EMBO/sd-character-level-ner huggingface dataset:
a,b) Confocal images of HeLa cells transiently co-transfected for 48 h with GFP-tagged MCUR1 and DsRed-Mito plasmids (a) or mRFP-tagged MCUR1 and EYFP–Mito (b).figf3(a,b) Confocal images of HeLa cells transiently co-transfected for 48 h with GFP-tagged MCUR1 and DsRed-Mito plasmids (a) or mRFP-tagged MCUR1 and EYFP-Mito (b). Scale bar, 20 μm. (c) Immunoblot analysis of Flag-tagged MCUR1 in the HeLa cell crude mitochondrial fraction, outer mitochondrial membrane (OMM) and mitoplasts, using antibodies against VDAC1 (outer mitochondrial membrane protein), COII (integral membrane marker) and Flag. (d) Immunoblot analyses of mitochondria-containing pellet and cytosolic fractions from plasma-membrane-permeabilized HeLa cells. Permeabilized cells were treated with or without tBid (50 nM) for outer mitochondrial membrane permeabilization and the appearance of cytosolic cytochrome c was verified. Intact and outer mitochondrial membrane permeabilized samples were exposed to proteinase K for 10 min. These samples were probed using antibodies against HSP60, OXA1, Flag and MCUR1. (e) Reciprocal co-immunoprecipitation (IP) of MCU-GFP and MCUR1-Flag transiently expressed in COS7 cells. Representative of four independent experiments. WB, western blot. (f) Co-immunoprecipitation of MICU1-Flag with MCU-GFP but not with MCUR1-GFP transiently expressed in COS7 cells. Representative of four independent experiments. (g) Immunoprecipitation of LETM1-GFP with anti-LETM1 failed to pull down MCUR1-Flag in transiently transfected COS7 cells. (h) Immunoprecipitation with Flag antibody pulled down LETM1-Flag or MCU-Flag transiently expressed in COS7 cells (immunoprecipitation lanes 2 and 3, lower panel), but only co-immunoprecipitated MCUR1-V5 in the MCU-Flag-expressing cells (immunoprecipitation lanes 4 versus 5, upper panel), despite lower expression of MCU-Flag in MCUR1-co-transfected cells (compare MCU-Flag and LETM1-Flag expression in MCUR1-co-transfected cells in lysate lanes 4 and 5, bottom panel). Representative of three independent experiments. Uncropped images of blots/gels are shown in Supplementary Fig. S6.
, in vitro.Figure 2A. Up: The schematic diagram of PGCs induced from chicken ESCs in vitro. Down: Morphological observation of the number of embryoid bodies in the in vitro PGCs induction model after LncBMP4overexpression and interference; oeLncBMP4 indicates the overexpression of LncBMP4; shLncBMP4 indicates the interference of LncBMP4; BMP4 induction was regarded as the control. Scale bar: 60 µm (n = 3 independent experiments). B. The expression of reproductive marker genes (Cvh and C-kit), LncBMP4, and BMP4 were detected by qRT-PCR after LncBMP4 overexpression and interference, in vitro (Data are shown as mean ± SEM, n = 3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA) . C. The efficiency of PGCs differentiation was detected by IF 4 d after induction, using C-KIT and CVH antibodies. Scale bar: 60 µm (n = 3 independent experiments). D. Detection of PGCs formation efficiency after LncBMP4 overexpression or interference, in vitro, by flow cytometry using the CVH antibody (Data are shown as mean ± SEM, n = 3 independent experiments, **** p < 0.0001, one-way ANOVA.
Figure 2A. Timeline for establishment of differentiated macrophage-like THP-1 cells (D-THP-1) from monocytic THP-1 cells. B. ELISAs were used to analyze the inhibitory effect of OSMI-1 (10 µM, final) on cytokine and chemokine production from THP-1 cells exposed to Stx2a (10 ng/mL) for 9 h (n = 3 biological replicates). Data information: For graphs error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001. The effects of OSMI-1 were compared with those of the vehicle (DMSO) control at each time-point. C. Real-time qRT-PCR analyses of fold changes in transcript levels for each cytokine/chemokine gene in D-THP-1 cells following Stx2a (10 ng/mL) intoxication for various time-points in the presence or absence of OSMI-1 (10 µM, final). Data are normalized using GAPDH as a loading control (n = 3, biological replicates). Data information: For graphs error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001. The effects of OSMI-1 were compared with those of the vehicle (DMSO) control at each time-point.
Organism-wide proteasome activity mapping reveals sexual dimorphism in a number of tissues.Figure 1A-D Proportional distribution of chymotrypsin-like activity of the proteasome across nine tissues from female (n=9), and male (n=10) mice. Proportional distribution of trypsin-like activity of the proteasome across nine tissues from female (n=9), and male (n=10) mice. Graphical representation of color code of tissues analyzed. Chymotrypsin-like activity of the proteasome across nine tissues in individual mice. Each data point (n=9 females and n=10 males) indicates data from a single mouse performed in triplicate. Each central line and error bar indicate mean +/- SEM . * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples. trypsin-like activity of the proteasome across nine tissues in individual mice. Each data point (n=9 females and n=10 males) indicates data from a single mouse performed in triplicate. Each central line and error bar indicate mean +/- SEM . * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.
Figure 5(A, B) Immunoblot analysis of AGIA-MmPLZF (A) or -MmSALL4 (B) in FLAG-MmCRBN-WT, -MmCRBN-I391V or -MmCRBN-V380E/I391V expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h. Data information: Data in bar graphs were calculated as relative Mm) PLZF or SALL4 band intensity with Mm) PLZF or SALL4, respectively. The band intensity of DMSO was 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (C, D) Immunoblot analysis of AGIA-GgPLZF (C) or -GgSALL4 (D) in FLAG-GgCRBN-WT or -GgCRBN-I390V expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h. (E, F) Immunoblot analysis of AGIA-Mm PLZF (E) or -MmSALL4 (F) in FLAG-MmCRBN-WT expressing CRBN-/- HEK293T cells treated with indicated concentration of DMSO, thalidomide (Tha) or 5-hydroxythalidomide (5-HT) for 16 h. (G, H) Immunoblot analysis of AGIA-GgPLZF (G) or -GgSALL4 (H) in FLAG-GgCRBN-WT expressing CRBN-/- HEK293T cells treated with indicated concentration of DMSO, thalidomide (Tha) or 5-hydroxythalidomide (5-HT) for 16 h. Data information: Data in bar graphs were calculated as relative (Gg or Mm) PLZF or SALL4 band intensity with (Gg or Mm) PLZF or SALL4, respectively. The band intensity of DMSO was 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).
PPM1G-B56δ is a new phosphatase holoenzyme complex.Figure 1(A) Interaction network of B56δ with its associated proteins identified in this study. Blue line indicates B56δ interaction with PPM1G. Red dashed line represent interaction with PP2A complex proteins. (B) HEK293T cells were transfected with SFB control, SFB-tagged PR130 and SFB-B56δ, and the cell lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot (WB). (C) Glutathione Sepharose beads bound with bacterially expressed recombinant GST, GST-B56δ or GST-PPP2R1A proteins were incubated with bacterially purified recombinant MBP-PPM1G and interaction of B56δ with PPM1G was detected by immunoblotting with MBP antibody. Interaction of B56δ-PPP2R1A was shown similarly using MBP- B56δ and GST-PPP2R1A proteins. Recombinant protein expression is shown by Coomassie staining. (D) HEK293T cells were transfected with SFB control, SFB- PPP2CA, SFB-PPP2CB, SFB-PPP2R1A, SFB-PPP2R1B and SFB- B56δ, and the lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot using PPM1G antibody. (E) Cell lysates from HEK293T cells expressing SFB vector, SFB-B56α, SFB-B56β, SFB-B56γ, SFB-B56δ and SFB-B56ε were pulled down and interaction with PPM1G was detected by western blotting using PPM1G antibody. (F) Schematic representation of B56δ full length (FL), along with its various deletion and point mutants. (G) Cells expressing B56δ full length (FL), Δ91-102, L183A and H282A mutants were pulled down with streptavidin beads and interaction with PPM1G was detected by western blotting. (H) Various mutants of B56δ were transfected along with myc-PPP2R1A and pulled down using streptavidin beads and interaction with myc-PPP2R1A was detected by immunoblotting. (I) Binding affinities of the B56δ with PPM1G and PPP2R1A subunit were determined by Bio-layer interferometry. 6xHis-tagged PPM1G and PPP2R1A were immobilized on Ni-NTA biosensors and incubated with the GST-B56δ wild type (WT) or Δ91-102 mutant at various concentrations (100-400 nm). Curves represent experimental trace obtained from the BLI experiments. Binding affinities (Kd) (+SD; n=3 independent experiments) for B56δ-PPM1G and B56δ-PPP2R1A are shown.
Rab12 regulates mTORC1 activity and autophagy by controlling the PAT4 protein concentration.Figure 4(A) Lysates of MEFs transfected with the siRNAs indicated were analysed by immunoblotting with the antibodies indicated. (B) Quantification of the phospho‐S6K levels shown in (A). (C) MEFs transfected with the siRNAs indicated were cultured under nutrient‐rich (N) or starved (S) conditions and were immunostained with anti‐LC3 antibody. The mean numbers of LC3‐positive dots per cell are shown. (D) A schematic model of inhibition of autophagy followed by increased activation of mTORC1 in Rab12‐depleted cells. MEFs, mouse embryonic fibroblasts; N, nutrient‐rich; PAT4, proton‐coupled amino‐acid transporter 4; S, starved; siRNA, short interfering RNA. *P0.05; **P0.01; ***P0.005.
Figure 2Figure 2. Cells-of-origin transcriptional-memory predicts survival in AML patients. (A-B) Number of identified genomic variants in indicated MLL-ENL transformed cells using RNA-Seq (A) A two-tailed t-test was performed in (A). LSK, wildtype LSK cells; GMP, wildtype GMP cells. Figure 2. Cells-of-origin transcriptional-memory predicts survival in AML patients. (A-B) Number of identified genomic variants in indicated MLL-ENL transformed cells using genomic sequencing (B). LSK, wildtype LSK cells; GMP, wildtype GMP cells. (C, D) Manhattan plots indicating estimated length of CNVs (C) on the y-axis in the respective chromosomal positions shown in the x-axis. (C, D) Manhattan plots indicating estimated number of genes in CNV areas (D) on the y-axis in the respective chromosomal positions shown in the x-axis. (E) MA-plots showing the log2-fold gene expression changes in the normal (left panel) and MLL-ENL transformed (right panel) cells as indicated. (F) Transcriptional memory signature; the overlap of differentially expressed genes in GMP-LSK in normal vs MLL-ENL transformed cells is significantly enriched using a hypergeometric test. (G) Survival differences between patients clustered using transcriptional memory signatures with a log-rank test. See also Appendix figure S2.
Figure 7A The procedure of CHAF1A depletion-mediated HIV-1 reactivation in primary CD4+ T cells. On Day -4, primary CD4+ T cells were isolated from PBMCs of healthy donors, and activated by PHA. On Day 0, activated CD4+ T cells were washed and infected with pseudotyped HIV-1. Another two days later, cells were treated with shluc and shCHAF1A lentiviruses respectively. Two weeks later, one part of naïve CD4+ T cells was treated with αCD3/αCD28/IL-2. One part of shluc-treated cells and one part of shCHAF1A-treated cells were further treated with 500 nM SAHA. On day 16, all the cells were proceeded to FCM analysis. B The flow cytometry figures of each group. The reactivation efficiency was indicated by the GFP-positive cells percentages which were labelled on the top right corner. C The statistical scatter plot of the results in (B). D The amounts of intracellular HIV-1 RNAs of latently infected primary CD4+ T cells which were isolated from PBMCs of HIV-1-infected individuals. αCD3/αCD28/IL-2 treatment was performed as positive control. shCHAF1A lentiviruses were packaged to knock down endogenous CHAF1A. shluc lentiviruses treatment was performed as negative control. SAHA was added as LRA supplement. E Envelope V1 to V3 region of HIV-1 RNAs in (D) was reverse-transcribed and PCR-amplified. The PCR products were proceeded to TA-cloning. At least 60 single clones were picked from each group and sequenced. These sequences and the standard HIV-1 sequence HXB2 were aligned. Genetic diversity index of each sequence was calculated for each group. F The schematic of CAF-1 body-mediated HIV-1 latency. Detailed information for the schematic was indicated in discussion. The schematic of CAF-1 body-mediated HIV-1 latency. Detailed information for the schematic was indicated in discussion.
Figure 3(A) Schematic representation of the main metabolic pathways. (B) LC-MS/MS was used to examine the metabolites in SGC7901 cells with or without UHMK1 knockdown. The data are shown in the heatmap. (C) (upper panel) LC-MS/MS analysis was performed to measure 15N-glutamine-labeled purine synthesis intermediates in BGC823 cells transfected with or without the WT-UHMK1 or UHMK1-K54R constructs. (lower panel) LC-MS/MS was used to analyze metabolites labeled with 13C-glycine in BGC823 cells transfected with or without the WT-UHMK1 or UHMK1-K54R constructs(three biological replicates). Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance. (D) RNA and DNA with incorporated 14C-glycine in BGC823 cells transfected with or without the WT-UHMK1 or UHMK1-K54R constructs were examined using LC-MS/MS(three biological replicates). Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance. (E) qRT-PCR assays were used to analyze the effects of WT-UHMK1 or UHMK1-K54R on the genes controlling purine metabolism. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance. (F) UHMK1 silencing significantly decreased SGC7901 cell proliferation, while ATIC overexpression or purine supplementation markedly reversed this inhibition. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. . Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance. (G) Treatment with the ATIC inhibitor significantly reversed the proliferation of BGC823 cells induced by UHMK1 overexpression. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.
Figure 6 A, B mRNA expression in scWAT of mice fed a high-fat diet (HFD) for 11 weeks followed by 5 weeks of HFD with 0.0075% Rosi (qRT-PCR). t-test Cited4-/- vs. Cited4+/+, (A) n=8/7, *P=0.029 (Ucp1), **P=0.009 (Elovl3), **P=0.006 (Cpt1b), **P=0.004 (Cyc1), *P=0.016 (Ndufb3), **P=0.003 (Cd36), **P=0.009 (Gyk), (B) n=7/8, *P=0.026 (Pck1)   C, D Ucp1 protein expression in scWAT of mice treated as in (A), determined by Western blot with VCP as loading control (n=8/7 for females and n=7/8 for males). Arrow indicates the band specific to Ucp1 (see Appendix Fig 4F)
Figure 1A. SDS-PAGE analysis of the D391N DIS3L2-RNA complexes. RNAs were radioactively labeled at the 5' end and DIS3L2-bound RNAs were detected by autoradiography. The negative control was performed with HEK293T-Rex cells (Control) B. DIS3L2 CLIP reads possess oligo(U) 3' termini. Histograms of reads with 3'-terminal stretches of identical nucleotides. Only oligo(U) tails longer than 4 nucleotides are observed. C. The untemplated uridylation is mostly found in the proximity of mature 3' RNA ends. The heat map shows the coverage of various RNA types by uridylated reads. RNA class and number of uridylated genes/transcripts for each category are indicated on the left. The length of each mature RNA was scaled to 100%. D. List of DIS3L2-bound uridylated miRNAs with the number of uridylated reads identified in all three replicate CLIPs. Members of the let-7 family are shown in bold. E. Identification of hybrid reads consisting of the 5' and 3' arm of pre-let-7 miRNA. Profile of read coverage along the pre-let-7 genomic locus is indicated above. F. Secondary structure of pre-hsa-let-7i, with both 5p and 3p mature products highlighted in bold. The intramolecular ligation position is indicated in dash line between nt 28 and 61. It corresponds to cleavage positions of RNaseT1, which leaves 3'-terminal guanosine residues. G. D391N DIS3L2 overexpression does not cause abundance changes of CLIPed uridylated RNAs. Scatter plot of transcript abundances estimated from RNA-seq from the cell line overexpressing D391N DIS3L2 and control HEK293T-Rex cells. CLIPed transcripts are marked in white.
Expression of PIWI proteins and piRNAs in the locust brain.Figure 2A PIWI genes are present in the locust brain. Rp49 served as a loading control (n=4 biological replicates). Data information: data are presented as mean ± SEM. B Western blot analysis of PIWI proteins in the locust brain. Tubulin was used as an endogenous control (n=2 biological replicates). The bars represent the mean of band intensity C The fluorescence signals of PIWI proteins were determined in locust brains. Hochest33342 was used to label cell nuclei in locust brains. The scale bars represent 20 µm. D Size distribution of genome-matching small RNAs among total RNAs and oxidized smRNA-seq libraries. CPM, counts per million. E Size distribution of transposon-matching sense (blue) and antisense (red) reads in oxidized smRNA-seq libraries. F The nucleotide identity at each read position of transposon-matching piRNAs shows a first-position nucleotide bias for uracil. G The pairwise read comparison of the oxidized small RNAs demonstrated an overlap of 10 nt between their 5′ ends.
Figure 7Sacculi were incubated with lysozyme and purified MepH (A), MepS (B), PBP4 (C), and PBP7 (D). The relative peak areas of Tri-Tri and Tri-Tetra containing 3→3 cross-links and that of Tetra-Tri and Tetra-Tetra containing 4→3 cross-links were combined. MepH preferentially hydrolyzed dimers containing 4→3 cross-links. MepS hydrolyzed dimers containing 4→3 and 3→3 cross-links with similar efficacies. PBP4 and PBP7 only hydrolyzed dimers containing 4→3 cross-links. The insets show variations in the relative peak areas of the Tri and Tetra monomers. MepH and MepS displayed L,D-carboxypeptidase activity, but not PBP4 and PBP7.
Rab7 and Arl8b mediate spatial segregation of late compartments via their effectors RILP and SKIP.Figure 2Effects of select transport route activation on the spatial organization of late compartments. A. Representative confocal images of fixed HeLa cells ectopically expressing Arl8b-GFP or Arl8b-Myc (green), in combination with HA-RILP (cyan) and/or Myc-SKIP, PLEKHM1-FLAG, or GFP-FYCO1 (red), immunolabeled against endogenous Rab7 (eRab7, magenta) and the indicated epitope tags. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3.5x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bar: 10µm. B. Colocalization (Mander's overlap) between the indicated pairs of proteins, n≥3 images (3≥ cells per image) analysed per condition from 2 independent experiments. C. Plot of eRab7- or Arl8b-GFP / Myc-Arl8b-positive pixel distribution in response to the indicated effector perturbations, expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n≥6 cells per condition analysed from 2 independent experiments. D. Colocalization (Mander's overlap) between Arl8b and Rab7 as a function of the indicated effector perturbations, n≥5 images (3≥ cells per image) analysed per condition from 2 independent experiments. E. Graphical summary of late compartment segregation as mediated by Rab7 and Arl8b.
Fig.Figure 3(A) Quantification of EMSA assays evaluating the binding of (PR)20 to 19 nt-long ssDNA, ssRNA, dsDNA and dsRNA molecules. Each probe (0.2 μM) was incubated with increasing concentrations of (PR)20 for 10´. Curve-fitting was performed using non-linear regression with the Hill equation. (B, C) Percentage of GAPDH or 5.8 rRNA levels quantified by qPCR in reactions containing increasing doses of (PR)20 (n=3).Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. (D) HTM-mediated analysis of 5-ethynyl-deoxyuridine (EdU) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 4 h. EdU was added 30´ prior to fixation. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm. (E) HTM-mediated analysis of γH2AX levels per nucleus in U2OS cells exposed to 3Gy of IR in the presence or absence of 10 μM (PR)20 for the indicated times. Note the accumulation of cells with persistent DNA damage (γH2AX) 10 h after IR in the presence of the DPR. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm. (F) Efficiency of CRISPR-mediated gene deletion quantified in mouse embryonic stem cells stably expressing EGFP, a doxycycline-inducible Cas9 and an EGFP-targeting sgRNA. The percentage of EGFP-positive cells was quantified by flow cytometry 72 h after inducing Cas9 expression with doxycycline in the presence or absence of 10 μM (PR)20 (n=3). Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. (G,H) Turbidity of solutions containing (PR)20 mixed with the different dNTPs measured by the optical density at 600 nm (O.D. 600) (G) (n=3). Data represent mean values ± SEM. A representative image of these assays is provided in (H). Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test.
Fig.Figure 2A H3122 cells were treated with XMU-MP-5 or crizotinib for 72 hours. Cell viability was measured by MTS assay. Each concentration was performed in triplicate. Data were presented as Mean ± SEM from three independent experiments (n = 3) B XMU-MP-5 inhibited colony formation of H3122 cells. H3122 cells were treated with XMU-MP-5 or crizotinib for 18 days and then stained with crystal violet. C XMU-MP-5 suppressed ALK signaling pathway in H3122 cells. D XMU-MP-5 induced cell apoptosis in H3122 cells. Cells were treated as indicated for 72 hours and then assayed by Annexin-V-FLUOS/PI staining. E Comparison of tumor volume of different treatment groups in 11 days. BALB/c nude mice bearing H3122 xenograft tumors were treated with vehicle, XMU-MP-5 and crizotinib by tail vein injection once daily. Data were shown as Mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test. N.S., P = 0.1063; **P = 0.0097. F Comparison of tumor weight of each group at end point. Data were shown as Mean ± SEM (n = 6). Statistical comparasions were performed using a one-way ANOVA with Dunnett test. *P = 0.0177, **P = 0.002. G Representative images of tumors analyzed by H&E and IHC staining. Scale bars, 100 μm.