EMBO/sd-character-level-ner huggingface dataset:
Figure 7(A) Schematic for EVs-based adjunctive immunotherapy and moxifloxacin-based chemotherapy in M.tb-infected mice. Representative histopathological analysis for lung sections of WT (B) mice that were infected with M.tb and subsequent left untreated (Mock) or treated with EVs from uninfected BMMs (EVs_Control), EVs from M.tb-infected BMMs (EVs), moxifloxacin (MXF), or combination of EVs and MXF (EVs+MXF). Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). Scale bar, 100 μM M.tb CFU in the lung and spleen of WT (C) mice treated with EVs, MXF or combination of both. Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed). Representative histopathological analysis for lung sections of Mavs -/- (D) mice that were infected with M.tb and subsequent left untreated (Mock) or treated with EVs from uninfected BMMs (EVs_Control), EVs from M.tb-infected BMMs (EVs), moxifloxacin (MXF), or combination of EVs and MXF (EVs+MXF). Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). Scale bar, 100 μM M.tb CFU in the lung and spleen of Mavs -/- (E) mice treated with EVs, MXF or combination of both. Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed). ELISA analysis for, IFN-β, TNF-α, IL-1β and IFN-γ protein level in serum isolate from M.tb-infected WT (F) or Mavs -/- (G) mice treated with EVs, MXF or a combination of both. Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).
Mfn2 ablation promotes abnormal ER expansion during ER stress conditions.Figure 1(A) WT or Mfn2 KO MEFs (Mfn2 KO cells) were treated with 1 μM Tg for 12 h and then processed for EM visualization of the ER morphology. Scale bar: 1 μm. (B) EM images of Tg‐treated Mfn2 KO cells show accumulation of ER membrane stacking. Scale bar: 1 μm. (C) WT, Mfn2 KO or Mfn1 KO cells were transfected with the Sec61β‐GFP plasmid and treated with 1 μM Tg for 24 h. Confocal microscopy images show ER vacuolization in Mfn2 KO cells treated with Tg. Scale bar: 10 μm. Insets show × 10 zoomed images. Scale bar: 5 μm. (D) WT and Mfn2 KO cells were treated with Tg 1 μM for 3 h and then incubated with brefeldin A‐bodipy to stain ER and Golgi. Representative flow‐cytometry histograms (upper panel). Mean fluorescence intensity was used to quantify ER expansion (n=5) (lower panel). Data are mean±s.e.m. *P0.05 versus WT group.
Figure 3Endocytosis of VE-cadherin in 17 h TNF-α treated HUVEC was induced by adding HL60-derived neutrophils for 20-30 min at 37°C and monitored by the internalization of preincubated mAb BV6, which was visualized in fixed cells with a secondary fluorescent antibody (white). Total VE-cadherin was stained with another mAb (red), nuclei stained with Hoechst (white). (A) HUVEC had been transfected prior to the experiment with control or PECAM-1-specific siRNA. Cell lysates were analyzed for expression levels of PECAM-1 and tubulin (below). Relative VE-cadherin internalization efficiency is given on the right. Endocytosis of VE-cadherin in 17 h TNF-α treated HUVEC was induced by adding HL60-derived neutrophils for 20-30 min at 37°C and monitored by the internalization of preincubated mAb BV6, which was visualized in fixed cells with a secondary fluorescent antibody (white). Total VE-cadherin was stained with another mAb (red), nuclei stained with Hoechst (white). (B) PECAM-1 siRNA treated HUVEC were transduced with WT PECAM-1-EGFP or PECAM-1-Y663,686F-EGFP. Expression levels of transduced PECAM-1 forms and of EGFP and VE-cadherin are shown in immunoblots (on the right). Relative VE-cadherin internalization efficiency is given on the right.
OVA257-264 -specific T-cell response and cytokine production.Figure 1A Specific CD8+ T-cell (OT-I) proliferation in response to immature BMDCs incubated with fdOVA, fdOVA/sc-αDEC or OVA257-264 peptide chemically coupled to anti-DEC-205 antibody (NLDC:pOVA). Percentage of CFSE-labeled proliferating T cells was evaluated on CD8+-gated cells after 72 h of DC:T co-culture. The mean ±SD of three independent experiments is reported. B Proliferation of adoptively transferred OT-I T cells in C57BL/6mice (n = 3/group) immunized with vehicle (PBS), NLDC:pOVA, NLDC:pOVA plus CpG, fd, fd/sc-αDEC or LPS-free ovalbuminprotein. Percentage of CFSE-labeled proliferating OT-I T cells was evaluated after three days on Vα2+CD8+-gated cells. The mean ± SD of two independent experiments is reported. Comparative analyses were performed using Student's t-test for unpaired samples. C Percentage of IFN-γ-producing CD8+ splenocytes isolated from mice immunized as in (B). The mean ± SD of two independent experiments is reported. Comparative analyses were performed using Student's t-test for unpaired samples. D-F IL-6 (D), IL-18 (E) and IFN-α (F) release in supernatants of BMDCs obtained from C57BL/6 mice. Supernatants of BMDCs incubated with NLDC145 antibody, wild-type phage particles (fdWT) or scFv αDEC-205 phage particles (fdsc-αDEC) for 20 h were analyzed by ELISA for cytokine production. Unstimulated culture (medium) and LPS-treated culture were, respectively, used as negative and positive controls. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples. D-F IL-6 (D), IL-18 (E) and IFN-α (F) release in supernatants of BMDCs obtained from C57BL/6mice. Supernatants of BMDCs incubated with NLDC145 antibody, wild-type phage particles (fdWT) or scFv αDEC-205 phage particles (fdsc-αDEC) for 20 h were analyzed by ELISA for cytokine production. Unstimulated culture (medium) and LPS-treated culture were, respectively, used as negative and positive controls. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.
Figure 7Representative electron microscopy of ER-mitochondria associations in NSC34 cells transfected with EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and treated with vehicle or 1 mM AR-A014418 for 16 hours. Arrowheads with loops show regions of association; scale bar=200 nm. Bar charts shows % of the mitochondrial surface closely apposed to ER in the different samples.
Figure 4Domain structure of the human SprT proteases SPRTN and ACRC, showing location of SprT protease domains and additional motifs, including a ubiquitin-binding UBZ domain in SPRTN and an N-terminal cluster of SIMs (sequence highlighted) in ACRC. Alanine substitutions introduced to disrupt the functionality of the ACRC SIMs (SIM*) are indicated. Representative images of U2OS cells stably expressing GFP-SPRTN or GFP-ACRC that were transfected with control (CTRL) or UBC9 siRNAs, exposed to formaldehyde in the presence or absence of ubiquitin E1 enzyme (UBA1) inhibitor TAK-243 (Ub-E1i) as indicated and fixed one h later. Scale bar, 10 μm. Quantification of data showing proportion of U2OS/GFP-SPRTN cells displaying nuclear GFP-SPRTN foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments). As in (C), but showing proportion of U2OS/GFP-ACRC cells displaying nuclear GFP-ACRC foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments). U2OS cells expressing GFP-ACRC were exposed to the indicated genotoxic agents (formaldehyde: 600 μM, 1 h; UV: 20 J/m2, 6 h recovery; hydroxyurea (HU): 2mM, 24 h; mitomycin C (MMC): 40 ng/mL, 24 h), preextracted and fixed, and analyzed by microscopy. Representative images are shown. Scale bar, 10 μm. HeLa cells transfected with plasmids encoding GFP alone or indicated GFP-ACRC alleles were subjected to GFP IP under denaturing conditions. Beads were incubated with recombinant polySUMO22-8 chains, washed extensively and processed for immunoblotting with SUMO2/3 and GFP antibodies. Representative images of U2OS cells expressing indicated GFP-ACRC alleles that were treated with 5-azadC in the presence or absence of SUMO inhibitor (SUMO-E1i), fixed 2 h later and immunostained with DNMT1 antibody. Scale bar, 10 μm.   Quantification of data in (G), showing proportion of cells displaying GFP-ACRC co-localization with DNMT1 foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments).
Figure 5(A) Maltoheptaose ubiquitylation by HOIL-1 was assayed at 30°C in the presence of Met1- or Lys63-linked ubiquitin dimers for the indicated times and visualised by Coomassie staining. (B) Maltoheptaose ubiquitylation by HOIL-1 was assayed at 30°C in the presence of Met1- or Lys63-linked ubiquitin tetramers for the indicated times and visualised by Coomassie staining. (C) HOIL-1[T203A/F204A/R210A] was assayed at 30°C in the absence or presence of the indicated ubiquitin dimers and tetramers. These mutations have been reported to impair the binding of ubiquitin to the NZF domain and their approximate location in HOIL-1 is indicated (schematic not to scale). (D) Assay of HOIL-1[Δ194-222] lacking the NZF core domain at 30°C in the absence or presence of the indicated ubiquitin oligomers. (E) Allosteric activation of HOIL-1[233-510] (lacking the LTM, UBL and NZF domains at the N‑terminus) by ubiquitin oligomers.
Figure 2(A) Quantification of total number of GnRH neurons during embryogenesis, at E12.5, E14.5 and E18.5. More GnRH neurons were found in E14.5 and E18.5 mutant embryos. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=4 to 6 mouse embryos from at least 3 litters. (B) Schematic representation of the areas containing GnRH migrating cells during embryogenesis at E14.5 and representative immunofluorescence showing migrating GnRH neurons at the level of the nose, the olfactory bulb (OB) and the ventral forebrain (VFB) in control and mutant embryos. Scale bar: 50μm. VNO, vomeronasal organ; OE, olfactory epithelium; OB, olfactory bulbs; VFB, ventral forebrain. (C) Quantitative analysis showing distribution of GnRH neurons in the nose, OB and VFB of mutant and control embryos. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=5 and 6 mouse embryos for mutants and controls, respectively, from at least 3 litters. (D) Representative image of cleared brains and immunolabeling for GnRH at the level of the olfactory bulbs, in a control (left panel) and a mutant (right panel) postnatal brain. Knock-out animals show an accumulation of GnRH neurons in the accessory olfactory bulb (circle formed by the GnRH labelling, right panel). AOB, accessory olfactory bulb. Scale bar: 300μm. (E) Representative image of cleared brains and immunolabeling for GnRH at the level of the organum vasculosum of the lamina terminalis (OVLT) in the median preoptic area, from a control (left) and a mutant (right) female postnatal brain. Scale bar: 500μm. Oc, optic chiasma. (F) Quantitative analysis of the distribution of GnRH neurons in cleared brains of mutant and control littermates. OB, olfactory bulb; DBB, diagonal band of Broca; 3V, periventricular region of the preoptic area; Tot, total. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=3 adult mice from 3 litters.
Figure 3Time course analysis of BES1 protein accumulation levels. pBES1::BES1-GFP seedlings were grown under short days for 6 days and samples were harvested each 2 hours for a 24 h interval. Total protein extracts were used for western blot analysis with an anti-GFP antibody to detect the BES1-GFP protein. Hybridization with anti-RPT5 antibody is included as a loading control. Dark and light periods are represented as black and white bars, respectively. The experiment was repeated twice with similar results Diurnal oscillation of BES1 and BZR1 transcripts. Col-0 seedlings were grown for 6 days under short days and samples were harvested every 3 hours. BES1 and BZR1 expression levels were determined by qRT-PCR analyses and normalized to PP2A. Relative levels are the mean of three technical replicates and error bars represent standard deviation. The experiment was performed three times with similar results. Dark periods are indicated by shading Western blot analyses of 35S::BES-GFP seedlings grown for six days under short days. Samples were collected every two hours, starting two hours before lights on (ZT-2) to ZT6. Western blots were hybridized with an anti-GFP antibody to detect the BES1-GFP protein, and anti-RPT5 as a loading control BES1 but not BZR1 is stabilized in the light. 35S::BES1-GFP and 35S::BZR1-GFP seedlings were grown for 6 days in continuous white light (WL), red light (RL), and continuous dark (DARK), and the BES1 and BZR1 proteins analyzed by western blot using an anti-GFP antibody. Hybridization with an anti-RPT5 antibody was used as a loading control BES1 accumulation is independent of brassinosteroid levels. Six day-old 35S::BES-GFP seedlings were treated overnight with 100 nM epi-brassinolide (BL) or 0.5 µM brassinazole (BRZ). Material was collected at the indicated time points and analyzed by western blot hybridization with anti-GFP and -RPT5 antibodies In vivo analysis of PIF4-BES1 complex formation. Six day-old pPIF4::PIF4-HA seedlings grown under short day conditions were collected at ZT0, ZT1 and ZT8. PIF4-HA was immunoprecipitated with an anti-HA antibody and the pulled-down BES1 protein immunodetected with an anti-BES antibody. * Indicates a non-specific band by the primary antibody used to immunoprecipitation.
Inactivation of SAGA/TREX-2 affect type II telomere recombination.Figure 5A Mean senescence profiles of the est2∆ (n=7) and est2∆ sus1∆ (n=8) clones isolated from the same heterozygous diploid. The error bars are SDs. B Mean senescence profiles of the est2∆ (n=10) and est2∆ sac3∆ (n=10) clones isolated from one heterozygous diploid. The error bars are SDs. C Duration of the short telomere-induced crisis of the clones analyzed in A). The crisis period was determined as the number of days the cell population stayed arrested without notable increase in cell density. The average crisis period is plotted for each genotype. est2∆ ada2∆ data are from and are shown for comparison. The error bars are SD of n independent clones. est2∆ (n=17), est2∆ ada2∆ (n=11), est2∆ sus1∆ (n=8) and est2∆ sac3∆ (n=10). The p values are from two-tailed Student's tests. p=0,000087 for ada2∆, p=0,047 for sus1∆, p=0,049 for ada2∆.*P<0,05, ***P<0,0001 D Frequencies of the telomerase-independent survivor types formed by the clones shown in A.
cKO mice.Figure 1A) Electron microscopy of abnormal mitochondria in p32cKO hearts of 6 months old mice. These two figures are also presented in Figure EV1A along with other EM figures.　Scale bars, 1 µm. B) Western blot analysis of autophagy marker proteins (LC3-II and p62) in 6 months old WT and p32cKO hearts. The bands of LC3-II and p62 are increased in the p32cKO heart. GAPDH was used as an internal control. Quantification is shown on the bottom. These values are presented as mean ± SD (n = 6 mice per group). Student's t-test was performed on WT vs p32cKO, **p < 0.01. C) Immunostaining of LC3, p62 and multi-ubiquitin in heart tissues of 6 months old. The magnified image (right panel) shows the ring-like staining pattern in the p32cKO heart (indicated arrows). Scale bar, 20 µm. The panel related to LC3 immunostaining in p32cKO is also presented in Figure EV1B (top left) along with other immunostaining pictures. D) In the 6 months old p32cKO heart, p62 and multi-ubiquitin staining patterns were co-localized. Scale bar, 5 µm. E) In the 6 months old p32cKO heart, the staining patterns of p62 and the mitochondrial marker protein, pyruvate dehydrogenase (PDH), were co-localized. Scale bar, 5 µm. (F) Western blot analysis of ubiquitinated proteins using an anti-multi-ubiquitin antibody in the 6 months old WT and p32cKO heart (n = 4 mice per group). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, ***p < 0.005. (G) Western blot analysis of phosphorylated p62 (Se351 and Ser403) and non-phosphorylated p62 in the 6 months old WT and p32cKO heart. Relative expression of phosphorylated p62 is shown on the right. Error bars are presented as mean ± SD. Student's t-test was performed on eight WT mice vs seven p32cKO mice, ***p < 0.005.
Figure 1A. Schematic outline of genome-scale CRISPR-Cas9 screen for genes whose KO sensitizes human RPE-1 cells to MMC. LD20, 20% lethal dose; NGS, next generation sequencing. B. DrugZ analysis of sgRNA depletion in CRISPR screen in (A) following low-dose MMC treatment (n=2 technical replicates). FA genes are highlighted in blue; SCAI is highlighted in red. C. Immunoblot analysis of U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO cells stably reconstituted with Strep-HA-SCAI (U2OS/SCAI KO/Strep-HA-SCAI). D. Clonogenic survival of U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO/Strep-HA-SCAI cells subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments). E. As in (D), except that cells were treated with Cisplatin for 24 h (mean±SEM; n=3 independent experiments). F. U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO/Strep-HA-SCAI cells were treated or not with MMC (9 nM) for 48 h, fixed and co-stained with PCNA antibody and DAPI. Cell cycle distribution was analyzed by quantitative image-based cytometry (QIBC) (≥2000 cells analyzed per condition). Data from a representative experiment are shown. G. U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO/Strep-HA-SCAI cells were treated or not with MMC (90 nM) for 1 h, fixed 24 h later and co-stained with RPA2 antibody and DAPI. RPA2 foci were quantified by QIBC (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; *, p<0.05; ns, not significant, two-tailed paired t-test). H. As in (G), except that cells were co-stained with γH2AX antibody and DAPI (≥3000 cells analyzed per condition; mean± SD; n=3 independent experiments; *, p<0.05; **, p<0.01; ns, not significant, two-tailed paired t-test). I. Experimental workflow for metaphase chromosome morphology analysis (top) and representative images of metaphase spreads from indicated cell lines treated or not with MMC (bottom). DNA was stained with DAPI. Scale bars, 10 µm. J. Quantification of radial chromosomes in (I) (mean±SD; 180 cells analyzed per condition; n=3 independent experiments; *, p<0.05; **, p<0.01, ns, not significant, two-tailed t-test). K. Quantification of chromosomal breaks/gaps in (I) (mean±SEM; 99 metaphase cells analyzed for each condition pooled from 3 independent experiments; **, p<0.01; ****, p<0.0001, ns, not significant, Mann-Whitney U test).
Identification of oligonucleotides that change alternative splicing of the serotonin 2C receptor.Figure 1A. Pre-mRNA processing of the serotonin 2C receptor. Exons are shown as boxes, introns are lines. Due to the use of two alternative splice sites (PS, DS: proximal and distal splice site) exon Vb is alternatively used, resulting in two RNAs: RNA1 lacking and RNA2 including exon Vb. Alternative splicing patterns are indicated by dashed and dotted lines; constitutive splicing patterns are indicated by solid lines. The start codon is indicated using a line with circle. Alternative usage of exon Vb introduces a frameshift, leading to different stop codons being used (lines with rectangles in exon VI, red box in exon VI indicates a different reading frame). The proteins formed by RNA1 (truncated 5HT2C) and RNA2 (full-length 5HT2C) are indicated. Circles in the second intracellular loop of RNA2-protein depict three amino acids changed by RNA editing. The location of the epitope against the truncated 5HT2C is indicated with a Y. B. The truncated receptor encoded by RNA1 is endogenously expressed. HEK293 cells were transfected with a cDNA encoding the human truncated 5HT2C (RNA1). SDS-lysates from these transfected cells and from mousehypothalamus were analyzed by Western blot using an antiserum made against RNA1 (Figure EV1). Mousehypothalamus was exposed 10 times longer. In brain, a band around 75kDa of unknown origin is detected by the antiserum (indicated by a star). C. The minigene used for the oligo-walk is schematically shown; the arrow indicates the CMV promoter. The boxed area corresponds to the sequence shown below. The secondary structure of the serotonin 2C receptor pre-mRNA determined by SHAPE (Shen et al, 2013) is shown and the oligonucleotides used are indicated as lines. The binding site for SNORD115 (Kishore Stamm, 2006) is boxed and five adenosines undergoing RNA editing are highlighted. The exon/alternative exon border in the proximal splice site (GG) and the alternative exon/intron border (Gg, distal splice site) are underlined and marked in red. Dots indicate base pairing, including non-canonical G-U base pairs. D. RT-PCR analysis of the serotonin 2C receptor minigene after oligonucleotide addition. Below is a quantification of three independent experiments. A statistical evaluation showed significant differences between oligo#5 (p=0.00008) and oligo#5-3 (p= 0.0019) and control.
mutations show perturbations in the CoA synthesis pathway and diminished mitochondrial respiration.Figure 4Relative quantification of COASY mRNA in human PKAN primary cell lines. n= 5 for both genotypes and cell types Relative quantification of COASY/Coasy in fresh, blood-derived lymphocytes from human and mouse compared by genotype. n=51,35 for human (control, PKAN, respectively) and n=5 for both mouse genotypes. Extracellular flux analysis in human fibroblasts showing differences in OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) by genotype. Oligom; Oligomycin, FCCP; Carbonyl cyanide-ptrifluoromethoxyphenylhydrazone, 2-DG; 2-deoxy-glucose. Quantification of baseline OCR and ECAR is shown, as well. n=3 per genotype with four technical replicates. Complex I activity assay using fibroblast lysates. Cells were either treated with vehicle or 50μM pPanSH for three days and total protein extract was used for the Complex I Dipstick assay n=4 for both genotypes and treatment groups. Relative mRNA expression of mitochondrially-encoded genes important for oxidative phosphorylation from complex I (MT-ND1), complex III (MT-CYB), cytochrome C (MT-CO1), and ATP synthase (MT-ATP6) in human mutant fibroblasts versus control cells. n=3 for both genotypes. Calculation of active mitochondria using the JC-1 assay by genotype in control and PKAN lymphoblasts.
Left: when glucose is sufficient, AMPK is inactive and mTORC1 is active.figf8Left: when glucose is sufficient, AMPK is inactive and mTORC1 is active. The active mTORC1 phosphorylates Ulk1 on Ser 757 to prevent Ulk1 interaction with and activation by AMPK. Right: when cellular energy level is limited, AMPK is activated and mTORC1 is inhibited by AMPK through the phosphorylation of TSC2 and Raptor. Phosphorylation of Ser 757 is decreased, and subsequently Ulk1 can interact with and be phosphorylated by AMPK on Ser 317 and Ser 777. The AMPK-phosphorylated Ulk1 is active and then initiates autophagy.