EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) NMR structure of Bax (α2-α5) dimer and lipid bilayer-interacting residues. α helices are shown as cylinders. In the α4 and α5 helices, some of the lipid bilayer-interacting nonpolar, polar and positively charged residues are shown as orange, green and blue sticks, respectively, including the R89, F93, F114, A117 and S118 that were mutated to negatively charged residues to disrupt the interaction with lipid headgroups (red ovals) or acyl chains (orange curves). The longest axis (L) of the dimer is tilted 60° from the bilayer normal (N) to maximize the nonpolar interaction with the lipid acyl chains while allowing R89 to interact with the polar lipid headgroups. (B-C) Fluorescent dye release from the mitochondria-mimic liposomes by the indicated wild-type (wt) or mutant Bax in the presence (B) or absence (C) of tBid or by tBid alone (C) was measured by quenching the fluorescence of the released dyes by the dye-specific antibodies outside the liposomes during a time course. The fraction of dye release was normalized to that by detergent. The data shown were obtained from n = 3 independent replicates using the same preparations of the proteins and liposomes. (D) Digitonin permeabilized BMK Bax-/-/Bak-/- cells expressing SMAC-mCherry in the mitochondria intermembrane space (50 μL, 500,000 total cells) were incubated with 25 nM of Bax and 2 nM cBid for the indicated time at 37°C in a 96-well plate. The samples were centrifuged for 10 min and separated into supernatant and pellet fractions. SMAC-mCherry release was calculated as the fraction of total (supernatant + pellet) mCherry fluorescence coming from the supernatant fraction. The data was normalized to the percent SMAC-mCherry release of wt Bax and cBid at 60 min. Each symbol represents the normalized SMAC-mCherry release for one single replicate (n = 3 or more independent replicates). Where applicable the data was fit with the [Agonist] vs. response - Variable slope equation in Graphpad Prism 8.0.1. The dotted lines represent the 95% confidence interval of the fit.
Comparison of immunostained HeLa cells after fixation with either PFA or glyoxal.Figure 13 Figure 13 Comparison of immunostained HeLa cells after fixation with either PFA or glyoxal. The fluorescence intensity of a variety of proteins was compared between cells fixed with PFA or glyoxal. Quantification is shown as percentage of signal derived from PFA fixed cells. 8 out of the 18 target proteins which were stained show significantly brighter signal when fixed with glyoxal. Only 4 proteins show significantly reduced staining intensity. For LC3B the intensity of less than 5 cells was quantified, therefore single data points were plotted in addition to the bars. N = 3 - 44 cells per condition. Scale bar = 10 µm. ** p < 0.01, *** p < 0.001
Figure 1(A) expression of FUS does not alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in transfected NSC34 cells. Immunoblots of NSC34 cells transfected with EGFP as a control (CTRL), or wild-type or mutant EGFP-FUS. Transfected cells were purified via EGFP using a cell sorter and the samples probed on immunoblots as indicated. On the FUS immunoblot, samples were probed with FUS antibody to show endogenous and transfected proteins; tubulin is shown as a loading control. (B) representative electron microscopy of ER-mitochondria associations in NSC34 cells transfected with control EGFP vector (CTRL), EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K as indicated; arrowheads with loops show regions of association. Scale bar=200 nm. Bar chart shows % of the mitochondrial surface closely apposed to ER in the different samples. Data were analysed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. N=27-30 cells and 247-424 mitochondria, error bars are s.e.m.; ***p<0.001. (C and D) siRNA loss of FUS does not affect ER-mitochondria associations or alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in NSC34 cells. (C) immunoblots of cells either mock transfected or treated with control (CTRL) or FUS siRNAs; GAPDH is shown as a loading control. (C and D) siRNA loss of FUS does not affect ER-mitochondria associations or alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in NSC34 cells. (D) representative electron microscopy of ER-mitochondria associations in control (CTRL) and FUS siRNA treated cells. Arrowheads with loops show regions of association. Scale bar=200 nm. Data analysed by unpaired t-test. N=27-28 cells and 193-202 mitochondria.
Figure 2The research strategy to dissect the role of RBPs in regulating exit-from-pluripotency. Firstly, we constructed individual RBP KO ES cells and examined their ability to exit from pluripotency through forming EB and random differentiation by LIF withdrawal assay. AP staining of 11 RBP-KO ES cell lines maintained under ES cells culture conditions. Scale bar, 200 μm. EB formation from WT and RBP-KO ES cell lines. WT ES cell-derived EBs formed cystic structures by day 8 of differentiation; most of RBP-KO ES cell-derived EBs were more compact. Scale bar, 600 μm. Time-course analysis of Oct4 expression and three lineage differentiation markers (Hand1, Brachyury and FGF5) during EB formation. It was quantified by qPCR, the relative expression of genes was calculated by the double delta Ct (ΔΔCt) method, normalized to the expression value at day 0. The relative expression data was represented as the heatmap. AP staining of WT and 11 RBP-KO ES cell lines after LIF withdrawal. Scale bar, 200μm. RBP ranking based on ES cell abilities to self-renewal (determined by colony formation (±LIF) and Oct4 expression) and exit-from-pluripotency (determined by AP staining, EB formation and marker genes expression) in the context of RBP KO. The expression value of Oct4, FGF5, Hand1 and Brachyury was represented as the relative level of these genes compared to their expressions in WT ES cells. The quantitation of other phenotypes, such as colony formation, AP staining and EB formation was manually defined.
Assembly of OXPHOS complexes.Figure 2A) BN-PAGE and subsequent immuno-blotting of mitochondrial extracts from indicated HeLa cell lines showing the assembly of F1FO ATP synthase and binding of IF1. Monomers (M), dimers (D) and oligomers (O) were detected. Left panel: Coomassie-stained gel. Right panels: Immunoblotting and detection of ATP synthase and IF1. WT: wild type HeLa cells; IF*: cells expressing IF1-H49K. Stars indicate oligomeric IF1 forms. I: complex I; III: complex III. B) Relative levels of ATP synthase monomers, dimers and oligomers. Semi-quantification of immunoblot shown in (A). C) Assembly of F1FO ATP synthase SU e-sEcGFP (Sue#) into monomeric, dimeric and oligomeric ATP synthase (CV). Proteins were separated by BN-PAGE and immunoblotted as indicated. Left panel: detection of subunit β (α-ATP5B); right panel: detection of sEcGFP with α−GFP; O: oligomeric ATP synthase; D: dimeric (blue **); M: monomeric F1F0 ATP synthase (blue*). D) Assembly of sEcGFP-tagged subunits CoxVIIIa-sEcGFP (CIV#) into complex IV, and assembly of subunits γ-sEcGFP (SUγ#) and SU e-sEcGFP (Sue#) into ATP synthase. Proteins were separated by BN-PAGE and immunoblotted. Lanes #1-#10 were cut into three pieces for further evaluation. Lanes 1-2: complex IV assembly in WT and CoxVIIIa-sEcGFP-expressing cells, detection of CIV with anti-Cox1. Lanes 3-4: complex V assembly in WT and SUγ-sEcGFP-expressing cells, detection with anti-SU γ. Lanes 8-10: assembly of CIV# into CIV, SU γ-sEcGFP (SUγ#) into CV, and SU e-sEcGFP (SUe#) into CV. At lane #6 in the Coomassie-stained gel, the positions of molecular weight markers (M) are shown. SC: respiratory supercomplexes; CIV2: complex IV dimers; CIV: complex IV monomer; s1: F1-c8 subcomplexes; s2: subcomplexes of CV containing subunits b, e and g He et al., 2018(). Blue **: dimeric F1F0 ATP synthase; blue*: monomeric F1F0 ATP synthase. Red asterisks: respiratory supercomplexes
a) Bif-1−/− mice exhibit moderate splenomegaly.figf8(a) Bif-1−/− mice exhibit moderate splenomegaly. The weight of spleens extirpated from Bif-1+/+ (n = 27) and Bif-1−/− (n = 34) mice (6-18-month old) was measured. Statistical significance was determined by Wilcoxon rank test. (b) Hematoxylin and eosin staining of tumour tissues. Tumours were observed in 3 of 21 (14.3%) and 26 of 29 (89.7%) autopsied Bif-1+/+ and Bif-1−/− mice, respectively. The scale bars represent 50 μm.
Figure 1 (A) Relative mRNA levels of FX in CD45+ leukocytes in the peripheral blood of E0771 (abbreviated to E) or LLC tumour-bearing mice. The mean sizes of E0771 and LLC tumours were 9.8 mm and 9.5 mm, respectively. Shown are averages (N = 8 mice/group) with SEM and one-way ANOVA  (B) Relative mRNA levels of FX in CD45+ leukocytes in various organs such as lung (Lu), Liver (Li), spleen (Sp), bone marrow (BM) and lymph node (Lymph) derived from no tumour-bearing or E0771-bearing mice. "Tumour" stands for mRNA levels of FX in E0771 tumours. Shown are averages (N = 6: organs from no tumour-bearing mice, N = 6: organs from E0771-bearing mice) with SEM and one-way ANOVA  (C) Immunohistochemical quantifications of FX expression in CD45+ leukocytes in the liver and lungs of no tumour-bearing or E0771-bearing mice. Shown are averages (N = 6) with SEM and Welch"s t-test  (D) Immunohistochemical quantifications of fibrinogen deposition in no tumour-bearing- and tumour-bearing- mouse lungs. Shown are averages (N = 7/ group, 14 random fields) with SEM. Welch"s t-test  (E) Representative photos of immunohistochemical co-localisation of FX+CD45+ cells and fibrinogen deposition in tumour-bearing mouse lungs. Circles show high fibrinogen deposition areas (scale bar, 50 μm)
a) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions isolated from resting cells TH1 cells and TH1 cells activated for 24 h with anti-CD3 and anti-CD28; LAMP-1 serves as a control.figf3(a) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions isolated from resting cells TH1 cells and TH1 cells activated for 24 h with anti-CD3 and anti-CD28; LAMP-1 serves as a control. (b) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions of TH1 cells activated for 24 h as in a with (+) or without (−) inhibition of lysosomal proteases during the final 3 h with NH4Cl and leupeptin (Lys inh); LAMP-1 serves as a control. (c) Immunoblot analysis of Itch and RCAN1 in resting TH1 cells and TH1 cells activated for 24 h as in a in the presence (NAc) or absence (Veh) of N-acetylcysteine. (d) Immunoblot analysis of lysates of TH1 cells transfected with GFP-Itch or GFP-mu-Itch (top) or with Myc-RCAN1 or Myc-mu-RCAN1 (bottom) and cotransfected with a GFP expression plasmid (to assess transfection efficiency), then activated for 24 h as in a. (e,f) Proliferation (e) and IL-2 production (f) of TH1 cells transfected with plasmid expressing GFP (Ctrl) or plasmids as in c and left unstimulated (Rest) or activated for 48 h (e) or 24 h (f) as in a (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.019 and **P = 0.0034 (e) or *P = 0.046 and **P = 0.044 (f) (t test). Data are representative of four (a,b), two (c) or three (d) independent experiments or are from three independent experiments (e,f; mean and s.e.m.).
Nhp2 is an epigenetic reader of H2AQ105me.Figure 3A) ChIP-qPCR for Nhp2-myc using anti-myc antibody across the rDNA locus. Samples were normalized to input. n=3, biological replicates, error bars are standard error of the mean. *, p<0.05, **, p<0.001, based on unpaired, 2-tailed t-test B) Peptide pulldown on H2AQ105 peptides from yeast expressing myc-tagged Nhp2. C) Peptide pulldown using recombinantly purified Nhp2. Eluted peptides were slot blotted and detected using streptavidine-HRP. D) Peptide pulldown on H2AQ105 peptides from lysates of human cells (HEK293T). E) ChIP-seq profile of Nhp2 (orange) and its respective input (black) across the entire chromosome XII of yeast. H2A-normalised H2AQ105me (grey) was directly taken as comparison. Magnification shows the two annotated rDNA loci in the yeast genome indicated by the black bars below the tracks. Scale bar is for the magnification.
Figure 2(A) The Venn diagram depicts the identification of 2511 QKI-targeted genes overlapping between three RBP-binding sites databases (POSTAR, EuRBPDB and doRiNA). (B) KEGG enrichment analysis was performed by clusterProfiler in R language. Top 20 interested KEGG enrichment terms of 2511 QKI-targeted genes shown in left panel. A total of 22 genes were enriched in TGF-β signaling pathway, which were presented in right panel. (C) Western blot analysis of QKI-5 expression in nuclear and cytoplasmic fractions extracted from A549 and H1299 cells. Lamin B and β-actin were used as internal controls in nuclear and cytoplasmic extracts, respectively. (D) Anti-QKI-5 RIP was conducted in nuclear and cytoplasmic extracts from A549 cells and H1299 cells, and followed by RT-PCR and gel staining analyses to examine TGFβR1 mRNA enrichment. The input and anti-IgG antibody were used as positive and negative controls, respectively. GAPDH served as an internal RNA control in RT-PCR analysis. (E) After RIP assay, endogenous TGFβR1 mRNA was enriched from A549 cells and H1299 cells and determined by qRT-PCR. Results were shown as the percentage of the enrichment to input. (F) A conserved consensus QKI-5 response element (QRE) was presented. (G) Schematic drawing shows two predicted QREs in TGFβR1 mRNA 3' UTR (upper panel). Boxed areas indicate several TGFβR1 3' UTR segments (positions, 2,058-2,557) containing wild-type (TGFβR1 3' UTR-WT) or mutants of QRE-1/2 (TGFβR1 3' UTR-Mut1, TGFβR1 3' UTR-Mut2, and TGFβR1 3' UTR-Mut1*2) (lower panel), which are prepared to subclone into pGL3-Luc reporter vector. Numbers represent the nucleotide position of TGFβR1 mRNA. (H) Western blot analyses of QKI-5 expressed in A549 and H1299 cells stably overexpressing Flag-tagged QKI-5. (I) Left panel: schematic drawing of the pGL3-TGFβR1 3' UTR luciferase reporter assay system. Middle and right panels: QKI-5-overexpressing and vector A549 and H1299 cells were transfected with pGL3-Luc reporter plasmids that were made by fusing the above-mentioned TGFβR1 3' UTR segments. Relative luciferase activities were determined as described in Materials and Methods and normalized to those in vector cells. (J) In vitro RNA pull-down assays were performed using biotin-labeled TGFβR1 3' UTR segments in A549 and H1299 cells. After pull-down, endogenous QKI-5 enrichments were detected by western blot analyses. β-actin was used as the protein control.
Figure 4(A) Mouse CCIN induced by cancer chemotherapy drug cyclophosphamide (CPA) consists of both neutrophil decrease and neutrophil recovery stages. ANC, absolute neutrophil count. (B) Equivalent low, medium, and high doses of Am80 and GCSF between human and mouse were calculated following FDA dose-conversion guidelines. (C) Neutrophil decrease model with low dose treatment. After 4 hr of CPA injection, mice were treated with regimens for 3 days. On day 2, mice were infected with 6 x 106 CFU of S. aureus via intravenous injection for up to 16 hr before euthanasia. Control mice without CPA. N, numbers of mice. (D) Vetscan counting PBleukocytes. (E) Neutrophils induced by low dose treatment in PB (section i) and BM (section ii). (F) Bacterial killing by PBneutrophils was assessed at 3 and 16 hr post-infection and in spleen (sections i-iii), using blood agar analysis of total extracellular viable bacteria. (G) Neutrophil decrease model with medium dose treatment, using similar procedures described in panel C. (H) Vetscan counting PBleukocytes. (I) Neutrophils induced by medium dose treatment in PB (section i) and BM (section ii). (J) Similar to panel F, bacterial killing by PBneutrophils was evaluated 3 and 16 hr post-infection and in heart.
Figure 3A The A3B3DF complex shown with the subunits unique to the eukaryotic V1 removed, highlighting the core conserved catalytic complex labeled as "Catalytic core". The three peripheral stalks and inhibitory subunit H are found only in the eukaryotic V1, whereas A3B3DF represents the full complement of subunits in the prokaryotic V1 complex. Notably, the prokaryotic V1 is an active ATP hydrolase (Arai et al, 2009). The orientation of the A and B subunits with respect to the membrane is indicated by the arrow on the left. B Conformational differences in the catalytic A subunits. The catalytic A subunits are shown aligned by their N-terminal β barrels, highlighting the change in conformation of C-terminal α helical bundles associated with the closed ((AB) 2 and (AB) 3; dark and light gray, respectively) and open ((AB) 1; magenta) catalytic sites. Movement of this domain in response to nucleotide occupancy is thought to drive rotation of the central rotor during catalysis. C ScV1's catalytic hexamer contains one open and two closed catalytic sites. Side view of the three catalytic hexamer A/B pairs (shown in surface representation). Note that the two closed pairs (AB) 2 and 3 are non-equivalently closed. D Alignment of the A3B3DF from ScV1 and nucleotide-free EhV1 by the catalytic hexamers. The A3B3DF from ScV1 is shown in the same color scheme as in Fig 2, EhV1 is in blue. The alignment shows the difference in position of the central rotor subunit, D. Note that the C-terminus of subunit D (DCT) is longer in eukaryotic V-ATPase (see electron density contoured at 1.2 σ in the box to the right).E ScV1 and EhV1are halted in different rotational positions. Alignment of the A3B3DF of ScV1 and nucleotide-free EhV1 (blue, with dashed lines drawn through the A subunits) by the central stalk subunit D, reveals that the two catalytic cores are halted in different rotational positions. The autoinhibited ScV1 requires 40° rotation to overlay its catalytic hexamer with that of the EhV1. F Comparison of the D subunits of ScV1 (red) and nucleotide free EhV1 (blue). HCT from ScV1 is shown in yellow and A subunit from EhV1 is shown in dark blue.
Figure 1RNA was isolated from control HuH7 cells or cells infected with HCV for 6 days in three independent experiments. These RNAs were hybridized to a Sure Print Agilent array and the number of coding or lncRNA (LncR) genes whose expression is up- or down-regulated (B>2) is shown (A). RNA was isolated from control HuH7 cells or cells infected with HCV for 6 days in three independent experiments. A heatmap of coding (B) genes relevant for this study is also shown. RNA was isolated from control HuH7 cells or cells infected with HCV for 6 days in three independent experiments. A heatmap of lncRNA (C) genes relevant for this study is also shown.
In situ base editing creates allelic series of oncogenic driver mutations.Figure 2Sanger-sequencing chromatograms showing the target regions of sgPik3caE542K, sgPik3caE545K and sgPik3caE453K in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacers that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgPik3caE542K, Lenti-sgPik3caE545K and Lenti-sgPik3caE453K. EditR was used to calculate the frequency (%) of C-to-T conversion at the indicated target cytosines of the protospacers in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors. Overview of the intraductal injections performed in WB1P-BE3 females with high-titer lentiviruses encoding Myc and either a non-targeting sgRNA (Lenti-sgNT-Myc) or the different sgRNAs targeting Pik3ca (Lenti-sgPik3ca-Myc). Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WB1P-BE3 females injected with Lenti-sgPik3caE542K-Myc (n=10), Lenti-sgPik3caE545K-Myc (n=10) and Lenti-sgPik3caE453K-Myc (n=10) showed a reduced mammary tumor-specific survival compared to WB1P-BE3 female mice injected with Lenti-sgNT-Myc (n=11) vectors (respectively 47, 44 and 49 days after injection vs 72 days after injection, P < 0.0001 by Mantel-Cox test). Sanger-sequencing chromatograms showing the target region of sgPik3caE542K, sgPik3caE545K and sgPik3caE453K in 3 independent tumors from WB1P-BE3 females injected with the corresponding Lenti-sgPik3ca-Myc vectors. Arrowheads highlight cytosines of the protospacer that show base editing. EditR was used to calculate the average frequency (%) of C-to-T conversion at the indicated target cytosines of the protospacers in tumors from WB1P-BE3 females injected with Lenti-sgNT-Myc or Lenti-sgPik3caE542K-Myc, Lenti-sgPik3caE545K-Myc and Lenti-sgPik3caE453K-Myc.
Network models consistent with naïve state maintenance predict the effect of TF forced expression in resetting from primed pluripotency.Figure 1(A) Flow-chart describing the methodology. Network components were identified based on functional studies from the literature, and possible interactions between components defined based on pairwise gene expression correlation. A set of experimental results served as constraints. The software RE:IN synthesises all possible interaction networks consistent with the constraints, which is termed the cABN. The cABN is used to formulate predictions to be tested experimentally. Importantly, predictions do not overlap with imposed constraints. If predictions are falsified, the cABN can be further refined by incorporating new experimental results as constraints. The refined cABN is used to generate further predictions (B) cABN derived from a Pearson coefficient threshold of 0.832, consistent with constraints previously defined for ESC self-renewal (Dunn et al. 2014). Solid arrow, required interaction; dashed arrow, possible interaction; black arrow, activation; red arrow, inhibition. There is no regulation hierarchy associated with component positioning (C) Illustration of EpiSC resetting constraints. See Appendix Fig S1F (D) Predicted number of regulation steps required for all models to stabilise in the naïve state under forced expression of a single network component. The red dashed line indicates the number of steps required under empty vector control (E) Fold increase of Oct4-GFP+ colony number over control under forced expression of individual factors. n ≥ 5, where each dot indicates an independent experiment. Box-plots show median, 1st and 3rd quartile values. One-sample Wilcoxon test p-values are as indicated, with p values < 0.05 shown in red (F) cABN derived from a Pearson coefficient threshold of 0.782 (G) Predictions from the 0.782 cABN. Light green regions indicate where some, but not all, concrete networks allow stable conversion to the naïve state. Sall4 is indicated in green, as this was imposed as a constraint and therefore is not a model prediction