EMBO/sd-character-level-ner huggingface dataset:
Mithramycin induced morphological changes are dependent on SWI/SNF eviction and the induction of H3K27me3.Figure 3A, B: Mithramycin displaces SMARCC1 and SMARCE1 SWI/SNF subunits from chromatin in a time-dependent manner in BT12 rhabdoid tumor (A) but not U20S osteosarcoma (B) cells. Western blot analysis showing whole cell lysate (Total), cytoplasmic soluble (CS), nuclear soluble (NS), and chromatin bound (Chr) fractions collected after exposure to solvent (S) or 100nM mithramycin for 8 or 18h and probed for the SWI/SNF subunits (BRD9, SMARCC1 or SMARCE1) or H3 (chromatin fraction control) and GAPDH (soluble fraction control). C: SMARCC1 and SMARCE1 mRNA expression does not change following 100nM mithramycin treatment as measured by qPCR fold-change relative to GAPDH (2ddCT). Data represents mean with standard deviation derived from three independent experiments. D: Addition of the proteasome inhibitor (bortezomib) rescues the loss of protein expression following 8 or 18-hour mithramycin treatment. BT12 cells were treated for 8 or 18-hours with solvent (S), mithramycin (M, 100nM), bortezomib (PI, 2.5μM), or combination of 100nM mithramycin and 2.5μM bortezomib (C). E: Loss of SWI/SNF occupancy at defined loci in the genome as measure by chromatin immunoprecipitation qPCR (ChIP-qPCR) at previously described SWI/SNF target genes, MYT1 (8-hour, P=0.0001; 18-hour P=0.0001) and CCND1 (8-hour, P=0.0001; 18-hour P=0.0001). ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons. F: Chromatin immunoprecipitation qPCR (ChIP-qPCR) of H3K27me3 at MYT1 (8-hour, P= 0.0001; 18-hour P=0.0001) and CCND1 (8-hour, P=0.02; 18-hour P=0.0001). H3K27me3 occupancy is increased in a time-dependent manner. ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons. Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in BT12 cells relative to loading control (H3). Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in G401 cells relative to loading control (H3). Re-expression of SMARCB1 following doxycycline treatment inhibits the mithramycin-dependent effects on H3K27me3 amplification
a–c) Knockdown of pro-autophagic proteins restores Δ133p53α expression.f2(a-c) Knockdown of pro-autophagic proteins restores Δ133p53α expression. MRC-5 fibroblasts at late-passage (PDLs 51) were transfected with three independent siRNA oligonucleotides (lanes 1-3) against ATG5 (a), ATG7 (b) and Beclin-1 (c), along with their control siRNA (lane C), for 4 days. Knockdown of ATG5 was confirmed by the loss of ATG5-ATG12 conjugate (detected by anti-ATG5 antibody). Knockdown of the other two proteins was confirmed using the corresponding antibodies. Δ133p53α, full-length p53 and LC3B (LC3-I and LC3-II) were examined in all samples. The relative expression levels of Δ133p53α and full-length p53 (normalized with β-actin) are shown below the images. (d) Knockdown of pro-autophagic proteins and treatment with bafilomycin A1 show similar effects on Δ133p53α and p62/SQSTM1. MRC-5 fibroblasts at late-passage (PDLs 51) were transfected with control siRNA (c), ATG5 siRNA (A5, no. 1 in a), ATG7 siRNA (A7, no. 1 in b) and Beclin-1 siRNA (B1, no. 2 in c) as in a-c, untreated (−) or treated with bafilomycin A1 (+, 100 nM for 4 h), and examined in immunoblots for indicated proteins. The relative expression levels of Δ133p53α and p62/SQSTM1 (normalized with β-actin) are shown below the images.
Figure 6Effects of KYA1797K and PTD-BBR on RAS degradation IB assay of MT-HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr, respectively Effects of KYA1797K and PTD-BBR o cell growth MTT assays of MT-HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr, respectively. Data are presented as the mean ± SD (n = 3) Effects of overexpression of GFP-BBR-WT or -2E mutant on KYA1797K-induced growth and transformation of CRC cells. MT-HCT116 or SW48 cells were transfected with GFP-BBR-WT, -2E, or GFP-Mock for 24 hr and then treated with KYA1797K via indicated conditions MTT assays were performed for measurements of cell growth Effects of overexpression of GFP-BBR-WT or -2E mutant on KYA1797K-induced growth and transformation of CRC cells. MT-HCT116 or SW48 cells were transfected with GFP-BBR-WT, -2E, or GFP-Mock for 24 hr and then treated with KYA1797K via indicated conditions foci formation assay were performed to detect cellular transformation Effects of cotreatments with KYA1797K and PTD-BBR on xenografted tumor growth. MT-HCT116 cells were subcutaneously injected in nude mice with subsequent i.p. injection of vehicle, 20 mg/kg of KYA1797K, or a cotreatment of PTD-BBR (25 mg/kg) and KYA1797K (20 mg/kg) for 20 d. Tumor volumes (upper panel) were measured every 4 days, and tumor weights (lower panel) were measured at the times of sacrifice. Data are presented as the mean ± SD (four mice per group) WCLs prepared from tumor tissues were immunoprecipitated with anti-β-catenin antibody. WCLs or co-IP samples were subjected to IB analyses IHC analyses of tissue sections incubated with the indicated antibodies and then counterstained with DAPI. Scale bar, 20 µm
a) Co-immunostaining for Cx43 and the indicated Atgs in NRK cells maintained in the presence or absence of serum (top).figf7(a) Co-immunostaining for Cx43 and the indicated Atgs in NRK cells maintained in the presence or absence of serum (top). Single and merged channels of the 3D reconstruction of the outlined regions are shown (bottom). Nuclei are highlighted with DAPI. (b,c) Immunoblot for the indicated proteins of immunoprecipitates (IP) of Cx43 in NRK cells maintained in the presence or absence of serum (b) or treated or not with 3-methyladenine (c). Atg5 is shown as the negative control for immunoprecipitation. (d) Co-staining for Atg9 and e-cadherin in control NRK cells and those knocked down for Cx43. Single and merged channels at higher magnification areas are shown. (e) Immunofluorescence for endogenous Atg9 and Cx43 in control NRK cells and those knocked down for Eps15. (f,g) Immunofluorescence for endogenous Cx43 (green) in control NRK cells or those knocked down for Atg14 (f) maintained in the presence or absence of serum. (f,g) Immunofluorescence for endogenous Cx43 (green) in control NRK cells or those knocked down for Atg9 (g) maintained in the presence or absence of serum. E-cadherin staining (red) is shown to highlight PM in g and h. (h) Immunofluorescence of endogenous Cx43 in control NRK cells or those knocked down for Atg14, treated (or not) with tamoxifen or lindane (left). E-cadherin staining (red) is shown to highlight PM in g and h. Full fields are shown in Supplementary Fig. 6h. Quantification of intracytoplasmic Cx43-positives vesicles (right; n = 3 wells, 3 independent experiments, >30 cells per experiment). Values are mean + s.e.m and significant for *P 0.01 and **P 0.001 using analysis of variance + Bonferroni test. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.
uORFs and oORFs regulate translation in mammals(A) uORFs repress translation in HeLa cells.Figure 3(A) uORFs repress translation in HeLa cells. Plot displays the cumulative distribution of translation efficiency in expressed (>0.5RPKM) transcripts containing 1, 2, or >2 uORFs versus transcripts lacking uORFs. Transcripts containing oORFs are excluded from this set. Two-sided Wilcoxon p-values are provided for each uORF set compared to the control. (A) uORFs repress translation in HeLa cells. Plot displays the cumulative distribution of translation efficiency in expressed (>0.5RPKM) transcripts containing 1, 2, or >2 uORFs versus transcripts lacking uORFs. Transcripts containing oORFs are excluded from this set. Two-sided Wilcoxon p-values are provided for each uORF set compared to the control. (B) oORFs repress translation in HeLa cells. Plot displays the cumulative distribution of translation efficiency in expressed (>0.5RPKM) oORF-containing transcripts versus transcripts lacking oORFs. Transcripts containing uORFs are excluded from this set. Two-sided Wilcoxon p-value is provided for the oORF set compared to the control. (C) uORFs repress translation in murine embryonic stem cells. Plot displays the cumulative distribution of translation efficiency in expressed (>0.5RPKM) transcripts containing 1, 2, or >2 uORFs versus transcripts lacking uORFs. Transcripts containing oORFs are excluded from this set. Two-sided Wilcoxon p-values are provided for each uORF set compared to the control. (D) oORFs repress translation in murine embryonic stem cells. Plot displays the cumulative distribution of translation efficiency in expressed (>0.5RPKM) oORF-containing transcripts versus transcripts lacking oORFs. Transcripts containing uORFs are excluded from this set. Two-sided Wilcoxon p-value is provided for the oORF set compared to the control. (E) uORF translation is correlated with CDS repression. Scatterplot displays the per-sample mean repression of uORF-containing transcripts versus the mean translation efficiency of uORFs in single-uORF transcripts. Repression is determined by calculating the difference between the mean TE of CDSs in uORF-containing transcripts versus the mean TE of CDSs in transcripts lacking uORFs/oORFs in their TLS. Only expressed transcripts (>0.5 RNA RPKM across samples per organism) were counted. Labels indicate the sample name. For more information on individual samples, see Expanded View Table EV3.
Caldendrin inhibits myosin Va processivity in in vitro F-actin gliding assay.Figure 3A Upper: Coomassie blue stained SDS-PAGE showing purified full-length myoVa (arrow) from HEK293T cells using a StrepTag-GFP-tag. Lower: Western blot of StrepTag-GFP-myoVa shows that myosin is associated with HEK293T-cell endogenous calmodulin (CaM) upon purification. B Schematic representation of in vitro F-actin gliding assay. CaM = Calmodulin, cald = caldendrin, PLL-PEG = Poly-L-lysine-polyethylene-glycol C Visualization of F-actin gliding caused by myoVa activity. The first (red) and last (green) frame of a 30 second time-lapse image stack were color-coded and overlaid to visualize movement. Yellow = actin filament did not move during 30 seconds of imaging. In the presence of only CaM (left), the majority of actin filaments are gliding. In the presence of both CaM and cald (right), a large number of filaments are immobile. Scale bar = 10 µm. D Quantification of immobile filaments as shown in C. In the presence of cald, the fraction of immobile actin filaments is significantly increased compared to CaM only (* p = 0.031, n=6 experiments, Wilcoxon matched-pairs signed rank test. In each experiment, between 189 to 491 individual filaments were observed. Individual actin filament numbers per experiment: 275, 309, 189, 362, 390, 491). E Relative frequency distribution of recorded gliding velocities. In the presence of cald, the velocity distribution is shifted towards slower velocities compared to CaM only (mean ± SEM, n=5 experiments, 8 filaments for each experiment and condition). F Quantification of F-actin gliding velocity. In the presence of cald, the velocity of F-actin gliding is significantly reduced compared to CaM only (mean ± SEM, ** p = 0.0052, paired t-test, n=5 experiments).
Figure 2A HA-tagged CHAF1A and CHAF1B were co-overexpressed in HEK293T cells. Forty-eight hours post transfection, cells were lysed and immunoprecipitated (IP) with anti-HA beads. The control groups were transfected with empty vector. The IP samples were proceeded to SDS-PAGE and developed with silver staining. Data showed both short and long development results. The whole lane of each group was cut into several gel slices and proceeded to in-gel digestion and LC-MS/MS. B The heatmap of CHAF1A and CHAF1B enriched proteins. Grey bar-covered genes represented nucleus proteins. Pink bar-covered genes represented chromatin binding proteins. C Gene Ontology (GO) analysis of significantly enriched proteins utilizing PANTHER classification system. D STRING network analysis of enriched proteins with the interaction confidence of 0.7. E-H k-means clustering of significantly enriched proteins. Four major clusters were classified: chromatin binding, epigenetic modification, chromatin remodeling, ubiquitination and SUMOylation. siRNAs targeting each CAF-1 subunits and corresponding enriched genes were transfected into TZM-bl cell lines respectively. The fold change of luciferase expression of each groups was normalized to siNC.
Figure 7. CRISPR-deletion of 18qp53-binding site reveals local changes in transcription and γH2AX formation in response to DNA damage. (A) Schematic showing the deletion of ~70 bp region covering non-canonical p53 binding site and adjacent (TG) n repeat in chromosome18q. (B) Western blot analysis of CRISPR deleted cell lines 18q WT and 18q Δp53 treated with DMSO (D) or 50 µM etoposide (E) for 24 hr. Blots were probed with pan-p53, phospho-p53, γH2AX, or GAPDH (as loading control) (C-E) RT-qPCR for p21 (C), PARDG6 (D), or 18q subtelomeric RNA (E) in 18q WT or18q Δp53 treated with DMSO (black) or 50 μM etoposide (red) for 24 hr. (F) ChIP-qPCR assay for γH2AX at 18q subtelomere in 18q WT or 18q Δp53 treated as in panels B-E.
Figure 6A. Principal component analysis (PCA) of wild-type, Ts6, Ts8, Ts11 and Ts15 ES cells, based on the whole gene expression signature except for those resides on aneuploid chromosomes and Y chromosome. B. The phylogenetic tree of the two replicates of wild-type, Ts6, Ts8, Ts11 and Ts15 ES cells based on the unsupervised hierarchial clustering of all protein-coding genes without those encoded from aneuploid chromosomes and Y chromosome.. C. Different trisomic ES cells exhibited overlapping gene expression changes. Gray bars indicated the percentage of up- or down-regulated genes among all genes in the indicated cell line. White bars indicated the percentage of the overlapping genes that was up- or down-regulated among all the up- or down-regulated genes in this cell line when compared with the gray bar indicated cell line. * P <0.05, hypergeometric test. D. Enriched GO terms of up- and downregulated genes across the trisomic ES cell lines. E. Enriched pathways of up- and downregulated genes across the trisomic ES cell lines identified by KEGG pathway analysis. F. RT-qPCR analysis of some gene expression in trisomic and wild-type ES cells. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.
Identification of the LC3-interacting region (LIR) in ALFY.Figure 2A Alignment of the potential LIR in human ALFY to the corresponding homolog sequences of representatives of mammals, birds, fish and insects. Ω indicates an aromatic residue, while Ψ indicates an aliphatic residue. B 35S−labelled in vitro−translated GFP−ALFY (aa 2981-3526) wild−type and different LIR mutants were incubated with GST−GABARAP or −LC3C and binding evaluated by ARG. 10 and 2% of the in vitro−translated proteins used were loaded to illustrate binding affinity. CBB staining shows equal amounts of GST proteins used. Data are representative of three independent experiments. C MBP, MBP−ALFY (aa 3255-3526) or the ALFY−LIR mutant (F3346A) conjugated to amylose resins was incubated in the absence or presence of purified GABARAP. The pulled−down complexes were subjected to SDS-PAGE and visualized by CBB staining. Data are representative of three independent experiments. D Thermodynamic parameters of ALFY−LIR peptide (aa 3341-3354) to Atg8 homologs. All titrations were performed at 25°C as described in Supplementary Methods. ITC data were fitted to a one−site binding model. ND, not detected. E 35S−labelled in vitro−translated GFP or GFP−Bchs (aa 3326-3341) were incubated with GST or GST−dAtg8a and binding evaluated by ARG. 5% of the in vitro−translated protein used was loaded. Ponceau staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.
Model for intrinsic regulation of Mif2 during kinetochore assembly Upper panel: In a wild type situation, Mif2 does not bind to the Mtw1 complex in the nucleoplasm as a consequence of adopting an auto-inhibitory conformation.Figure 9Upper panel: In a wild type situation, Mif2 does not bind to the Mtw1 complex in the nucleoplasm as a consequence of adopting an auto-inhibitory conformation. Binding of Mif2 to the Cse4 nucleosome allows a conformational change that promotes its interaction with the Mtw1c and thus triggers full kinetochore assembly. Lower panel: Exchanging the N-terminus of Mif2 with the N-terminus of Ame1 prevents the adoption of an auto-inhibitory conformation. As a consequence, Mif2 forms interactions with the Mtw1c already in the nucleoplasm. These interactions might promote the assembly of soluble kinetochores that are able to bind to microtubules or chromatin away from centromeres and are therefore not available for proper kinetochore assembly on Cse4 nucleosomes.
FBXO7 ubiquitinates PSMA2.Figure 4A. Lysates of HEK293T cells, transfected with empty control vector, myc-FBXO7 or myc-FBXO7 ΔF-box plasmids together with the FLAG-PSMA2 expression plasmid, were immunoblotted with the FLAG, myc or pan 14-3-3 antibody. B. Lysates of HEK293T cells, transfected with the indicated plasmids, were subjected to boiling, followed by IP with the GFP antibody (PSMA2), followed by IB with the ubiquitin (DAKO) antibody (upper panel). Inputs were immunoblotted with the GFP or myc antibody (lower panels). Three independent blots were quantified and band intensity normalized to control average. (ANOVA, *p<0.05, **p<0.001, mean + s.e.m) C. and D. Lysates of HEK293T cells, transfected as in B, were first subjected to a denaturing protocol, then to IP with the GFP antibody, followed by IB with the K63-specific ubiquitin antibody (C) or the K48-specific antibody (D). Inputs were immunoblotted with the GFP or myc antibody (lower panel). Three independent blots were quantified and band intensity normalized to control average. (ANOVA, *p<0.05, mean + s.e.m). C. and D. Lysates of HEK293T cells, transfected as in B, were first subjected to a denaturing protocol, then to IP with the GFP antibody, followed by IB with the K63-specific ubiquitin antibody (C) or the K48-specific antibody (D). Inputs were immunoblotted with the GFP or myc antibody (lower panel). Three independent blots were quantified and band intensity normalized to control average. (ANOVA, *p<0.05, mean + s.e.m). E. Lysates of HEK293T cells, transfected with the indicated HA-ubiquitin, GFP-PSMA2 or myc-FBXO7 plasmids, were subjected to boiling, followed by IP with the GFP antibody (PSMA2), followed by IB with the HA antibody (upper panel). Inputs were immunoblotted with the GFP, myc or HA antibody (lower panels). Three independent blots were quantified and band intensity normalized to control (lane 2) average. (ANOVA, Dunnett's multiple comparison ***p<0.001, mean + s.e.m). F. Brain lysates from FBXO7+/+ and FBXO7-/- mice immunoblotted with the PSMA2 or γ-tubulin antibody. The latter served as loading control. 10 brains from each genotype were included in the quantitative analyses (paired t-test, mean + s.e.m.). G. cDNA generated from cortex isolated from FBXO7+/+, FBXO7+/- and FBXO7-/- mice, was analyzed with quantitative PCR using FBXO7- and PSMA2-specific primers along with the housekeeping gene β-actin. n=4, 2 and 4, respectively (unpaired t-test, mean + s.e.m.) H. For the in vitro ubiquitination assay, purified PSMA2, tagged and immunopurified FBXO7 and SCF core components from HEK293T cells, E1 enzyme, ubiquitin and ATP together with indicated E2 enzymes were incubated for 1 h at 37 °C. The reactions were immunoblotted with the PSMA2, ubiquitin or FBXO7 antibodies.
Figure 4A. Overview of the dBRD9-A transcriptomics experiment in A549 cells. B. Up- (red) and down- (blue) regulated genes identified in A549 cells following treatment with 100 IU/mL IFN-α2 for 6h: ISGs were defined as induced by IFN-α2 with FC>2, p-value ≤ 0.001. C. Impact of dBRD9-A pre-treatment on the induction of ISGs identified in (B). Colors indicate IFN-induced transcripts significantly affected (blue) or unaffected (gray) by dBRD9-A pretreatment (FC>2, p-value ≤ 0.001). D. Heatmaps highlighting the gene expression changes observed with each treatment. Left: the top 50 ISGs, ranked by IFN-induced expression (B), were compared with respect to the impact that dBRD9-A pre-treatment had on induction. Right: comparative fold change in induction for each ISG in the presence or absence of dBRD9-A. E. ISG expression level following IFN-α2 stimulation, grouped by whether they were affected or not by dBRD9-A pretreatment F. RT-qPCR confirmation of transcriptomics results for selected transcripts (MX1, MX2, IFITM1, ISG15 and IFIT1) in A549 cells treated as outlined in (A). GAPDH transcript levels were used for normalization. Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by unpaired 2-tailed t-test on ΔCt values (*p-value < 0.05; n.s. not significant).
Figure 7A. Representative spinning disk confocal time series of U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), with indicated treatments (left). White arrowheads highlight poleward flux of the photoactivated regions. Scale bar, 10 µm. Time, min:sec. Right panels display corresponding kymographs of the photoactivated spindles used for quantification of the flux rates (red dotted lines highlight MT-flux slopes). Scale bars, 2 µm (horizontal) and 30 sec (vertical). B, C. Quantification of MT-flux in prometaphase (B) and metaphase (C) bipolar spindles subjected to indicated treatments. Graphs represent MT flux of individual cells with mean ± SD. siNDC80, siNDC80 + GSK923295, early prometaphase, early prometaphase + GSK923295, siControl, and siKIF4A N (number of cells, number of independent experiments): siNDC80 + KIF15 + STLC (33, 5), siNDC80 + KIF15 + STLC + GSK923295 (25, 3), early prometaphase + KIF15 + STLC + GSK923295 (11, 2), siKIF15 + STLC (29, 3), siKIF4A + siKIF15 + STLC (10, 3). D. Representative spinning disk confocal time series of U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), with indicated treatments (left) and corresponding kymographs (right) as described in (A). Scale bars, 2 µm (horizontal) and 30 sec (vertical).
Cross-reactivity and neutralization efficiency of SARS nAbs against SARS-CoV-2.Figure 3.Binding of SARS nAbs to SARS-CoV S1 protein were tested by ELISA. Recombinant S1 protein of SARS-CoV were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein.B. Binding of SARS nAbs to SARS-CoV-2 S1 protein were tested by ELSIA. Recombinant S1 protein of SARS-CoV-2 were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein. Neutralization of SARS-CoV nAbs against SARS-CoV-2 PSV. Antibody competition with SARS-CoV RBD binding to ACE2. Recombinant SARS-CoV RBD protein was coated on plates, nAbs and recombinant ACE2 were then added for RBD binding competition measurements.