EMBO/sd-character-level-ner huggingface dataset:
Alvocidib induces cell death through the mitochondrial apoptotic pathway.Figure 4A) Indicated cell lines were treated with DMSO, 200nM alvocidib or 25nM dinaciclib for 48h, then cells were stained with Annexin V/PI. Flow cytometry was then used to quantify Annexin V+ cells (n=2). Alvocidib: CrizR1 P=0.005, AlecR P =0.001, PC9 P =0.05, A549 P =0.002, H1299 P =0.03, H460 P =0.07; P dinaciclib: CrizR1 P=0.02, AlecR P =0.005, PC9 P =0.06, A549 P =0.08, H1299 P =0.09, H460 P =0.01. (B) CrizR1 cells were treated with DMSO or 200nM alvocidib for 24h and cell extracts were hybridized to a 43-antibody array and analysed by immunoblotting. Graphs depict the significant changes from two independent experiments. C) CrizR1/CeritR/AlecR cells were treated with DMSO or 200nM alvocidib or 50nM dinaciclib for 6h and cell extracts were analysed by western blotting. D) (Top) Western blot analysis of CrizR1 cells treated with siScrambled or siRNA for CDK7 or CDK9 for 72h. (Bottom) CrizR1 were treated as above, or with DMSO/alvocidib/THZ1 and cells were stained with Annexin V/PI and analysed by flow cytometry for Annexin V+ cells 72h post-transfection (n=2). Annexin siCDK7 P=0.2, siCDK9 P=0.015, alvocidib P=0.0001, THZ1 P =0.004. E) H3122 parental and CrizR1 cells were treated with DMSO, 200nM alvocidib or 250ng/ml actinomycin D for 6 hours. RNA was extracted and the mRNA levels of the indicated genes were quantified by RT-qPCR. CDK1: DMSO vs H3122 DMSO P=0.002, Actinomycin vs DMSO P=0.0003, Alvocidib vs DMSO P=0.0005; CDK2: DMSO vs H3122 DMSO P =0.0001, Actinomycin vs DMSO P<0.0001, Alvocidib vs DMSO P=0.0005; CDK6: DMSO vs H3122 DMSO P =0.0005, Actinomycin vs DMSO P =0.0004, Alvocidib vs DMSO P =0.0004; CDK9: DMSO vs H3122 DMSO P=0.0003, Actinomycin vs DMSO P =0.0002, Alvocidib vs DMSO P =0.0003; CCNB1: DMSO vs H3122 DMSO P=0.02, Actinomycin vs DMSO P=0.0002, Alvocidib vs DMSO P=0.0003; CCNE1: DMSO vs H3122 DMSO P =0.0004, Actinomycin vs DMSO P =0.0004, Alvocidib vs DMSO F) RT-qPCR analysis of MCL-1 and Survivin expression after treatment of CrizR1 cells with 100nM alvocidib, 25nM dinaciclib and 50 nM THZ1. RNA was extracted after 24h of treatment. MCL-1 alvocidib vs control P=0.0001, dinaciclib vs control P=0.0001, THZ1 vs control P =0.0001; Survivin: alvocidib vs control P =0.01, dinaciclib vs control P=0.01, THZ1 vs control P =0.0001. G) (Top) Western blot analysis of CrizR1 cells treated with siScrambled or with a pool of 4 different siRNAs targeting Survivin (siBIRC5). (Bottom) Cells were stained with Annexin V/PI and analysed by flow cytometry for Annexin V+ cells 72h post-transfection. Annexin: early apoptosis P =0.0003, late apoptosis P =0.5, alive P <0.0001. H) The CCLE RNA-seq dataset was used and RPKM values were plotted for the Cyclin D1 and Survivn genes, indicating high expression in H3122 cells compared with other LUAD cells.
Figure 5(A) Schematic in design of rRv2626c and its mutants. (B) Bacterially purified 6xHis-rRv2626c and analyzed by Coomassie blue staining (left) or immuno blotting (IB) with αHis (right). (C) BMDMs were incubated with rRv2626c-CA, rRv2626c-DN, or rVehicle for the indicated times and then cell viability measured with MTT assay. (D) BMDMs were pretreated with rRv2626c-WT, rRv2626c-CA, or rRv2626c-DN for 1 h, and stimulated with 100 ng/ml LPS for 18 h. Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA. (E) Myc-Rv2626c-expressing Raw264.7 or THP-1 cells were pretreated with rRv2626c-WT (2.5 μg/ml), rRv2626c-CA (10 ng/ml), rRv2626c-DN (2.5 μg/ml), for 1 h, and stimulated with 100 ng/ml LPS for 30 min, followed by IP with αMyc or αTRAF6, IB with αMyc and ubiquitin. WCLs were used for IB with αMyc, αTRAF6, αHis or αActin.
Figure 2(A) Mass spectrometry analysis uncovered that FAM134B was phosphorylated at serine 151. (B) Comparison of the self-interaction of FAM134B mono-phosphorylation mutants using co-IP. (C) Comparison of the self-interaction of FAM134 tri-phosphorylation mutants using co-IP. (D) Comparison of membrane scission activity using in vitro liposome fragmentation assay. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n≧3). (E) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co-expressing GFP-SEC61B and FAM134B (WT)-Flag, FAM134B (SA)-Flag or FAM134B (SD)-Flag. (F, G) Measurement of the intracellular ER-scission activity. FAM134B knockout (KO) U2OS cells was engineered to express EGFP-FAM134B (WT), EGFP-FAM134B (SA) or EGFP-FAM134B (SD) at endogenous levels, lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1). GFP-positive puncta were quantified for each cell in (G). For control, 25 cells were counted (n=25); For EGFP-FAM134B (WT), n=27; for EGFP-FAM134B (SA), n=29; for EGFP-FAM134B (SD), n=25. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM. (H, I) Measurement of the ER-phagy activity. FAM134B knockout (KO) U2OS cells was engineered to express mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (S151A) or mCherry-EGFP-FAM134B (S151D) at endogenous levels. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (I). For mCherry-EGFP-FAM134B (WT), 25 cells were counted (n=25); for mCherry-EGFP-FAM134B (SA), n=23; for mCherry-EGFP-FAM134B (SD), n=21. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.
Ptch1+/− MB allografts A Change of tumor volume during GlaB or vehicle treatment period (18 days).Figure 6A Change of tumor volume during GlaB or vehicle treatment period (18 days). B Representative flank allografts average volumes. Error bars indicate SD. *P 0.05. C Representative Masson's trichromestaining of tumors; immunohistochemistry of Gli1, Ki67 and TUNEL stainings. Scale bars represent 35 μm for Masson's trichromestaining, 25 μm for Gli1 and TUNEL, and 10 μm for Ki67 stainings. D Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in (C). Error bars indicate SD. *P 0.05. E qRT-PCR analysis show Hh and proliferation target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr. F Western blot analysis show Hh and proliferation target protein expression levels. Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.
A associates with large and active chromatin domains in ESCs A.Figure 3A. Western blot showing equal BAZ2A levels in wt-ESC and F/H-BAZ2A ESC line containing FLAG-HA sequences at the N-terminus of both Baz2a alleles. Whole cell lysates from equivalent amounts of cells were analysed. SNF2H, and Tubulin are shown as protein loading controls. B. Anti-FLAG ChIP of wt-ESCs and F/H-BAZ2A-ESCs cultured in 2i or serum. Data were measured by qPCR and normalized to input and a control region that is not bound by BAZ2A. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (* < 0.05, ** < 0.01). C. Representative images showing the association of BAZ2A at regions enriched in H3K27ac and low in H3K27me3. The grey rectangles highlighted some of BAZ2A associated regions. D. Pearson correlation heat map for the indicated ChIPseqs in ESC+2i. BAZ2A ChIPseqs were performed with FLAG antibodies in F/H-BAZ2A-ESCs cultured in 2i or serum and with HA antibodies in F/H-BAZ2A-ESC+2i. Data of H3K4me3, H3K27ac and H3K27me3 are from this work. H3K36me3, H3K9me3 and H3K4me1 values in ESC+2i were taken from published data sets E. Boxplot showing the mean of normalized BAZ2A occupancies in ESC+2i and ESC+serum at BAZ2A-bound regions. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (**** < 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes. F. Differential BAZ2A-binding in 2i and serum correlates with H3K27ac occupancies. Boxplot showing the mean of normalized H3K27ac and H3K27me3 read counts in ESC+2i and ESC+serum at all BAZ2A-bound regions and BAZ2A-bound regions only in 2i were calculated and their log2-fold changes are plotted. H3K27ac in ESC+serum Statistical significance (P-values) was calculated using the paired two-tailed t-test (*** < 0.001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes. G. Representative images showing 2i-specific BAZ2A-bound regions and H3K27ac and H3K27me3 in ESC+2i and ESC+serum. The grey box highlights strong TIP5-bound regions and corresponding histone modifications. H. Representative images showing that genes regulated by BAZ2A are enriched in H3K27me3, depleted of H3K27ac and not bound by BAZ2A. I. Heat map profiles of H3K27me3 and H3K27ac at ±5 kb from the TSS of genes differentially regulated by BAZ2A in ESC+2i and random genes. Differentially regulated genes are shown as upregulated or downregulated in ESC+2i upon BAZ2A knockdown. Data were ranked by H3K27me3 levels. Transcription start site (TSS). J. Boxplot showing the mean of normalized H3K27me3 and H3K27ac read counts over +/- 1kb of TSSs from upregulated, downregulated and random genes. Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.
Figure 4A. Schematic representation of the flow of the experiment. TCs were assembled on DNA-RNA scaffolds (DNA was biotinylated); RNA was labeled at 3' with radioactive UTPs (asterisks). The RNA length (including label) was 33 nt. B. Representative primary data - polyacrylamide gel (the experiment was performed 3x with the same results). Lane 1, the full length (33 nt) labeled RNA; lane 2, the same as lane 1 but it included also incubation with RNase J1 (J1), 17-18 nt long fragments (RNase J1 stopped by RNAP; indicated with asterisks) and <5 nt fragments (RNA released from TC into buffer - indicative of TC disassembly) are shown; lane 3, the same as lane 1 but included also incubation with RNase R (R); lanes 4 and 5, TCs were denatured by heat prior to RNase addition to demonstrate the activity and cleavage patterns of both enzymes; lanes 6 and 7, the same as lanes 2 and 3 but the buffer was washed off (TCs were retained by streptavidin beads) to demonstrate which RNA fragments were associated with TC; lanes 8, 9 (M1, M2) Mw marker generated by treating the 30 nt RNA with alkali and formamide (M1-4 min treatment, M2-7 min treatment). As reported in (Costanzo et al., 2016) [and references therein], the cleavage by alkali or formamide leaves the phosphate group of the attacked phosphodiester bond bound at 3', initially in the 2',3' cyclic form (upper band in the band couples). This successively opens (lower band in the band couples) yielding a double-banded pattern for short oligoribonucleotides.
Figure 3The top panel displays the ear length phenotype on the field (only for plants that were transcriptome profiled). The bottom panel displays the average z-scored gene expression profile of spatially autocorrelated gene cluster 2 (291 genes), mapped to the field. Shown on top are the Pearson's correlation (r) between the cluster 2 expression profile and ear length, the corresponding p-value (computed using cor.test in R) and the corresponding q-value (computed using the Benjamini-Hochberg method on all comparisons of cluster gene expression profiles with the ear length profile). The scales on the top and to the right of the field maps give field plot dimensions in cm.
Figure 4(A) Schematic representation of starvation experiment in BALB/c mice. (B) Change in miR-122 level in starved mouse liver. Endogenous miR-122 (left panel) and CAT-1 mRNA (middle panel) levels in mouse liver starved for 12h were quantified by qRT-PCR. miR-122 levels in the blood serum of corresponding animals were also measured (right panel). The Ct values of miR-122 and CAT-1 mRNA obtained for each individual animal liver and blood serum were plotted (mean+/- s.e.m., n=5). (C) Relative level of HuR in both liver and lung of Fed and Starved mice were analysed by western blots, β-actin was used as loading control . (D) Schematic representation of HA-HuR expression or Control plasmid injection experiment in BALB/c mice. (E) Western Blot analysis to check expression of HA-HuR in liver samples of injected mice. β-Actin was used as loading control. HA-HuR expressing Huh7 cells lysate was used as positive control. (F). Change in miR-122 level upon expression of HA-HuR in mouse liver. Endogenous miR-122 level in HA-HuR expression plasmid injected mouse liver were quantified by RT-PCR and compared against liver RNA samples collected from control (pCIneo injected) group of animals (mean+/- s.e.m., n=5). (G) Endogenous miR-122 target CAT-1 (left panel) and Aldolase (right panel) mRNA levels in HA-HuR or pCIneo (control) injected mouse liver were quantified by RT-PCR (mean+/- s.e.m., n=4). (H) miR-122 levels in the blood serum of pCIneo (control) or HA-HuR expression plasmid injected mice were measured. The Ct values of miR-122 and CAT-1 and Aldolase mRNAs obtained for each individual animal liver and blood serum were plotted (mean+/- s.e.m., n=5).
Analysis of the mitochondria-caspase dependent pathway.Fig 3A,B) Relative gene expression analysis of the pro- and anti-apoptotic markers Bax, Bak, Bcl-2, and Bcl-xl in Cal-78 and SW-1353 cells treated with the respective IC50 values of bortezomib for 24 h (n = 10). C) The human apoptosis antibody protein array revealed the downregulation of the heat shock proteins HSP27, HSP60, and HSP70 as well as HTRA, Livin, p27, and p53 in both cell lines. D) Western blot analysis showed an upregulation of cytochrome C in the cytoplasmic fraction after bortezomib treatment.
Fig.Figure 5(A) Schematic of the screening procedure. Compounds were dispensed in matrigel coated 384 well plates prior to seeding of murine myoblasts. Myoblasts were incubated for 24 h with 10 µM of the individual compounds, 5 µM of the positive control Trichostatin A or with the negative control DMSO. After fixation and staining with the IIH6 antibody, detecting glycosylated α-dystroglycan, the cells were imaged using the confocal PE Opera LX high-throughput microscope. The intensity of the α-dystroglycan glycosylation staining was determined, values normalized and a hit list generated. Scale bar, 20 µm. (B) Compound validation strategy. After the primary screen, the α-dystroglycan glycosylation staining pattern of initially identified compounds was examined to exclude autofluorescent as well as toxic compounds. From the remaining compounds 11 hit compounds were nominated after acquired images were compared to controls in the respective screening plate. A dose response assay followed by flow cytometry assays further narrowed down hits to 1 compound. (C) Dose response curve. H2K 2B4 cells were treated with serial dilutions of 11 hit compounds for 24 h and fixed, stained and imaged as described above. The average values and standard deviations of the mean fluorescent intensities of technical triplicates were calculated and a non-linear regression curve was fitted for the 3 compounds showing the highest increase in α-dystroglycan glycosylation (IIH6 immunostaining). Representative images of cells treated with 5 µM of the respective compounds as well as EC50 values are shown. HPFPTzone: (5Z)-5-[(3-ethoxy-4-hydroxy-phenyl) methylene]-3-(4-fluorophenyl)-2-thioxo-thiazolidin-4-one. 4BPPNit: 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile. DPPTzone: (5E)-5-[(2,5-dimethyl-1-phenyl-pyrrol-3-yl) methylene]-3-phenyl-2-thioxo-thiazolidin-4-one. Regression curve and EC50 values were calculated using GraphPad Prism 7 software. In images shown, green represents glycosylated α-dystroglycan and blue Hoechst staining. Scale bar, 50 µm. (D) Structures of the 3 compounds showing the highest increase in α-dystroglycan glycosylation. Structure 1: HPFPTzone. Structure 2: 4BPPNit. Structure 3: DPPTzone. (E) Flow cytometry analysis confirmed that 4BPPNit treatment (20 µM) significantly increased IIH6-reactive glycan in mouse H2K 2B4 cells, compared with untreated cells. There was no significant difference in the percentage of IIH6-positive cells. Values indicate mean ± s.e.m. (n= 6, triplicates of 2 independent experiments; t-test; NS, not significant; ** p<0.01).
Figure 1A Structure of the PAP8 locus, blue boxes: exons, lines: introns. Red box: inserted T-DNA as inverted repeats (IR1/IR2) in the first intron. White box with a diagonal red line: deletion at the left border of IR2 and part of second exon (italicized grey sequence). Green and red arrows represent forward and reverse primers respectively as o1: oPAP8_rtp_F ; o2: oPAP8_E3_R; o3: oPAP8_rtp_R; o4:op8i2_R; oLB: oLBb1.3. B PCR performed on genomic DNA with indicated primers as shown: o1: oPAP8_rtpF, o2: oPAP8_E3R, o3: oPAP8_rtpR, oLB: oLBb1.3, EF1α: ELONGATION FACTOR 1α, WT: wild type, pap8-1: Homozygous albino plant, Ht: Heterozygous green plant; T: T-DNA, arrowhead: 670-bp contaminant amplification product used as loading control. C RT-PCR on wild type and pap8-1 homozygous plants grown in the dark for 3 days followed with 72-h growth under white light to allow greening of the wild type; EF1α used as control. D Sequence alignment of predicted full length orthologous PAP8 protein found in representatives of major phylogenetic clades Arabidopsis thaliana, At1g21600; Oryza sativa Indica, EEC67529.1; Amborella trichopoda, XP_006827378.1; Selaginella moellendorffii, XP_002976643.2 Physcomitrella patens, XP_024396032.1. cTP, chloroplast transit peptide as predicted with ChloroP1.1 (www.cbs.dtu.dk) underlined in yellow; HCR shaded in grey, highly conserved region. (*), (:), (.), conserved, strongly similar, or weakly similar amino acid properties (standards from www.uniprot.org). amino acids colors as in Clustal Omega (Red (AVFPMILW): Small + hydrophobic [includes aromomatic - Y]); Blue (DE): Acidic; Magenta (RHK): Basic; Green (STYHCNGQ): Hydroxly + sulfhydryl + amine + G.) bNLS, bipartite NLS as predicted with NLS mapper E Half-open siliques of a heterozygous plant showing the embryo greening; scale bar equals 250 µm. The given number is the position rank of the silique from top to bottom of the inflorescence presenting the segregation of homozygous and heterozygous seeds based on their ability to transiently develop chloroplasts. F Mutant Rescue: WT and two representative pap8-1 plants were grown in vitro using sucrose and low white light intensity (of 10 µmol.m-2.s-1); scale bar equals 20 mm.
Figure 5a-b, Comparison of splenic CD8 Tn mitochondrial mass [Mitotracker (MT) green MF, a] and mitochondrial potential (MT Deep Red, b) of young and 45-wk-old Dj-1 KO and WT mice. c-d, Comparison of CD8 Tem mitochondrial mass (c) and mitochondrial potential (d) of young and 45-wk-old Dj-1 KO and WT mice. e-h, Oxygen consumption rate (OCR) of unstimulated (e) and stimulated CD8 Tn (f) by 2ug/ml of anti-CD3 and 1ug/ml of anti-CD28 for 24 hours. The CD8 Tn were isolated from 45-wk-old mice. OCR of unstimulated (g) and stimulated CD8 Tn (h) by anti-CD3 and -CD28 for 24 hours. The CD8 Tn were isolated from young mice. Left of each OCR measurement panel, representation plots following Mito stress test. Right of each panel, quantitation of basal and maximum respiration under Mito stress test conditions The dashed line indicates the time when the corresponding modulator Oligomycin (Oligo), Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) or Rotenone and Antimycin (AA/Rot) was added. i-k, Extracellular acidification rate (ECAR) of unstimulated and stimulated CD8 Tn by anti-CD3 and -CD28 antibodies for 24 hours (i). The CD8 Tn were isolated from 45-wk-old mice. ECAR of unstimulated (j) and stimulated (k) CD8 Tn by anti-CD3 and -CD28 for 24 hours. The CD8 Tn were isolated from young mice. Left of each ECAR measurement panel, representation plots following Glycolysis stress test. Right of each panel, quantitation of glycolysis and glycolytic capacity under Glycolysis stress test conditions. The dashed line indicates the time when the corresponding modulator glucose, Oligomycin (Oligo) or 2-deoxy-glucose (2-DG) was added.
Identification of novel, USP22-regulated RIPK3 ubiquitination sites A Schematic representation of the experimental strategy for quantitative analysis of ubiquitination sites in USP22 KO HT-29 cells versus HT-29 CRISPR/Cas9 control (ctr) cells under untreated and necroptosis-induced conditions.Figure 5A Schematic representation of the experimental strategy for quantitative analysis of ubiquitination sites in USP22 KO HT-29 cells versus HT-29 CRISPR/Cas9 control (ctr) cells under untreated and necroptosis-induced conditions. Control cells were grown in SILAC medium containing Arg0/Lys0 ("Light") labeled, whereas USP22 KO cells were labeled in SILAC medium containing Arg10/Lys8 ("heavy") labeled. In the treated conditions, cells were incubated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 6 h. Cells were lysed, and equal amounts of extracted proteins extracted from "Light" or "Heavy" were pooled and digested in-solution with trypsin. Ubiquitin remnant peptides were enriched using di‐glycine‐lysine‐specific antibodies, fractionated by Micro‐SCX and analyzed by LC‐MS/MS. B Bar graphs demonstrating the number of up‐, non‐, and downregulated ubiquitination sites in USP22 KO HT-29 cells versus control under TBZ-treatment. C Schematic representation of RIPK3. Indicated are the two major protein domains, the kinase domain (KD), the RHIM and the three different potential ubiquitination sites identified by ubiquitin remnant profiling. D HeLa cells expressing Dox-inducible RIPK3 WT, D160N, K42R, K351, K518R, 2xKR or 3xKR were pre‑incubated with 1 µg/ml Dox, 20 µM Dab, or 10 µM NSA overnight, followed by pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 4 h. Cell death was measured by quantification of PI-positive nuclei. E HeLa cells expressing Dox-inducible RIPK3 mutants were treated with 1 µg/ml Dox overnight. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. GAPDH served as loading control.