EMBO/sd-character-level-ner huggingface dataset:
Figure 6(A) HOIP is recruited to disease-associated protein aggregates formed by Ataxin-3-Q84, SOD1 G85R, TDP-43 Q331K or Optineurin R96L. SH-SY5Y cells co-expressing disease-associated proteins (green) and HOIP (red) were analyzed by immunocytochemistry; DAPI (blue). Scale bar, 20 µ Linear ubiquitin accumulates at disease-associated protein aggregates under endogenous LUBAC expression. M1-linked ubiquitin (red) and disease-associated proteins (green) were analyzed by immunocytochemistry in SH-SY5Y cells using the 1E3 antibody (Millipore) (B) DAPI (blue). Scale bar, 20 µm. Linear ubiquitin accumulates at disease-associated protein aggregates under endogenous LUBAC expression. M1-linked ubiquitin (red) and disease-associated proteins (green) were analyzed by immunocytochemistry in primary striatal neurons using the 1F11/3F5/Y102L antibody (Genentech) (C). DAPI (blue). Scale bar, 20 µm. (D) SDS-soluble SOD G85R and TDP-43 Q331K species are modified by linear ubiquitin chains. HEK293T cells were co-transfected with disease-associated proteins and either the plasmids indicated or with control siRNA or OTULIN siRNA. 48 h after transfection cells were lysed under denaturing conditions (1% SDS), followed by immunoprecipitation of SOD G85R and TDP-43 Q331K via the HA tag (1% Triton X-100, 0.1% SDS). Immunopurified proteins were detected by Western blotting using the M1 ubiquitin-specific antibody 1E3.
Figure 2A Plots of relative mRNA abundance vs circadian time (CT) for clock genes determined by qRT-PCR in whole head extracts of Nipped-A RNAi (timG4/+;Udcr2/UNipped-ARNAi-1/+) and control (timG4/+;Udcr2/+) collected on DD1 (tim/cry, n=5; Pdp1ε/per/clk/cyc, n=3; vri, n=6). For each time series, the value of the lowest time point was set to 1. B Western blots of proteins from whole head extracts of Nipped-A RNAi and control flies collected on DD1 and probed with TIM, PDP1ε and PER antibodies. C Quantification of TIM (n=5), PDP1ε (n=3) and PER (n=3) protein levels of blots in (B). TIM, PDP1ε and PER protein levels were normalized to that of HSP70. For each time series, the value of the control at the peak time point was set to 1. D Brains from Udcr2/+;cryG4-16/+ and Udcr2/+;cryG4-16/UNipped-ARNAi flies collected at CT0, 4, 8, 12, 16, 20 on DD1 were immunostained with TIM (green) , PDP1ε (red), PER (green) and PDF (red or green) antisera. The scale bar represents 10µm. E Quantification of TIM, PDP1ε and PER protein levels in the s-LNvs of images in (D) (TIM: CT8: Udcr2/+;cryG4-16/+, n=89; Udcr2/+;cryG4-16/UNipped-ARNAi, n=58; CT20: Udcr2/+;cryG4-16/+, n=130; Udcr2/+;cryG4-16/UNipped-ARNAi, n=80; PDP1ε: CT4: Udcr2/+;cryG4-16/+, n=46; Udcr2/+;cryG4-16/UNipped- RNAi, n=26; CT16: Udcr2/+;cryG4-16/+, n=65; Udcr2/+;cryG4-16/UNipped-ARNAi, n=41; PER: CT0: Udcr2/+;cryG4-16/+, n=90; Udcr2/+;cryG4-16/UNipped-ARNAi, n=58; CT12: Udcr2/+;cryG4-16/+, n=86; Udcr2/+;cryG4-16/UNipped-ARNAi, n=64).
Figure 3Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA. Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA. SUMOylation assay as in (A) with SUMO provided as XRCC1-SUMO instead of free SUMO. Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA. SUMO transfer as depicted in (B) with additional APE1, POLß, LIGIII, and TDG. Immunoblot analysis of XRCC1, TDG, TDG-SUMO in TDGwt (wt), TDGsnm (snm) and XRCC1null mESCs (null). Immunoblot analysis of TDG and TDG-SUMO in TDGwt (wt) and XRCC1null mESCs (null) exposed to H2O2 as indicated. Top: Schematic of the SUMO-mediated TDG-BERosome (grey oval) formation and DNA processing. Bottom: Reconstitution of active DNA demethylation on a 5caC containing synthetic DNA double-strands in presence of dynamic SUMO-conjugation/de-conjugation. Left panels lanes 3-14: BER reconstitution with XRCC1 or XRCC1-SUMO as indicated. Right panels lanes 15-21: BER reconstitution with free SUMO or XRCC1-SUMO as SUMO donor as indicated. ds59merDNA substrates: lane 1-2 CG/CG HpaII methylation sensitive restriction site, cleavable by HpaII; lane 3-6, 8-14, 15-17, 21 HpaII methylation sensitive restriction site restriction site with a caC (5caCG/CG), not cleavable by HpaII; lane 7 and 20 repaired Product (CC/CG) that is cleavable by HpaII after excision/repair of caC/CG to CG/CG by BER. Percentage of total signal of fully repaired product is indicated. ss59merDNA, single stranded DNA.
Figure 7A In CNS, viable cyst antigen and excretory-secretory products (CLys/CSN) promote a phagocytic phenotype from resident brain microglia expressing low Iba-1 but high CCL2, IP-10, IL-10 and PGE2, a potential synaptic inhibitor. After cyst degenerates, dead cyst materials and released vesicular fluid (CVF) interact with microglia and polarize an inflammatory and non-phagocytic phenotype with high expression of Iba-1 and the pro-epileptic TGF-ß. B In periphery, GDH/IDH from CLys/CSN license tolerogenic CD163hiCD206hi monocytes to express IL-10 and PGE2, which can then bind to IL-10R and EP2 and EP4 on CD4+ T cells driving the expansion of CD25hiFoxP3+CTLA4+ Tregs expressing IL-10 and the CNS homing receptors CCR6 and CCR7. When dead cyst products (CVF) reach the periphery, not yet identified active molecule promotes an inflammatory phenotype in monocyte and mature dendritic cells with high expression of CD68, CD80, CD86 and MHCII, while selectively inducing apoptosis in macrophages associated with significant synthesis of TNFα and the eicosanoid 12,13-DIHOME. M0=monocyte; M=macrophage; DC=dendritic cell; GDH=Glutamate dehydrogenase; IDH=Isocitrate dehydrogenase.
Figure 2A. HEK293 cells treated with non-targeting (Ctrl) or FIP200 siRNA were co-transfected with EGFP-LC3 and either empty vector control (EV), Myc-C9orf72S or Myc-C9orf72L. 24 h post transfection cells were treated with vehicle or 100 nM BafA1 for 6 h. Samples were lysed and subjected to SDS-PAGE and immunoblot. Autophagy levels were determined by immunoblot for EGFP-LC3-I and II. Expression of Myc-C9orf72 was confirmed using anti-Myc (* indicates a nonspecific band). FIP200 knockdown was confirmed using anti-FIP200 antibodies. α-tubulin was used as loading control. B. HeLa cells treated with non-targeting (Ctrl) or FIP200 siRNA were co-transfected with empty vector (EV), Myc-C9orf72S or Myc-C9orf72L (red) and EGFP-LC3 (green) to label autophagosomes. As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Autophagy was quantified as the number of EGFP-LC3 positive autophagosomes per cell from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test: **** P ≤ 0.0001, N (cells) = Ctrl/EV: 73; FIP200/EV: 76; Ctrl/EV/Torin1: 73, FIP200/EV/Torin1: 70; Ctrl/C9orf72L: 71; FIP200/C9orf72L: 74; Ctrl/C9orf72S: 72; FIP200/C9orf72S: 72). Scale bar = 20 µm. FIP200 knockdown was confirmed by immunoblot (Appendix Fig S2).
Optimization mutation analysis for D-arabinose evolution experiments.Figure 3Structural mutations observed in sequencing data of Experiments (Exp.) 1 and 2 (green) as well as residues previously identified as important for binding L-arabinose (blue) are highlighted on one chain of the AraC homodimer protein structure. The six base pair deletion observed in Exp. 1 appears to be most clearly linked to affecting substrate binding. Expression data (RNAseq) for significantly differentially expressed genes (q-value < 0.05, FDR-adjusted p-value, n=2 biological replicates for each condition). Scatter plot shows log2(fold change) of gene expression data comparing endpoint to initial populations for Exp. 1 and Exp. 2 (grey dots) with the location of the gene in the reference genome as the x-axis. Those genes that are associated with AraC transcription units are highlighted (red dots for Exp. 1 and blue dots for Exp. 2). Above the plot, the transcription units are labeled green if AraC activates expression (in the presence of arabinose) or red if AraC represses expression of those genes. The proposed two pathways for metabolizing D-arabinose. The pink pathway is enabled by the optimizing mutations observed in araC. Growth rate analysis of various weaned (starting point of static phase) and optimized (endpoint of static phase) strains with or without fucK or araB genes knocked out. Strains were grown in triplicate (n=3) on M9 minimal media with D-arabinose as the sole carbon source. The colored bars represent the calculated mean growth rate and the error bars represent the standard deviation. The p-values reported were calculated using a two-sided Welch's t-test.
PBX1 improves the differentiation of neural progenitor cells towards mDA fate.Figure 4(A) Scheme of the differentiation protocol for hNES cells. The AF22 cells (NES cells derived from induced pluripotent stem cells) were infected at day 2 with TET-on PBX1 lentiviral particles, and doxycycline (DOX) treatment started the day after. (B) Representative immunofluorescence microscopy/confocalimages showing that PBX1-overexpression increases the number of TH+neurons after 8 days of differentiation of hNES cells (AF22 line derived from induced pluripotent stem cells). (C) While the number of cells (Dapi+) in both conditions did not change, the number of TH+cells increased 2.3 fold in PBX1-overexpressing cells. (D) TH+cells derived from the PBX1-overexpressing hNES cells were also positive for mDAn markers such as LMX1A and PITX3. All scale bars, 40µm. Error bars represent mean ± standard deviation. Biological replicates per condition and p-value (T-test) are indicated in the graphs.
Figure 3Body weight after 16 h fasting and 6 h chow refeeding at 22°C (n = 6 animals per group) Food intake during 6 h refeeding (n = 6 animals per group). Body temperature assessed after 16 h fasting and subsequent refeeding for 6 h (n = 6 animals per group). Blood glucose (n = 6 per group). Serum insulin and corticosterone levels determined by ELISA (n = 6 animals per group). Serum NEFA and triglycerides concentration in Flox and GRBATKO mice after fasting and refeeding (n = 6 animals per group).
a) Pull-down experiments with semisynthetic HA-CK2α protein and recombinant Pin1.figf3(a) Pull-down experiments with semisynthetic HA-CK2α protein and recombinant Pin1. CK2α was immobilized using HA-specific (anti-HA) antibodies bound to protein G beads. Pin1 was incubated with immobilized CK2α for 20 min at 4 °C. (b) Coimmunoprecipitation of endogenous Pin1 with CK2α. Pfa-containing and unmodified semisynthetic HA-CK2α proteins were spiked into REF52 cell lysates and immobilized using HA-specific antibodies. Input samples are shown for loading control. (c) Pull-down experiments with Pfa344 HA-CK2α protein and recombinant Pin1 in the presence of Pin1-specific or nonspecific IgY. (d) Relative CK2α protein concentration over time following microinjection of Pfa344 CK2α with Pin1-specific IgY or nonspecific IgY (control). Data represent mean values ± s.d. Full gels corresponding to those in panels a-c are in Supplementary Figures 10 and 11.
Comparison and architecture of the human and yeast GID complexes.Figure 5A. ARMC8β (difference map shown as red surface) binds to the scaffold, distal from the WDR26 WD40 propeller (the 5-subunit hGID map is shown in grey). B. Homology models of RanBP9 (SPRY and LisH domains) (magenta), TWA1 (blue) and the WD40 domain of WDR26 (green) are shown fitted into the map of the 5-subunit hGID complex. A homology model of ARMC8β fitted into the difference density is shown in red. The approximate position of the RING-domain containing subunits, MAEA or RMND5a, is indicated with a dotted line. C. The yeast GID complex was superimposed on the cryo-EM map of the 5-subunit hGID complex. The Gid4 (orange), Gid1 (magenta), Gid8 (blue), Gid5 (dark red) and Gid9 (gray) subunits of the yeast structure are shown in the same orientation as the hGID complex. Gid9 (gray) subunits of the yeast structure are shown in the same orientation as the hGID complex. D. Spatial arrangement of yeast Gid4 with respect to WDR26 is shown in context of the hGID complex.
Figure 6A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm. B. Quantification of the number of CETN3 foci per spermatocyte (n = 30) at each meiotic prophase substage. C. Centrosome (PCNT) separation was assessed in Aurka; Plk1 dKO spermatocytes. Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation classifications D. Aurora A- and PLK1-mediated regulation of meiotic centrosome biogenesis during spermatogenesis. After spermatocytes enter meiotic prophase and complete the first round of centriole duplication, presumably stimulated by PLK4, the centrioles must mature in order to effectively act as MTOCs during the first meiotic division. PLK1 and AURKA regulate this maturation process by ensuring the recruitment of various maturation factors including TACC3, CEP164, and γ-tubulin (γ-TUB). PLK1 and AURKA are also required for the efficient degradation of β-catenin (β-CAT), a necessary step to ensure centrosome separation prior to bipolar spindle establishment at metaphase I. The mechanism by which the first and second rounds of centriole duplication are restricted to early meiotic prophase I and interkinesis, respectively, is not understood and warrants further investigation. However high levels of PLK1 are necessary to avoid a premature second round of centriole duplication, which insinuates that PLK1 counteracts the centriole duplication function of PLK4.
a, Survival of wild-type, rpd3&Dgr, hda1&Dgr and rpd3&Dgr hda1&Dgr strains after 4NQO, MMS and HU treatment.f5a, Survival of wild-type, rpd3Δ, hda1Δ and rpd3Δ hda1Δ strains after 4NQO, MMS and HU treatment. Error bars represent s.d. calculated from seven independent experiments. b, HO was induced in G2 wild-type, hda1Δ, rpd3Δ and hda1Δ rpd3Δ cells and western and Southern blot analyses were performed. c, Wild-type SAE2::PK and hda1Δ rpd3Δ SAE2::PK cells were grown as in b and western blot was performed as in Fig. 4b. d, e, Wild-type SAE2::PK and gcn5Δ SAE2::PK strains were grown as in b and after 30 min of HO induction either VPA (d) or rapamycin (e) was added or not. Western blot was performed. f, Percentage of viability of wild-type, atg1Δ, rpd3Δ hda1Δ and atg1Δ rpd3Δ hda1Δ strains. Error bars represent s.d. calculated from four independent experiments.
Figure 2(A) MITOL-HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild-type HeLa cells or HeLa cells stably expressing GFP-Parkin were transfected with MITOL-HA, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-HA and anti-Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. (B) Correlation statistics for the localization of MITOL-HA and Tom20 in the absence or presence of GFP-Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. (C) MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag-MITOL were transfected with a GFP-Parkin plasmid or the pEGFP-C1 vector, treated with 15 µM CCCP +/- 10 µM MG132 for 3 hours, and then immunoblotted with anti-Flag, anti-ubiquitin, and anti-tubulin antibodies. Black arrowheads indicate 3Flag-MITOL in the upper panel and mono-ubiquitin in the middle panel, respectively. (D) The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that Mitofusin2 (MFN2) underwent rapid degradation within 3 hours of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria-rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 hours 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor ZVAD-FMK (10 μM) was added to cells along with valinomycin to prevent cell death. (E) Quantification of 3Flag-MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 µM valinomycin + ZVAD-FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m relative to untreated samples in three biological replicates. (F) Pre-existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 hours to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 hours, cells were immunostained using anti-Flag, anti-Pex14 (peroxisomal membrane protein), and anti-Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 µm.
Figure 2a, DARPP-32 (D32) immunostaining shows that striatal neuron cultures consist mainly of D32-positive medium spiny neurons (MSNs). 40x magnification, scale bar = 50 µm. MAP2 marks neurons, Hoechst (Hoe) stains DNA. n = 3 cultures from independent mouse litters. b, 100x magnification images show intracellular distribution of D32 in D32-positive neurons. Scale bar = 10 µm. c, MSN cultures at DIV11 were treated for 2h with either 10 µM of the D1-agonist SKF-38393 (SKF) alone or with SKF and 10 µM of the selective D1-antagonist SCH-23390 (SCH). qPCR was performed to determine the relative mRNA levels of the immediate early genes cFos and Arc. Mean + SEM; one-way ANOVA with Holm-Sidak post-hoc correction; p** < 0.01, p**** < 0.0001; n = 3 independent cultures as biological replicates. d, MSNs were depolarized with potassium chloride (HiK, 55 mM) or treated with the D1-agonist SKF-38393 (10 µM) for 1h, 2h or 4h. Subsequently, qPCR was performed to assess immediate early gene mRNA levels. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test; indicated p-values for induction over control for each treatment; p* < 0.05, p** < 0.01, p***<0.001; n = 4 independent cultures as biological replicates. e, To assess differences in mRNA load induced by HiK vs SKF, the values from the experiments in panel d) were summed up over time for each treatment and the sums for each treatment were divided by each other to yield the ratio IHiK/ISKF. Values for this ratio were compared through one-way ANOVA with Tukey's post-hoc test; mean + SEM; p*<0.05, p*** < 0.001; n = 4 independent cultures as biological replicates. f, immunohistochemistry analysis shows that cells in human iPSC-derived forebrain organoids express the forebrain neuron marker FOXG1 (green) and the neuron differentiation marker MAP2 (red). Hoe = Hoechst. Scale bar = 30µm. g, Differential induction of Npas4 through HiK and not forskolin in hiPSC-derived forebrain organoids. Organoids were treated with HiK (55 mM) or the PKA activator forskolin plus TTX (Fsk, 10 µM; TTX 1 µM) for 1h before qPCR analysis was performed to assess mRNA levels of Npas4 and Arc. Mean + SEM; n = 7 organoids in control condition, n = 5 organoids for HiK, n = 5 organoids for Fsk/TTX. One-way ANOVA with Tukey's post-hoc test, ns = not significant, p* < 0.05, p***<0.001.
Figure 1A. ROCK2 immunohistochemistry stained sections of normal and stages I, II and III cases of human pancreas adenocarcinoma. Scale bar = 50 µm (left). Histoscores of ROCK2 staining (right) in normal pancreas (n=5), and pancreas adenocarcinoma stage I (n=22), stage II (n=46) and stage III/IV (n=4). One-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test. B. Overall survival of 107 patients without alterations versus 37 patients with ROCK1 and/or ROCK2 gene amplification, significantly increased mRNA or truncation mutation from TCGA research network. Survival p value determined by Log-Rank test. C, D. Log2 median-centered ROCK1 or ROCK2 RNA expression in normal (n=39) vs PDAC (n=39), normal (n=5) vs pancreatic adenocarcinoma (PAC) (n=12), or normal (n=6) vs pancreatic carcinoma (PC) (n=11) samples from indicated studies. Exact p values determined by Mann-Whitney test. E. ROCK1 and ROCK2 mRNA expression in human PDAC (n=146) from the TCGA research network. Significance (p value) of slope deviation from 0 determined by Deming regression. F. Representative hematoxylin and eosin (H&amp;amp;E) and ROCK2 immunohistochemistry stained sections of normal mouse pancreas, acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN) stages 1-3, and PDAC from indicated genotype mice. Scale bar = 50 µm. G. Quantification of ROCK2 staining in epithelial cells of normal, ADM/PanIN1 (KC), PanIN2 (KPC), PanIN3 (KPC) and PDAC (KPC) pancreatic tissue (n=5 mice per group). One-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test. H. Survival analysis of Pdx1-Cre; LSL-KRasG12D/+; LSL-Trp53R172H/+; LSL-ROCK2:ER (RKPC) mice without (n=21) or with conditional ROCK activation with tamoxifen citrate (n=19). Survival p value determined by Log-Rank test.