EMBO/sd-character-level-ner huggingface dataset:
Figure 6(A) Mean ± SEM body weight of ASMko and wt mice treated or not with Ca074Me at the indicated time during treatment (n = 7 mice per group). Performance in the Rotarod test of ASMko and wt mice treated or not with Ca074Me for 1 month expressed as mean ± SEM time spent in the rotating rod on each of four trials (B) Performance in the Rotarod test of ASMko and wt mice treated or not with Ca074Me for 1 month expressed as mean ± SEM time of the four trials (C) (D) Immunofluorescence staining against the PC marker Calbindin in the posterior lobules of the Cb in ASMko and wt mice treated or not with Ca074Me for 1 month. DAPI staining shows cell nuclei. Scale bar, 200 μm. (E) Mean ± SEM number of Calbindin positive cells in the posterior zone (lobules IX-X) of the Cb from the different mouse groups (n= 7 mice per group Two-way Anova, Bonferroni post hoc).
ULK1 interacts with FIP200.Figure 3.(A) HEK293T cells were cotransfected with FLAG-FIP200 and HA-ULKs. Cell lysates were subjected to immunoprecipitation (IP) using antibodies against FLAG. The resulting precipitates were examined by immunoblot (IB) analysis with the indicated antibodies. The asterisk indicates nonspecific band. (B) Lysates of MEFs were immunoprecipitated with anti-ULK1 or anti-FIP200 antibody or preimmune rabbit serum, and the resulting precipitates were subjected to immunoblot analysis with antibodies against ULK1 and FIP200. (C) Schematic representation of ULK1 mutants used in D. (D) HEK293T cells were cotransfected with FLAG-FIP200 and various ULK1 mutants and analyzed as in A using anti-HA and anti-FLAG antibodies. (E) NIH3T3 cells stably expressing GFP-ULK1 (left) and GFP-ULK1ΔC (right) were cultured in starvation medium for 120 min. Bar, 20 μm.
Wde controls Egg function by recruiting the protein to the chromatin in OSCs.Figure 6Heat map of Egg ChIP-seq within a 10kb window centered on Egg peaks. Note that ChIP peaks was called using the ChIPed and Input samples Metaplots showing Egg ChIP-seq signals for genomic regions around Egg peaks. Displayed are fold-changes in Egg occupancy normalized to input. Heatmaps of Egg ChIP-seq signal within 10 kb windows centered on Egg peaks in control (siEGFP) and Wde-depleted (siWde) OSCs. Metaplots showing Egg ChIP-seq signals for genomic regions around Egg peaks. Displayed are fold-changes in Egg occupancy normalized to input. Metapolts showing Egg ChIP-seq signals for genomic regions around transposon (TE) insertions. ChIP-qPCR was performed before (siEGFP) and after (siWde) Wde depletion. qPCR was performed for Piwi target transposons (Stalker2, mdg1, and gypsy) and normalized to %input of a PIwi non-target transposon (roo). Error bars represent SD from three independent experiments. Density plots for Egg ChIP-seq signal on Stalker2, mdg1, and gypsy in control (siEGFP) and Wde-depleted (siWde) OSCs. Input: input DNA was used as a control. RPM: reads per million mapped reads.
Stable Parkin wild-type or pathogenic mutants expressing SH-SY5Y cell lines were induced with doxycycline (DOX) for 72 h and treated with CCCP for the indicated times.figf2Stable Parkin wild-type or pathogenic mutants expressing SH-SY5Y cell lines were induced with doxycycline (DOX) for 72 h and treated with CCCP for the indicated times. Cells were stained with anti-Parkin (green), anti-TOM20 (red) and Hoechst33342 (blue). (a-b) CCCP treatment of stable SH-SY5Y cells for 0 h. Unstimulated control cells (- DOX) show relatively low levels of endogenous Parkin, compared to cells with induced expression of Parkin wild-type or K161N, R275W and G430D mutants (+ DOX). Immunostaining (a) and western blot analysis (b) of the induced cells demonstrate comparable levels of Parkin expression in the selected clones. β-Actin served as a loading control. (c, d) After 2 h of CCCP treatment, massive colocalization of wild-type Parkin and mitochondrial clusters can be observed in the induced cells when compared with unstimulated controls. Similarly, Parkin K161N and R275W mutants translocate to mitochondria, whereas G430D does not. (e, f) After 24 h of CCCP treatment, induced wild-type Parkin-expressing cells show full clearance of damaged mitochondria. In contrast, the pathogenic mutants analysed fail to execute mitophagy and, therefore, damaged mitochondria remain uncleared. Representative images of three independent experiments are shown. Scale bars, 10 μm (a, c and e). (d, f) Quantifications of the observed mitochondrial effects in Parkin-expressing SH-SY5Y cells (n = 3). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with induced wild-type Parkin. WT, wild-type.
Figure 6A, Schematic of Topologizer. B, Topological structure of WT MC subpopulations. C, Vector field of RNA velocity projected onto the Topologizer structure for MCs collected from uninjured WT hearts or WT hearts at 2, 7, and 14 dpi. Arrows indicate the direction and ''speed'' of the velocity at each node. D, Quantification of arrow length E, Transcriptional similarity between MC1 and MC2 subtypes from the uninjured (un) hearts or hearts at 2, 7 and 14 dpi, respectively. F, Hierarchical tree showing the relationship between the MC subtypes and the myeloid cells from kidney marrow. G. Three gene clusters identified associated with MC2 to MC1 transition. H, Feature significant gene ontology (GO) terms (adjusted p-value < 0.05) with representative genes. The number of genes is shown in parentheses. I, Topological structure of WT FB subpopulations. J, Vector field of RNA velocity projected onto the Topologizer structure for FBs collected from uninjured WT hearts or WT hearts at 2, 7, and 14 dpi. Arrows indicate the direction and ''speed'' of the velocity at each node. K, Topological structure of WT eEC subpopulations. L, Vector field of RNA velocity projected onto the Topologizer structure for eECs collected from uninjured WT hearts or WT hearts at 2, 7, and 14 dpi. Arrows indicate the direction and ''speed'' of the velocity at each node.
Fig.Figure 7(A) Quantification of Greatwall, ENSA/ARPP19 and PP1 phosphorylation based on phospho-proteomic data. (B) Confirmation of ENSA/ARPP19 S67 phosphorylation in mitotic extracts from Ctr and DIA treated cells, before and after ENSA/ARPP19 siRNA depletion. (C) Dynamics of mitotic substrate dephosphorylation triggered by Cdk1 inhibition in control and DIA treated B1dd/B2ko cells. The cells were enriched in mitosis by Thymidine release and proTAME/Apcin treatment and treated with 1µM Flavopiridol to trigger mitotic exit. Dephosphorylation was measured by probing the immunoblots with anti Cdk1 substrate (anti-pSP) antibody. (D) Three repeats of the experiment in (C) were quantified and corrected based on the corresponding GAPDH intensity values. The bar plots show the mean of three experiments and the standard deviation is indicated by error bars. (E) Dynamics of Greatwall, PP1, and Cdc27 dephosphorylation following Cdk1 inhibition in proTAME/Apcin treated mitotic cell extracts from Ctr or DIA treated cells. Cell extracts were prepared at the indicated times following treatment with 1µM Flavopiridol and analysed by immunoblotting
PAS targeting activity of ATG16L1 lies within its CCD.Figure 2Schematic presentation of ATG16L1 fragments and mutants used in this study. The following fragments encompassed the indicated amino acids: Wild type (WT): 1-623; ∆1: 39-632; ∆2: 120-623; ∆3: 206-623 and ∆4: 336-623. Mutant ∆2∆182-205 consists of the ∆2 fragment with an additional deletion in amino acids 182-205. All constructs contained a Flag-S tag at the N-terminal end. Fragments depicted in (A) were expressed in U2OS cells and amino acid starved for 5 hr followed by fixation and immunostaining against S tag to detect S-ATG16L1. Scale bar: 10 μm. U2OS cells expressing Flag-ATG16L1∆2 or Flag-ATG16L1∆2∆182-205 were treated as in (B) and stained using antibodies against Flag tag (to detect ATG16L1, green) and FIP200 (red). Scale bar: 10 μm. Protein-protein interaction assay in 293T cells transiently transfected with the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies. Homodimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies. Homodimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies. Flag-ATG16L1WT or Flag-ATG16L1∆182-205 were stably expressed in ATG5-/- cells and analysed by immunofluorescence using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm.
Figure 4Figure 4. Deletion of both β-catenin and Hoxa9 abrogates leukemia development in LSK-MLL-ENL cells. Keys and colour code in the left top corner indicate the origin and the β-catenin and Hoxa9 status of MLL-ENL transduced cells. (A) Colony numbers in serial replating assay of MLL-ENL transduced cells. Images of typical 4th round colonies shown above. Data are represented as mean ± SD. (B) Summary of immunophenotypic analysis (Figure S4D) of LSK-MLL-ENL Hoxa9-/-Ctnnb1fl/fl with or without 72h Tamoxifen treatment. Data are represented as mean ± SD. N=3, two-tailed t-test was performed. (C-E) % of apoptotic cells (AnnexinV assay) (C-E) % of apoptotic cells (AnnexinV assay) (E), cells in the indicated cell cycle phases (BrdU assay) (F) and CD45.2+ donor cells in the bone marrow of recipient mice at the indicated time points post-transplantation (G) of LSK-MLL-ENL transduced cells. Data are represented as mean ± SD. N=4, two-tailed t-test was performed. (F) Kaplan-Meier survival curve of mice transplanted with LSK-MLL-ENL carrying different Hoxa9 and Ctnnb1 genotypes. N=5 per genotype was used. (G) Summary of the LSC frequencies obtained from the in vivo limiting dilution experiments (Figure S4E-H) using the LSK-MLL-ENL leukemic cells with different genotypes. See also Appendix figure S4
Figure 1A. Schematic drawing of the mdi genomic locus showing the CG3249 (mdi) transcript (gray box) and coding region (black box), and the location of the gfp insertion generated by CRISPR/ Cas9-mediated recombination with 2 guide RNAs (arrows). The resulting fusion protein is referred to as MDI-GFP. The mdi1 mutation is a 2.4 kb deletion that removes most of the mdi coding region. B. Western blots of MDI protein in wild type (wt) and mdi1 flies, showing that mdi1 is a protein-null mutation. Tubulin was used as loading control. C. Ovarioles expressing MDI-GFP stained with a mitochondrial marker, ATP synthase (ATP-S). Overlapping signals (merge) indicate that MDI localizes to mitochondria. Note that MDI is highly expressed in germ cells at late germarium stages (left panels) and in egg chambers (right panels). Bars, 10 m.
CDC7 contributes to replication dependent chromosomal breakages.Figure 6A. Representative metaphase spread from BRCA2 depleted U2OS cell treated with HU. Scale bar = 5 μM. Arrows indicate the chromosomes enlarged in the insets, two of which show chromatid breaks (* B-C. The graphs show the average number of chromatid breaks per spread. In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance assessed by student t test (** p˂0.01, *** p<0.001 D-E. U2OS and AS-CDC7 cells were transfected with control (siCon) or BRCA2 (siBRCA2) targeting siRNAs. 48 hours post transfection cells were either mock-treated or treated with 10 μM XL413 or 10 μM 3MB-PP1 respectively for 24 hours with Nocodazole added for the last 16 hours of the experiment. The graph shows the average number of chromatid breaks per spread. In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance assessed by student t test (*p<0.05, *** p<0.001
Figure 2A. Left, time course of ear swelling elicited by topical application of 5% PVP-I solution in 0.15% Oxa-challenged WT and TRPA1-/-mice, or 5% povidone solution in Oxa-challenged WT mice. In this and subsequent similar figures, the number of mice is indicated. Right, bar graph highlighting the response at 24 h shown at left. B. Time course of ear swelling elicited by topical application of the indicated concentrations of iodine in 0.15% Oxa-challenged mice, and a bar graph highlighting the response at 24 h. C. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged WT and TRPA1-/-mice, and bar graph highlighting the response at 24 h. D. Hematoxylin and eosin stained tissue sections of iodine treated ear in 0.15% Oxa-challenged WT and TRPA1-/-mice (n ≥ 3). E. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged TRPA1-/-mice, following intraperitoneal injection of AMG517 or vehicle, and bar graph highlighting the response at 24 h. F. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged mice, following intraperitoneal injection of RP67580 or vehicle, and bar graph highlighting the response at 24 h.
High-performance liquid chromatography-electrochemical detection quantitation of (a) striatal DA and its metabolites (b) dihydroxyphenylacetic acid (DOPAC) (c) Homovanillic acid (HVA) and (d) 3MT levels at three different time points (1, 2 and 12-month-old) in Pgam5 WT or KO mice.f5High-performance liquid chromatography-electrochemical detection quantitation of (a) striatal DA and its metabolites (b) dihydroxyphenylacetic acid (DOPAC) (c) Homovanillic acid (HVA) and (d) 3MT levels at three different time points (1, 2 and 12-month-old) in Pgam5 WT or KO mice. (e) Representative images of TH-immunohistochemistry from WT and KO mice. The VTA region is indicated. Stereological quantitation of (f) TH and (g) Nissl-positive neurons in the SNpc (n=4 mice per group). Error bars represent the mean±s.e.m., n=4 mice per group. (two-way analysis of variance, ***P0.01, *P0.05, NS, not significant). mo, month old.
Figure 3(A) Scheme of WT HOIP and HOIP mutants. (B) Quantification of the co-localization of WT HOIP or HOIP mutants and Htt-Q97 aggregates. Data are displayed as mean ± SD and were analyzed by Mann-Whitney U-test, n = 3. Data information: *p ≤ 0.05, ***p ≤ 0.001. (C) Htt-Q97-GFP (green) was co-expressed with HA-tagged WT or HOIP mutants (red) in SH-SY5Y cells and analyzed by immunocytochemistry using an antibody against HA. DAPI (blue). Scale bar, 10 µm. (D) Recruitment of HOIP to Htt-Q97 is decreased in p97/VCP-deficient cells. p97/VCP-deficient SH-SY5Y cells co-expressing Htt-Q97 and HA-HOIP were reconstituted with either WT VCP or VCP∆PIM and analyzed by immunocytochemistry. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9. Data information: *p ≤ 0.05, ***p ≤ 0.001. (E) Recruitment of p97/VCP to Htt-Q97 is decreased in HOIP KO cells. WT HAP1 cells (control) or HOIP KO HAP1 cells reconstituted with either WT HOIP or HOIP∆PUB were analyzed by immunocytochemistry. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Bonferroni Multiple Comparison Test, n = 5. Data information: *p ≤ 0.05, ***p ≤ 0.001. (F) The interaction of soluble Htt-Q60 with p97/VCP is dependent on HOIP. WT HAP1 cells or HOIP KO HAP1 cells were transfected with Htt-Q60-HA. 48 h after transfection cells were lysed with 1% Triton X-100, followed by an immunoprecipitation of Htt-Q60 via the HA tag. Immunopurified proteins were detected by Western blotting using an anti-VCP and anti-HOIP antibody.
Aβ plaque pathology in the brains of APP23 mice.Figure 3Aβplaque pathology in the brains of APP23miceAβimmunostaining (CN3 antibody, dark blue) in 25-μm sagittal brain sections shows only sparse Aβ deposits primarily in the frontal cortex of 6- to 8-month-old APP23mice. At 12 months and thereafter, there is a progressive increase in plaque number and size and a progressive involvement of different brain regions. Insets highlight the plaque characteristics at the different ages. Scale bar, 100 μm.The mean number of Aβ plaques per section per hemibrain increased with age in 3- to 12-month-old mice. Only four mice were analyzed in the 3-month-old age group, as APP23mice do not develop plaques at this age (Sturchler-Pierrat et al, 1997). The 6-, 8-, and 12-month-old age groups included 12, 10, and 11 mice, respectively (these are the same mice that were used for CSF and brainAβ measurements). Note that the Aβ plaques became too numerous and often could no longer be individually distinguished in the age groups > 12 months of age. Data are represented as group means ± SEM.
trans-QTL regulate chromatin binding of transcription factors required for pluripotency and differentiation.Figure 5A. Scatterplot of difference in chromatin accessibility in the 33 BXD inbred versus difference in H3K4me3 modification in the parental strains for trans-caQTL (LOD ≥ 8). ATAC-seq signal represents the log2 ratio of the average signal based on the genotype at the QTL. B. H3K4me3 level is predicted by the genotype at the QTL versus local genotype. H3K4me3 ChIP-seq for B6 and D2 parental lines compared to two BXD strains (mean ± SEM). Genotypes for both the location of the peak and location of the QTL are indicated below. C. Scheme to determine whether local (peak) or distal (QTL) genotype predicts chromatin state. A ratio of ~ 1 (log2(ratio) ≈ 0) indicates similar chromatin modification. D. Log2 fold change of H3K4me3 level when comparing BXD75 or BXD87 to the parental genotype either at the location of the peak or based on the genotype at the QTL for all trans-caQTL for which the local genotype and QTL genotype differ (LOD score ≥ 7). E. Grey - Publicly available ESC ChIP-seq datasets were evaluated for significant overlap of QTL targets (Fisher's exact test, top ten factors with q-value cutoff < 0.01). To validate a subset of these results ChIP-seq was performed for TRIM28 (N = 3), POU5F1 (N = 2), and P300 (N = 2) in B6 (blue) and D2 (orange) ESCs. Each circle represents a biological replicate or independent ChIP-seq accession from Cistrome. F. Left - Coverage profiles (averaged biological replicates, N=3) for ChIP factor occupancy at Cd59a on Chr 2 under trans-regulation by the Chr 13d QTL. Distal ca-QTL target region used for quantification is indicated by a black box with LOD score below. Right - Scatterplots for ChIP factors comparing quantitative level of modification/binding for independent replicates between B6 and D2 ESCs. G. The difference in occupancy of P300 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 13 caQTL targets (LOD > 7, Pearson's r = -0.61). H. TRIM28 occupancy at the same targets of Chr 13d caQTL in F were negatively correlated with ATAC signal (Pearson's r = -0.64). I I. Similar to F, coverage profiles for ChIP factor occupancy for a putative regulatory element on Chr 5 near Zfp932, targeted by Chr 12 trans caQTL, showed greater occupancy of POU5F1 and increased open chromatin compared to D2. Distal ca-QTL target region used for quantification is indicated by a black box with LOD score below. J. The difference in occupancy of POU5F1 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 12 caQTL targets (LOD > 7, Pearson's r = -0.66). K. Similar to J showing negative correlation between TRIM28 occupancy and chromatin accessibility (Pearson's r = -0.51). D
E3‐mediated autoubiquitination with Ub and phosphoUb A Time course for a ligase reaction with GST‐tagged cIAP1 (aa 363–614), UBE2D1 and Ub or phosphoUb.Figure 6A Time course for a ligase reaction with GST‐tagged cIAP1 (aa 363-614), UBE2D1 and Ub or phosphoUb. See Supplementary Fig S11D for a reaction with UBE2D3. B Anti‐Ub Western blot of an identical reaction run for 3 h.C Comparison of the number of Ub chain linkages in the reaction in (B).D Ub chain linkage profile for the reaction in (B). E Reaction of GST‐tagged TRAF6 (aa 1-285) with UBE2D3 as in (A). See Supplementary Fig S11E and F for additional TRAF6 reactions. F Anti‐Ub Western blot of GST‐TRAF6/UBE2D1 run for 3 h.G Comparison of the number of Ub chain linkages in the reaction in (F).H Linkage composition in GST‐TRAF6‐UBE2D1 reactions reveals 58% Lys63 chains with wt Ub and UBE2D1. Significantly smaller quantities of linkages are assembled with phosphoUb. I-K Chain assembly as in (A) with UBE2L3 and (I) GST‐tagged NEDD4L (aa 576-955) (Mund Pelham, ), (J) NleL (aa 170-782) (Hospenthal et al, ) and (K) HOIP RBR‐LDD (aa 699-1072) (Smit et al, ). While the HECT E3 ligases NEDD4L and NleL are active with Ub and phosphoUb, HOIP is significantly less active with phosphoUb. See also Supplementary Fig S11G and H.
Figure 5(A) Stereoview of a superposition the structure of apo form of UgpB (blue) and the G3P-bound form of UgpB (orange; pdb: 4aq4) is shown. The apo form of UgpB was modeled by SWISS-MODEL program using the 1.25 Å crystal structure of the non G3P binding W169S, W172S variant of UgpB as a template. The C-terminal domains (residues 123-281 and 360-415) of apo UgpB and G3P-bound UgpB were superimposed to illustrate the G3P-induced repositioning of a domain relative to the other. (B) Apo form exists in a closed conformation and the G3P-binding site is buried in between the two domains (right). G3P-bound form adopts an open conformation and exposes a cleft in the domain interface (left). The surface of UgpB are colored cyan and a continuous groove that buried in closed form but exposed in the open conformation are highlighted according to the characteristics of residues (green, hydrophobic residues; orange, polar residues; blue, positive-charged residues; red, negative charged residues). The enlargement shows the position of residues E66, W169, W172 and R374.
Figure 4(A) A summary map of cytoscape-generated protein-protein interaction network for STXBP4, α-catenin and a group of Hippo pathway proteins. (B) STXBP4 forms a protein complex with α-catenin and a group of Hippo pathway proteins. (C) STXBP4 promotes the association of α-catenin with AMOT and LATS1. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay. (D) Loss of STXBP4 diminishes the association of α-catenin with AMOT, LATS1 and YAP. HEK293A and STXBP4 KO cells were transfected with the SFB-tagged α-catenin construct and subjected to the pulldown assay. (E) STXBP4 induces the co-localization between α-catenin and AMOT as well as LATS1 and YAP. HEK293A cells were transfected with the indicated constructs and immunofluorescence was performed. Scale bar, 20 μm. Identification of several STXBP4 missense mutations that disrupt its interaction with α-catenin and AMOT. The missense mutations within the STXBP4 α-catenin-binding region and the 9-amino acid sequence of the STXBP4 WW domain were indicated and annotated (F). Identification of several STXBP4 missense mutations that disrupt its interaction with α-catenin and AMOT. The identified missense mutations respectively disrupted the STXBP4- α-catenin (G) and STXBP4-AMOT (H) complex formation. (I) Inhibition of actin cytoskeleton promotes the STXBP4-associated protein complex formation. HEK293A and the STXBP4 KO cells were subjected to immunoprecipitation using pre-immune serum and anti-STXBP4 serum under the indicated treatments. (J) The missense mutations of STXBP4 (F) diminished the ability of STXBP4 to rescue YAP phosphorylation in the STXBP4 KO cells with low actin cytoskeleton tension. YAP phosphorylation was detected using phospho-tag gel, where the YAP phosphorylation level was indicated.