.i.
Figure 2Propidium iodide (PI) uptake in indicated wildtype THP-1 or ∆CASP-1, ∆CASP-4, ∆ASC cells, or THP-1 stably expressing GSDMD-targeting miRNA (GSDMDmiR) infected with the indicated strains of Toxoplasma gondii (Tg) for 24 h. Cells were untreated or treated with IFNγ as indicated. Mean area under the curve (AUC) ± SEM from n = 3 experiments are shown. ns, not significant, from two-way ANOVA following adjustment for multiple comparisons. Real-time apoptosis assay (Annexin V (AnnV)-Glo luminescence) from IFNγ-primed primary human MDMs transfected with non-targeting control (siCTRL; left) or GBP1 siRNA (siGBP1; right) and left uninfected or infected with type I or type II Tg. Depicted is the mean ± SEM of n = 4 donors. AU, arbitrary units. Immunoblots from primary MDMs infected with the indicated Tg strains for 12 h. Cells were untreated or pre-treated with IFNγ before infection. Treatment with TNFα and cycloheximide (CHX) served as positive control for apoptosis. Images are representative of n = 4 biologically independent experiments. Live time-lapse imaging to visualize apoptotic cell death and measure caspase-3/7 activities using a fluorescent caspase substrate. THP-1 WT were primed with IFNγ or left untreated and infected with either type I or type II Tg. Plot in (D) shows cumulative caspase-3/7 activity from an 18-hour time-course (mean ± SEM from n = 3 independent experiments). Representative phase-contrast images overlaid with fluorescence signal for caspase-3/7 activity (green) images are shown in (E) from IFNγ-primed THP-1 cells infected with type I Tg captured at indicated times post-infection. Scale bar 20 μm. AnnV-Glo assay from IFNγ-primed THP-1 transfected with siRNA against indicated human caspases and infected with type I or type II Tg. Area under the curve (AUC) from a real-time AnnV-Glo luminescence assays similar to those in (B) are plotted as mean ± SEM from n = 3 independent experiments. AnnV-Glo assay from IFNγ-primed primary human MDMs transfected with siRNA against CASP8, CASP10 and non-targeting control (CTRL) and infected with type I or type II Tg. Area under the curve (AUC) from real-time assays similar to those in (B) are plotted from n = 4 independent experiments. Matched shapes and color of symbols indicate donors.
Figure 1A. Viability of primary human PMNs following treatment with purified recombinant bi-component leukocidins using the viability dye CellTiter. Bars indicate Mean ± SEM, with n = 5 donors. B. Half maximal effective concentrations (EC50) of native and heterologous toxin pairings on primary human PMNs using the viability dye CellTiter. E = LukE, D = LukD, A = HlgA, B = HlgB, C = HlgC, S = LukS-PV, F = LukF-PV. Bars indicate Mean ± SEM, with n = 6 donors. C. Leukocidin octamer highlighting stem domain motifs deleted from LukE and LukD (LukEmut1, LukEmut2, and LukDmut), as shown from the side view (left), and top view (right) of the pore. The monomeric structures of LukE (3ROH) and LukD (4Q7G) were obtained from the Protein Data Bank, and superimposed and colored on the structure of the HlgA-HlgB octameric pore (3BO7) using PyMOL. The location of where the deleted residues would be in the pore structure is shown from a side view on the left, and the location within an oligomeric pre-pore structure are shown from a top view on the right. D. Viability of primary human PMNs following incubation with WT and mutant toxin pairings using the viability dye CellTiter. Insert: 2 μg per lane of purified recombinant toxins visualized by Coomassie Blue staining on a 12% SDS-PAGE gel. E = LukE, D = LukD, Emut1 = LukEmut1, Dmut = LukDmut. Bars indicate Mean ± SEM, with n = 4 donors. E. Viability of primary humanPMNs following simultaneous incubation with a lethal dose of WT LukED (78.12 nM per subunit) and a dose response of WT or dominant-negative (DN) toxin subunits using the viability dye LukE. E = LukE, D = LukD, Emut1 = LukEmut1, Emut2 = LukEmut2, Emut1Dmut = LukEmut1LukDmut, Emut2Dmut = LukEmut2LukDmut . Bars indicate Mean ± SEM, with n = 6 donors.
Expanded polyglutamine repeats selectively associate with mitochondrial GAPDH, inhibiting GAPDH-induced mitophagy.Figure 4Western blots showing co-immunoprecipitation of GAPDH from the total lysates and isolated mitochondria of PC12 cells with anti-GAPDH antibody. Anti-EGFP antibody detects the presence of polyglutamine repeats. Bottom panel shows co-immunoprecipitation of GAPDH in the total lysate of PC12 cells treated with formaldehyde to cross-link proteins. Ctr* = control. n = 3. Western blots showing co-immunoprecipitation of GAPDH from the total lysate (left) and isolated mitochondria (right) of brain tissues of wild-type (WT) and transgenic R6/2 (HD) mice with anti-GAPDH antibody. The presence of huntingtin was examined with anti-huntingtin antibody and anti-polyglutamine antibody, respectively. Co-immunoprecipitated huntingtin or polyglutamine (polyQ) was normalized to GAPDH. Left: n = 2. *P = 0.03; **P = 0.02. Right: One representative experiment from two repeats. Sub-mitochondrial localization of GAPDH and expanded polyglutamine repeats. Isolated mitochondria were left untreated or subjected to the proteinase K treatment in the presence or absence of detergent (Triton X-100). n = 3. Illustration describing association of oxidized GAPDH (represented by green circles) and expanded polyglutamine repeats (in mutant huntingtin protein (MtHtt) represented by purple circles) at the outer membrane of damaged mitochondria.
Figure 3Different constructs of RB were transfected in H1299-RBKO treated with doxorubicin 16 h to induce genotoxic stress; the RB constructs levels of expression were evaluated using HA antibody. The bar diagrams show the quantification of the western blots data. A) Evaluation of the small pocket expression in an MDM2 dose-dependent manner. B) Cells were transfected with the RB large pocket construct. Different constructs of RB were transfected in H1299-RBKO treated with doxorubicin 16 h to induce genotoxic stress; the RB constructs levels of expression were evaluated using HA antibody. The bar diagrams show the quantification of the western blots data. C) Cells were transfected with RB full length without the 5´URT (named RB-HA). D) Cells were transfected with the RB full-length plus 260 nt of its own 5´ UTR (named RB+5´UTR). E) The binding of MDM2 to the RB mRNA in H1299 cells as estimated using RNA CoIP. Cells were treated with doxorubicin (1 µM for 16 h) or DMSO. F) RT-qPCR of RB and GAPDH of the MDM2-immunoprecipitation from the polysomal fractionation (see EV2D). Data are representative of three similar independent experiments. G) PLA assay using anti-RB and anti-MDM2 antibodies, H1299 cell were treated or not with etoposide to induce stress, the endogenous proteins were visualised. Cell nuclei were visualised with DAPI blue. The red dots show the interaction between the two proteins. The scale bar corresponds to 20 μm. H) RNA-PLA assay using rabbit anti-MDM2 and a mouse biotin-antibody. H1299- RBKO was stable transfected with RB-HA or RB+5´UTR. Cell nuclei were visualised with DAPI blue. The red dots show the interaction between the protein and the mRNA. The scale bar corresponds to 20 μm.
Working models for Ccq1-mediated nucleosome stability and telomere maintenance.Figure 8A Ccq1 recruits CLRC and SHREC to subtelomeric chromatin, which counteracts nucleosome instability conferred by the DNA sequence of TAS. CLRC may contribute to nucleosome stability indirectly by depositing H3K9me, which prevents access to RNAPII and, thus, nucleosome eviction through transcription. Conversely, stabilized nucleosomes will contribute to maintaining critical levels of H3K9me at TAS. SHREC may contribute to nucleosome stability directly through nucleosome positioning via its ATP-dependent remodeler activity conferred by Mit1; indirectly through histone deacetylation via the HDAC moiety Clr3, thereby preventing open chromatin structure and access to RNAPII B TAS or other DNA sequences may represent fragile sites causing DNA breaks through replication fork collapse or the recruitment of unknown factors causing genome instability. Ccq1 may prevent genomic instability by binding to subtelomeric chromatin, thereby stabilizing fragile sites and/or competing with the binding of destabilizing factors. Homologous sequences present in the TAS may promote recombination by BIR (break-induced replication) or similar mechanisms
Figure 6(A-C) Ciliary length measured by fluorescence microscopic analysis at 1 year of age (1y). (A) Representative immunostaining of the ciliary marker Gt335 (green) in longitudinal cryosections of PRC ciliary regions in WT and USH1C retinas. Counterstaining by DAPI, scale bar 10µm. (B) Gt335 staining at higher magnification and principle of quantifying ciliary length by Gt335 staining. Scale bar 500nm. (C) Length of connecting cilia (CC) appear elongated in USH1C when applying a Fiji script to immunostained histological sections (2 pigs, 1y, 1 retina each, number of examined connecting cilia is indicated, error bars represent SD, two-tailed Student's t tests, ****p<0.0001). (D, E) Measuring CC length in TEM sections. (D) Representative images of longitudinal cross sections and principle of determining CC length. Scale bar 500nm. (E) Elongation of CC length is confirmed in TEM sections of USH1C PRC. (1 pig, 1y, 1 retina each, number of examined connecting cilia indicated, error bars represent SD, two-tailed Student's t tests, ****p<0.0001). Length of primary cilia in dermal fibroblasts. (F) Staining of ciliary structures in fibroblasts stained with ARL13B (ciliary shaft, green) and PCNT2 (ciliary base, red), DAPI (nucleus, blue). Scale bar upper panel 5 µm, lower panel 2.5 µm. (H) Similarly, ciliary length of fibroblasts from a human USH1CR31X/R80fs patient are elongated, compared to a healthy control (7 TR, number of cells examined indicated, KS-test, ****p<0.0001). (G) Left panel: The number of ciliated cells is decreased in porcine fibroblasts USH1C pigs, compared to WT controls (6 TR, number of examined cells indicated, error bars represent SD, two-tailed Student's t tests, *p<0.05). Right panel: The length of primary cilia in porcine fibroblasts is increased in USH1C (6 TR, number of cells examined indicated, Kolmogorov-Smirnov (KS)-test, ****p<0.0001). (I-M) Outer segment (OS) PRC architecture by TEM of longitudinal retinal sections. (I) At 3w, WT show normal sub-cellular organization, including parallel stacking of photosensitive discs and calyceal processes (black arrows, representative image from 2 pigs, 2 retinas, scale bar 600nm). (J) In 3w USH1C rods, membrane discs appear also in vertical orientation (white arrows, representative image from 2 pigs, 2 retinas, scale bar 750nm). At 1y, interstitial gaps appeared in disc stacks of the OS in USH1C pigs (K, arrow, scale bar 550nm) and vesicles are found at the OS base (L, asterisk, scale bar 500nm). (M) The disc architecture in 1y animals was substantially distorted (arrows, representative image from 2 pigs, 2 retinas, scale bar 850nm). (N) Localization of cellular anomalies in an USH1C rod highlighted in orange.
Histone H3K9ac profiles after ACC in Col-0 and hsfa2 at HSP18.Figure 4Figure 4: Histone H3K9ac profiles after ACC in Col-0 and hsfa2 at HSP18.2, HSP22.0, HSP70 and APX2 as detected by ChIP-qPCR.Col-0 (blue bars) and hsfa2 (orange bars) seedlings were subjected to ACC or control treatment (NHS) 4 d after germination. At the indicated time points after the treatments, ChIP-qPCR was performed with antibodies against H3K9ac or H3. Schematics (grey bars, UTR; black bar, exons) show positions of regions analyzed. Intergenic control region 1 is 3123 bp (APX2) or 2570 bp (HSP22.0) upstream of the TSS, or 5311 bp (HSP18.2) or 6725 bp (HSP70) downstream of the TSS, respectively. Data are averages over four biological replicates. Amplification values were normalized to input and H3 and the Col-0 4h NHS region 2 (HSP18.2, HSP22.0and HSP70) or 3 (for APX2). *, p<0.05; **, p<0.01 for differences between genotypes at the same time point and treatment; squares and triangles within bars mark significant differences (p<0.01 and p<0.05, respectively) between ACC and NHS samples of the same time point and genotype, Student´s t-test. Error bars indicate SE.
Figure 3HCC4006 cells were (A) transfected with two siRNA against RHOB (siB1, siB2) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells. HCC4006 cells were (A) transfected with two siRNA against RHOB (siB1, siB2). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments. HCC4006 cells were (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells. HCC4006 cells were (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments. HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells. HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments. Erlotinib IC50 values were quantified in RHOB-overexpressing or RHOB-depleted (D) HCC827 cells, (E) HCC2935 cells and (F) H3255 cells, as determined by an MTS assay after 72 h treatment. RHOB overexpression or inhibition was monitored by western blotting for each condition. G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI), then erlotinib IC50 values were determined after 72 h by an MTS assay and a correlation analysis was performed. G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.
Figure 6A) Schematic representation of the minor intron-containing p120, SCN4A and mSCN8A minigenes and their promoters (rous sarcoma virus [RSV] or cytomegalovirus [CMV]). HnRNP H binding sites are depicted as GGGA. B) Western blot analysis of FUS levels under control knockdown (lanes 1, 4, 7), FUS knockdown (FUS KD; lanes 2, 5, 8), and FUS rescue (lanes 3, 6, 9). HeLa cell extracts were subjected to SDS-PAGE and western blotting with anti-FUS antibodies (green) and anti-FLAG (red, upper part). The yellow signal in the upper part is due to the overlay of the red and the green channels. Tyrosine tubulin was detected as loading control (red, lower part). C) RT-qPCR results indicating the ratio of spliced to unspliced RNA from the indicated minigenes under control knockdown (Ctr KD), FUS KD, and FUS KD followed by a rescue with an RNAi-resistant cDNA expression construct (Rescue). Average values and standard deviations are shown of nine biological replicates for p120 and SCN4A (five including the rescue condition) and of eight biological replicates for mSCN8A (all including the rescue). Single and triple asterisks indicate p-values of <0.05 and <0.001, respectively.
Figure 3(A) Top: Schematic drawing of the (CGA-CCG) dicodon reporter mRNA used for in vitro translation experiments. Bottom: Scheme outlining the principle of ribosome nascent chain (RNC) purification. Stalled and colliding ribosomes are pulled down via an affinity tag on the nascent chain. Both RQC and NGDRQC+ are triggered by a (CGA-CCG) dicodon containing arrest sequence in vivo. (B) Western blot showing the arrest products derived from the GFP-X-FLAG-HIS3 reporter. Translation products were detected by western blot using an anti-GFP antibody. Both RQC and NGDRQC+ are triggered by a (CGA-CCG) dicodon containing arrest sequence in vivo. (C) Northern blot for the 5' NGD-IM derived from the (CGA-CCG) reporter in ski2∆ cells. 5'-NGD-IMs were detected with a DIG-labelled GFP probe. SCR1 was used as a load control. (D) Western blot of test translations using the (CGA-CCG) dicodon stalling mRNA reporter shown in (A). The mRNA reporter was added to a yeast in vitro translation extract obtained from a ski2∆uS10-3HA strain. Expression of the translation products (free His- and V5 tagged truncated uL4 protein and the same protein attached to tRNA) was visualized with an anti-V5 antibody. (E) Sucrose gradient fractions (top) and western blot analysis (bottom) of the (CGA-CCG) dicodon-stalled RNC samples. After the translation reaction, the RNCs were affinity-purified as indicated in (C). The eluate was loaded on a 10-50 % sucrose gradient and fractionated. Each collected fraction was analysed using anti-HA antibody to detect uS10-HA. Note that disomes are preferentially polyubiquitinated over monosomes.
Figure 2BMDMs were stimulated with TNF (100 ng/ml) in combination with TAK1i (125 nM) for the indicated time points. LDH release BMDMs were stimulated with TNF (100 ng/ml) in combination with TAK1i (125 nM) for the indicated time points. mixed supernatant and cell extracts were analysed. Representation of known caspase cleavage site and molecular weight of corresponding cleavage fragment in mouse GSDMD. BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h in the presence or absence of KCl (50 mM). Where indicated, cells were pre-incubated with MCC950 (10 μM) 20-30 min prior to TNF/TAK1i stimulation. BMDMs were costimulated with TNF (100 ng/ml) and SM (250 nM; 6 h) and mixed supernatant and cell extracts were analysed by immunoblot BMDMs were costimulated with TNF (100 ng/ml) and SM LDH release in the cell culture supernatant were quantified at the indicated time points. BMDMs were costimulated with TNF (100 ng/ml) and SM
Figure 6(A) Survival model #1 with low dose treatment after 4 hr of CPA injection. Mice were infected with 5 x 106 CFU of S. aureus via intravenous injection on day 3. Moribund mice were euthanized and recorded as deceased on the day of euthanasia. Control mice without CPA. (B) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii). Data represent 3 independent experiments with similar results. (C) Gram and Giemsa stains analyzed neutrophil phagocytosis and bacterial clearance in mice surviving on day 9. White arrows indicate phagocytosed S. aureus in neutrophils. (D) Survival model #2 with low dose treatment after 24 hrs of CPA injection. Other schedules/procedures were the same to those described in panel A. (E) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii). Data represent 2 independent experiments with similar results. (F) Survival model #3 with medium dose treatment after 24 hrs of CPA injection. Other schedules/procedures were the same to those described in panel A. (G) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii).
Figure 5Clustering of GO terms enriched in S3-LCEs. GO terms were clustered using the Markov cluster algorithm. GO clusters are named by the GO term with the lowest P-value in each cluster. Node size represents the number of genes in the GO term, and the edge width represents the similarity coefficient between two GO terms. P-values of the top 3 GO terms contained in each GO cluster indicated in (A). GO terms were color-coded according to the corresponding colors in (A). Box plot showing lengths of S3-LCEs in each GO cluster. **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to "organelle organization" by Steel-Dwass test. The box plot shows the interquartile range (boxes), the median (central band) and the minimum and maximum except for the outliers at the ends of whiskers (n = 8,115 for "Other RBP-dependent exons", n = 3,078 for "All SRSF3-dependent exons", n = 595 for "ORGANELLE ORGANIZATION", n = 57 for "DNA-BINDING TRANSCRIPTION FACTOR BINDING", n = 27 for "LYSINE-ACETYLATED HISTONE BINDING", n = 513 for "POSITIVE REGULATION OF TRANSCRIPTION", and n = 75 for "HISTONE MODIFICATION").
TBL1 controls PI3 kinase signaling by direct transcriptional regulation.Figure 3Gene expression microarray from Capan-1 cells transfected with siRNA. Heatmap shows genes annotated to KEGG pathway hsa04110 "Cell Cycle" and/or gene ontology cluster GO:0007049 "Cell Cycle". Protein expression of Panc02 cells with stable shRNA expression prior to implantation into mice. Chromatin immunoprecipitation from Capan-1 cells with or without siRNA-mediated knockdown of TBL1. IgG and primers for PIK3CA coding region served as a negative control; histone H3 served as a positive control. Proliferation time course assay in control and TBL1 shRNA-transfected Panc02 cells overexpressing constitutively active PI3K p100α mutant E545K. Cells were stained with crystal violet, and absorbance was measured at 595 nm. n = 9 cell culture wells per group; statistically significant at P < 0.05 for indicated comparisons: shNC pBabe-puro vs. shNC pBabe-PI3K (*), shTBL1 pBabe-puro vs. sbTBL1 pBabe-PI3K (#), shNC pBabe-puro vs. shTBL1 pBabe-puro ($), shNC pBabe-PI3K vs. shTBL1 pBabe-PI3K (†); P-values: day 2: 2.01 × 10−3 (#), 4.11 × 10−9 ($), 5.77 × 10−3 (†); day 3: 4.50 × 10−5 (*), 1.01 × 10−9 (#), 1.71 × 10−30 ($), 1.22 × 10−6 (†); day 4: 5.61 × 10−22 (*), 1.10 × 10−45 (#), 6.65 × 10−62 ($); two-way ANOVA with Bonferroni post-test. Matrigel invasion assay in same cells as in (D). Invading cells per microscopy field were counted. n = 6 microscopy fields per group; statistically significant at P < 0.05; P-values: 6.64 × 10−4 (*), 1.35 × 10−7 (#), 6.94 × 10−5 ($); two-way ANOVA with Bonferroni post-test. mRNA expression of PIK3CA in human patient samples; significant difference (P < 0.05) between tumor and normal tissue *P = 2.13 × 10−4 determined by paired two-tailed Student's t-test, n = 18 patients. Correlation of mRNA expression of TBL1 and PIK3CA in human patient tumor tissue (normalized to 18S rRNA and relative to a pooled organ donor sample); n = 17 patients; P-value determined by testing for slope of regression line ≠ 0. Survival analysis in 105 patients with PDAC: High PIK3CA expression is significantly associated with shorter post-resection survival. P-value determined by log-rank test as described in Materials and Methods.