EMBO/sd-character-level-ner huggingface dataset:
Genomewide gene expression analysis of iPSC‐derived DAn from PD patients and controls.Figure 3Heatmap showing the 437 differentially expressed genes (DEGs) detected in PD patients with either L2PD (L2) or sPD (s) as compared to controls (C) (n = 14), and density color code for gene expression levels. This analysis indicates that L2PD and sPD share similar gene expression changes with respect to controls. A total of 254 DEGs were upregulated in PD whereas 183 showed significant downregulation (Linear model with empirical bayes moderation of the variance similar to ANOVA with FDR‐adjusted P < 0.05). Venn diagram representing the number of DEGs in iPSC‐derived DAn from PD patients with respect to healthy subjects (n = 14) showing that most DMCpGs in L2PD were common to sPD. Technical validation of 10 top DEGs in PD iPSC‐derived DAn as compared to controls (n = 14) identified by genome wide gene expression study (upper heat‐map) and validated by real‐time qPCR (lower heat‐map). Relative gene expression and statistical significances were calculated using the ΔΔCt method and GAPDH, ACTB, and PPIA as endogenous controls. Pearson correlation analysis between microarray and real‐time qPCR data showed a high degree of correlation (r 2 = 0.99, P = 7.7 × 10−7) (bottom graph). L2, LRRK2‐associated PD; s, sporadic PD.
Figure 5(A) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with increasing concentrations of H2O2 as specified and probed with antibodies as indicated. (B) WB analysis of HeLa and TRIM16KO lysates of cells treated with 400 µM of H2O2 for different durations as indicated and probed with antibodies as shown. The red arrows indicate the time point where the cells were retreated with 400 µM of H2O2. L.E, Low exposure; H.E, High Exposure. (C) Densitometric analysis of NRF2 band intensity normalized with actin. (D) WB analysis of HeLa and TRIM16KO lysates of cells treated with 2.5 µM (2 h) of As2O3. (E-G) RNA isolated from HeLa and TRIM16KO cells, untreated or treated with MG132 (20 µM, 2h) were subjected to qRT-PCR with primers of genes as indicated. The fold induction in MG132 treated samples is calculated relative to untreated samples. Mean ±SD, n=3, *p < 0.05, **p < 0.005 (Student's unpaired t-test). RNA isolated from control and NRF2 knockdown HeLa cells transfected with control vector or Myc-TRIM16 subjected to qRT-PCR with primers of genes as indicated. Mean ±SD, n=3, **p < 0.005, ***p < 0.0005 (Student's unpaired t-test). RNA isolated from control and NRF2 knockdown HeLa cells transfected with control vector or Myc-TRIM16 subjected to qRT-PCR with primers of genes as indicated. Mean ±SD, n=3, **p < 0.005, ***p < 0.0005 (Student's unpaired t-test). (K) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with h increasing concentrations of H2O2 as specified and probed with antibodies as indicated. (L) Densitometric analysis of Ub smear intensity normalized with actin. Mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test). (M) Representative confocal images of control, Ubb and Ube2n siRNA transfected cells treated with H2O2 (200 µM, 2h) and IF was performed with Ub and p62 antibodies. Scale bar: 10 µm. (N) The graph shows the percentage of cells with protein aggregates. Data from ≥ 10 microscopic fields for each condition (40X), n=2, Mean ±SD. (O) Western blot analysis of Ubb and Ube2n siRNA transfected cell lysates with antibodies as indicated. (P) Pictorial representation of results
Inhibition of microglial phagocytosis ameliorates photoreceptor degeneration in rd10 mice.Figure 7A, B Effect of phagocytosis inhibition on retinal microglia ex vivo. Retinal explants acutely isolated from P21-23 rd10/CX3CR1GFP/+ mice were incubated in Ringer's solution (control), or Ringer's solution containing either the vitronectin receptor inhibitor cRGD peptide or its inactive analog cRAD (400 μM) for 1 h. GFP-labeled (green) ONL microglia in explants in cRGD transitioned from amoeboid morphologies containing multiple phagosomes to more ramified morphologies with fewer phagosomes. No morphological changes were detected in microglia incubated in cRAD (insets show morphologies at higher magnification). Scale bar, 40 μm. Quantifications of total number of phagosomes per 40× field (left) and the mean number of phagosomes per microglia (right) demonstrate significant reductions in phagocytic activity of cRGD, relative to cRAD, exposure (n = 8 imaging fields from in each condition). C-N Structural effects of in vivo inhibition of microglial phagocytosis. P20 somatamice were injected intravitreally in one eye with the phagocytosis inhibitor cRGD, and in the control contralateral eye with the inactive analogue, cRAD. At P23, prominent microglia infiltration in the ONL observed in control-injected eyes (C) was decreased in the contralateral cRGD-injected eyes (D). ONLmicroglia in control eyes demonstrated more numerous phagosomes (inset, arrows) (E) compared with cRGD-injected eyes (F). Pairwise comparisons of control- vs. cRGD-injected eyes demonstrated that phagocytosis inhibition significantly reduced the densities of infiltrating microglia (G) and microglialphagosomes (H), and mean phagosome number per microglia (I). ONL atrophy in control eyes (J) was more advanced compared to cRGD-injected eyes (K), with significantly greater mean ONL thickness (L), and ONLnuclear layers (M) in cRGD-injected eyes. Mean density of TUNEL+ONLnuclei was lower, with marginal significance (N) (n = 13 animals; measurements in cRGD-injected eyes normalized to contralateral control eyes, two-sided paired t-test). Scale bar, 40 μm. O-T Functional effects of in vivo inhibition of microglial phagocytosis. Representative dark-adapted ERG recordings at P26 (following intravitreal injections at P20 and P23) in a cRGD-injected eye (red) relative to the contralateral control eye (black) (O) showing that mean a- (P) and b-wave (Q) amplitudes were significantly greater in the cRGD-injected vs. control eyes at higher flash intensities. Representative light-adapted responses (R) illustrating that while mean light-adapted a-wave amplitudes (S) were slightly but not significantly greater in cRGD-injected eyes, mean b-wave amplitudes (T) were significantly greater at higher flash intensities (n = 13 animals, two-sided paired t-test, *P < 0.05, with exact values alongside).
Figure 7(A) Immunofluorescence staining against Iba-1 in Cx, Hip and Cb of 2 month-old Npc1nmf164 and wt mice. DAPI staining shows cell nuclei. Scale bars, 30 μm. (B) 3D rendered images obtained with Imaris Surface software from high magnification z-stack images of immunofluorescence staining against Iba-1 showing representative microglial morphology in Cx, Hip and Cb of Npc1nmf164 and wt mice. Scale bars, 10 μm. (C) Mean ± SEM number (upper panel) and area (lower panel) of Iba-1 positive cells in different brain regions from Npc1nmf164 and wt mice (n = 5 mice per group, >30 cells per mouse, Student´s t-test). (D) Immunofluorescence staining against Iba-1 in the Cb of a NPC affected and a control child. Scale bars, 50 μm. Graph shows mean ± SEM area of Iba-1 positive cells (n = 30 cells, Student´s t-test). (E) Immunofluorescence staining against MBP in Cb of Npc1nmf164 and wt mice. Scale bar, 50 μm. Graph shows mean ± SEM number of MBP aggregates per area (n = 5 mice per group, Student´s t-test). (F) High magnification images and their 3D rendered replicates from cerebellar Npc1nmf164 microglia stained against Iba-1 and MBP containing myelin debris (arrowheads). Scale bars, 10 μm (up), 2 μm (down). The orthogonal projection of the cells in the right panels confirms the presence of myelin debris inside the cell and underneath the plasma membrane. DAPI staining shows cell nuclei. Scale bars, 10 μm. (G) Mean ± SEM percentage of microglia showing internalised myelin debris in wt and Npc1nmf164 mice (n = 5 mice per group). (H) Immunofluorescence staining against F4/80 and CathB in Cb of Npc1nmf164 mice. Scale bar, 30 μm. The table shows mean ± SEM percentage of microglia presenting CathB staining in Npc1nmf164 and wt mice. Below, magnified images of the selected areas. Scale bar, 20 μm. (I) Immunofluorescence staining against Iba-1 and CathB in Cb of a NPC patient. DAPI shows cell nuclei. Scale bar, 50 μm. A merged magnified image of the selected area is shown in the right down panel, scale bar 10 μm. Table shows mean ± SEM percentage of microglia presenting CathB staining in NPC and control children.
Figure 2(A-H) Expression of Tecrl in mouse development. Expression is not observed in (A) the cardiac crescent at E7.5 but is prominent in (B) the inflow tract (ift) region at E8.5, particularly in (C) the left sinus venosus (lsv). (D) At E9.5, Tecrl expression is observed in all four chambers of the heart but is strongest in the inflow tract. (E-G) From E10 onwards, Tecrl is expressed in the somites. (H) At E14.5, Tecrl expression is present in the entire myocardium; [atrium (a), ventricle (v), neural tube (nt), lung (lu), right atrium (ra), left atrium (la), right ventricle (rv) and left ventricle (lv)]. (I) mRNA expression analyses of TECR and TECRL by RT-qPCR in human tissues demonstrates preferential expression of Tecrl in the heart; [Bone marrow (Bone M.), heart, skeletal muscle (Sk.Muscle), uterus, liver, spleen, thymus, thyroid, prostate, brain, lung, small intestine (Small I.) and colon] (J) Localization of MYC-Tecrl in COS-1 cells. The accumulation of MYC-Tecrl is perinuclear consistent with it being localized to the ER. (K-M) Co-localization of MYC-Tecrl and the ER marker Calnexin in H10 cells. Nuclei stained with DAPI. Scale bar: 10 μm.
Figure 4A-E. PP4c gene silencing promotes HDAC3 phosphorylation in the presence of KCl (A-C, arrows), both in the nucleus and the cytoplasm (D). Scale bar, 20 μm. (C,D) Data is expressed as mean fold change vs scrambled (scr) siRNA of fluorescent intensity of pHDAC3 or PP4c in GFP positive cells ± s.e.m. N=3 biological replicates. (**p<0.01; ***p<0.005) indicate significant difference versus scrambled siRNA (Student's t-test). (E) PCR against PP4c demonstrates silencing of PP4c mRNA. F-G. PP2a gene silencing inhibits the KCl induced dephosphorylation of HDAC3 as shown by immunoblotting of pHDAC3 (F). (G) Data is expressed as mean fold change of immunoblot band intensity ± s.e.m. N= 3 biological replicates (*p<0.05; ****p<0.001) indicate significant difference versus control siRNA (Student's t-test). H-I. Genetic modification of activity-dependent phosphorylation of serine 424 of HDAC3 affects DRG neurite outgrowth. (H) DRG neurite outgrowth after transfection of control (V5 control), WT HDAC3-V5, phospho-dead HDAC3 (S424A)-V5 or phospho-mimetic HDAC3 (S424D)-V5 and after 24h in culture. Scale bar, 50µm. (I) Data is expressed as mean ± s.e.m. N= 8-9 biological replicates, approximately 35 V5 positive cells each. * p<0.05, ** p<0.01, *** p<0.005 indicate significant difference (One way ANOVA followed by Bonferroni).
Figure 6Brcc3 expression confirmed by RNA-seq [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)]. Brcc3 expression confirmed by RT-qPCR [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)]. Brcc3 expression confirmed by western blotting [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)]. Confirmation of Brcc3 expression from knockdown samples in by RT-qPCR (technical duplicates) Confirmation of Brcc3 expression from knockdown samples in western blotting Nanog HR assay with Brcc3 knockdown (three biological replicates). The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown, as shown in Figure 2C. The combined number of GFP-positive cells and total number of colonies are shown (please note that total cell number of #A13 for drug screening was 3.67×107 because of contamination of 1 out of 12 dishes, compared with the other colonies for which 4×107 cells were used).
Figure 4(A - D) BACE1 endocytic trafficking followed in siBin1- and siControl-treated neurons expressing BACE1-GFP N-terminally tagged with FLAG using a pulse/chase assay with M1, an anti-FLAG antibody, analysed by epifluorescence microscopy.(A) Endocytosed BACE1 detected with M1 (15 min pulse; top panels) and BACE1-GFP (bottom panels). Arrowheads identify axons (Ax) and dendrites (Dd) magnified on the right. Scale bars, 10 µm.(B) The amount of endocytosed BACE1 per cell body, dendrite and axons was normalized to BACE1-GFP expression in the cell body and quantified as percentage of siControl (n=3, NsiControl=17, NsiBin1=19, mean ± SEM).(C) Recycled BACE1 detected with M1 (15 min pulse, acid stripping and 20 min chase; top panels) at the plasma membrane of non-permeabilized neurons and BACE1-GFP (bottom panels). Arrowheads identify axons (Ax) and dendrites (Dd) magnified on the right. Scale bars, 10 µm.(D) The amount of recycled BACE1 analysed as in (b) (n=4, NsiControl=31, NsiBin1=38, ***PCB=0.0002 and ****PAx<0.0001 siBin1 vs. siControl, t-test; ##P=0.0014 Axon vs. Dendrite, 1-way ANOVA with Tukey multiple comparisons test; mean ± SEM). (E and F) Bin1 (green) and BACE1 (magenta) localisation in axons (E) and dendrites (F) of neurons expressing BACE1-GFP immunolabelled at 11 DIV with anti-Bin1, and analysed by spinning-disk confocal microscopy. Scale bars, 10 µm. The white squares are magnified on the right. Scale bars, 1 µm.(G) Quantification of colocalisation between Bin1 and BACE1 in axons and dendrites (n=3, NDd=17, NAx=21; ***P=0.0006 Axon vs. Dendrite, t-test; mean ± SEM). (H and I) Bin1 (green) and Rab5 (magenta) localisation in axons (H) and dendrites (I) of neurons expressing Rab5-GFP analysed as in (E and F). Scale bars, 10 µm. The white squares are magnified on the right. Scale bars, 1 µm.(J) Quantification of colocalisation between Bin1 and Rab5-positive endosomes in axons (Ax) and dendrites (Dd) (n=3, NAx=39, NDd=16, ****P<0.0001, t-test, mean ± SEM).
Structure of the MSP domain of MOSPD2 in its unbound form, and in complex with a conventional FFAT and a Phospho-FFAT.Figure 8A-C: Ribbon diagram of the MSP domain of MOSPD2 in its unbound form (A), in complex with the conventional FFAT of ORP1 (B), and in complex with the Phospho-FFAT of STARD3 (C). D: Surface representation of the MSP domain of MOSPD2 in complex with the conventional FFAT of ORP1. Data information: Phosphorous, nitrogen, and oxygen atoms are colored in orange, blue, and red, respectively. Carbon atoms are shown in slate blue, grey and rose in the MSP domain, the classic FFAT motif, and the Phospho-FFAT motif, respectively. the protein surface is colored according to the YRB scheme, showing hydrophobic, negatively and positively charged atoms in yellow, red and blue, respectively; the other atoms are in white E: Close-up view of the structure near the conventional FFAT motif highlighting critical residues (in stick model) of human MOSPD2 present in the binding interface. Data information: Phosphorous, nitrogen, and oxygen atoms are colored in orange, blue, and red, respectively. Carbon atoms are shown in slate blue, grey and rose in the MSP domain, the classic FFAT motif, and the Phospho-FFAT motif, respectively. red spheres are water molecules. F: Surface representation of the MSP domain of MOSPD2 in complex with the phosphorylated FFAT of STARD3. Data information: Phosphorous, nitrogen, and oxygen atoms are colored in orange, blue, and red, respectively. Carbon atoms are shown in slate blue, grey and rose in the MSP domain, the classic FFAT motif, and the Phospho-FFAT motif, respectively. the protein surface is colored according to the YRB scheme, showing hydrophobic, negatively and positively charged atoms in yellow, red and blue, respectively; the other atoms are in white G: Close-up view of the structure near the Phospho-FFAT motif highlighting critical residues (in stick model) of human MOSPD2 present in the binding interface. Data information: Phosphorous, nitrogen, and oxygen atoms are colored in orange, blue, and red, respectively. Carbon atoms are shown in slate blue, grey and rose in the MSP domain, the classic FFAT motif, and the Phospho-FFAT motif, respectively. red spheres are water molecules. H,I: Close-up view of the hydrophobic pockets interacting with the 2nd residue (F207) (H) and the 9th residue (F214) (I) of the Phospho-FFAT of STARD3; residues of MOSPD2 constituting the pockets are indicated. Data information: Phosphorous, nitrogen, and oxygen atoms are colored in orange, blue, and red, respectively. Carbon atoms are shown in slate blue, grey and rose in the MSP domain, the classic FFAT motif, and the Phospho-FFAT motif, respectively. the protein surface is colored according to the YRB scheme, showing hydrophobic, negatively and positively charged atoms in yellow, red and blue, respectively; the other atoms are in white J: Superposition of the structures shown in (E) and (G) showing the similar trajectories of the peptides.
AAV-BR1-iCre-mediated gene recombination in Ai14 Cre reporter mice.Figure 6Two weeks after vector injection (1.8 x1011 genomic particles/ animal), into 16-weeks old mice, Cre-mediated gene recombination driven by the CAG promoter was observed mainly in brain endothelial cells (lower panel, red). No recombination was observed in control animals without BR1-iCre-virus injection (upper panel). CD31 (green) was used as a marker for brain endothelial cells. Panels show representative examples of n = 3 animals. Scale bars represent 250 µm.
Figure 4A. Northern blots showing levels of β-globin reporter mRNAs in wild-type HeLa Tet-off cells and UPF3B knockout HeLa Tet-off cells. β39 is a tetracycline (Tet)-inducible reporter with a PTC at codon 39 whose levels are shown at different timepoints after transcriptional shut-off (chase) as indicated above each lane. β-GAP is a stable, constitutively-expressed, longer β-globin mRNA used as transfection control. Proteins overexpressed (OE) in each condition are indicated on top and reporter mRNA half-lives (t1/2) along with standard errors of means are on the bottom. B. Schematic of human UPF3A, UPF3B and the UPF3A chimeric proteins where UPF3A domains are replaced by the corresponding domains from UPF3B (see material and methods for detailed domain definition). Previously characterized UPF2 binding domain (UPF2-BD) and EJC-binding motif (EBM) are shown. C. Northern blot showing steady-state levels of β39 NMD reporter and β-GAP control in HeLa Tet-off UPF3B knockout cells upon transient overexpression of wild-type UPF3 proteins or different UPF3A chimeric proteins indicated above each lane. Below each lane, relative fold-change (Rel FC) indicates β39 reporter levels (normalized to β-GAP control) as compared to the normalized β39 reporter levels in UPF3B expressing cells. D. Immunoblot showing levels of EJC proteins or HNRNPA1 in input or EIF4A3-IP from 3DKO#2 cells stably expressing different UPF3 proteins or EGFP as a control as indicated above each lane. Relative IP of FLAG-tagged proteins are quantified against EIF4A3.
Figure 9Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days. A Phenotypic presentation (top) and H&E staining (bottom) of dorsal skin. Scale bar: 100 μm. Left: H&E staining data; right: statistical data (mean ± SEM). Data information: Data are represented mean ± SEM. P values are determined by two-tailed Mann-Whitney U test *P<0.05, **P<0.01. Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days. B Clinical scores plotted with mean ± SEM. *denotes statistical significance when compared with the Tlr7wt/wt group. Data information: Data are represented mean ± SEM. P values are determined by Tukey multiple comparison test *P<0.05, **P<0.01. Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days. C Body weight change plotted with mean ± SEM. *denotes statistical significance when compared with the Tlr7wt/wt group. Data information: Data are represented mean ± SEM. P values are determined by Tukey multiple comparison test *P<0.05, **P<0.01. Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days. D Quantitative PCR analysis of mRNA encoding IL-23/IL-17A axis cytokines and other psoriasis-related cytokines in the dorsal skin. Results were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed Mann-Whitney U test *P<0.05, **P<0.01. Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days. E Representative p-p65 staining of the dorsal skin. Scale bars: 100 μm. Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days. F The graphic illustration of the mechanism of SHP2 accelerating the development of psoriasis. In macrophages of normal skin, when stimulating by TLR7 agonist ssRNA (e.g., IMQ, R848), TLR7 is phosphorylated and SHP2 is recruited to dephosphorylate TLR7, then TLR7 is ubiquitinated, next activates downstream NF-κB signaling. However, in infiltrated macrophages of psoriatic skin, SHP2 is overexpressed. Hyper-dephosphorylation of TLR7 at Tyr1024 by SHP2 leads to hyper-ubiquitination of TLR7 and overactivation of NF-κB activation, resulting in unrestrained skin inflammation.
Figure 7A. Loss of Exo1 suppresses srs2Δ defect in multiple DSB repair mechanisms involving extended resection of broken ends: SSA (left), de novo telomere addition (middle) and BIR (right). Average ± SD (n=4) is shown for each genotype for all experiments presented. Strains used: SSA: NK4691-NK4693; NK4805-4808; NK5070-5073; NK5074-5080; de novo t.a: NK1264; NK2375, NK2376; NK2016, NK2017; NK5244, NK5245; BIR: NK4070, NK4079; NK5321, NK5322; NK5446, NK5447; NK5448, NK5449. B. The effect of HU on the survival of SRS2 and srs2Δ cells after DSB induction (top panel). The control experiment involving similar HU treatments in the absence of DSBs is shown in the bottom panel. Average ± SD (n=4) is shown for each genotype. Strains used in B-C: NK4691-4693; NK4805-4808. C. Formation of fragment L in SRS2 and srs2Δ cells in the presence and absence of 25 mM HU. Average ± SD (n=3) is shown for each time point.
Figure 6Bub1 CR cells complemented with the indicated Bub1 constructs and time from NEBD to exit in nocodazole measured by time-lapse microscopy. Mean (red line) and standard error of mean (black bar) indicated. Kinetochore levels of Mad1 in Bub1 CR cells complemented with the indicated YFP tagged Bub1 constructs. Mad1 levels are normalized to YFP signal. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown. Still images of unperturbed mitosis of HeLa cells transfected with Venus-Bub1 4xCD1. CFP-Histone 3 was used as the chromosome marker (the middle panel) and Venus-Bub1 4xCD1 was shown in the bottom panel. Scale bar, 5 μm. HeLa cells transfected with the indicated Bub1 constructs and time from NEBD to exit determined during an unperturbed mitosis. The time from NEBD to exit was analyzed in each condition. Mean (red line) and standard error of mean (black bar) indicated. (Mann Whitney test, ns: non-significant). A representative result from at least 3 independent experiments is shown.