EMBO/sd-character-level-ner huggingface dataset:
Figure 4A Volcano plot depicting down-regulated (left) and up-regulated (right) genes in Mettl5 KO mESCs compared with WT mESCs in Ribo-seq. Significantly altered genes are defined as those ≥1.5-fold change and P value < 0.05 (shown in red dots). Blue dots represent genes with <1.5-fold change and P value < 0.05. Green dots represent genes with ≥1.5-fold change and P value ≥ 0.05. Black dots represent genes with ＜1.5-fold change and P value ≥ 0.05. P values were inferred from two-sided Wald tests (DESeq2) and P value adjustments were made for multiple comparisons. Examples of up- or down-regulated or non-significantly changed genes are annotated in the graph. B Track plot showing translational changes of Fbxw7 in Mettl5 KO mESCs compared with controls. C RT-qPCR analysis of Fbxw7 mRNA levels in WT and Mettl5 KO cells during the indicated time points course of EB induction. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant. D Western blot analysis showing FBXW7 expression in control and Mettl5 KO cells during the indicated time points of EB induction. GAPDH was used as a loading control. E RT-qPCR analysis showing Fbxw7 mRNA levels in Day7 KO, KO+HA-MT5WT, and KO+HA-MT5Mut EBs. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant. F Western blot analysis showing FBXW7 expression in Day7 KO, KO+HA-MT5WT, and KO+HA-MT5Mut EBs. GAPDH was used as a loading control.
Fig.Figure 5(A) ATP concentration in liver homogenates obtained from 34-week-old wild-type, untreated or treated LmnaG609G/+ mice. (B) Mitochondrial calcium measured in liver mitochondria isolated from the indicated experimental mouse groups. (C) Magnesium concentration in isolated mitochondria. (D) Activities of the indicated mitochondrial complexes (I, II, III, IV, and V) in the absence or presence of rotenone (2 µmol/L), 2-thenoyltrifluoroacetone (TTFA; 1 mmol/L), antimycin A (10 µmol/L), potassium cyanide (KCN; 1 mmol/L), or oligomycin (10 µmol/L). (E) The boxed scheme shows the five mitochondrial complexes involved in the electron transport chain and their known inhibitors.Data information: Results are presented as mean ± S.D. (n=16). One-way ANOVA and Tukey's multiple comparisons post-hoc test were used for statistical analysis. *p<0.05; **p<0.01; ***p<0.001.
Compartment-specific localization of neuronal transcripts and proteins.Figure 1(A) Schematic of the workflow for the culture of primary hippocampal neurons and 48 hours of PTX-treatment. (B) Quantification of miniature EPSC amplitudes in mock- and PTX-treated hippocampal neurons. (n=10 neurons per condition from 3 independent experiments, independent two-sample Wilcoxon-Mann-Whitney-U test, *p<0.05). (C) Volcano-plots demonstrating enrichment of transcripts in either the somatic (red) or process (yellow) compartment with representative genes highlighted. (D) Top Gene Ontology Pathway Analysis for transcripts enriched in the somatic and process compartment. (E) Volcano-plots demonstrating enrichment of proteins in either the somatic (red) or process (yellow) compartment with representative genes highlighted. (F) Quantification of the highlighted genes enriched in either the somata or processes at the transcript or protein level. (Upper row: Scatterplots of transcript level; n=2; crossbar represents mean. Lower row: Boxplot of protein levels; n=4).
Figure 4A. Box plot showing the distribution of fold enrichment values per genes sorted by exon number, n= 66, 247, 389, and 574 genes with 1, 2, 3, and 4 exons, respectively. Dashed line represents an IP/Input ratio of 1, taken as a reference. B. Reads from input and IP counted in unstimulated and PMA stimulated triplicates give significant enrichment values of 1.4 and 0.7 on introns and exons, respectively, shown as mean and s.d., n=3 biological replicates. Dots represent individual data points. C. Box plot of reads belonging to spliced transcripts versus all reads in input or RNAchIP samples pooled between + and -PMA, calculated as a ratio between split reads and total reads, n=3 biological replicates. Dots represent individual data points. Data information: All box boundaries represent 25th and 75th percentiles; center line represents median; whiskers indicate ±1.5×IQR; points are values of outliers. All P-values are * P < 0.01, ** P < 0.005, ***P < 0.001; two-tailed Student's t-test. D, E. Average distribution profiles of the depicted hexameric motifs (D), or SINE and LINE repeats (E) on RefSeq genes, computed over exons (black box) and on ±0.7kb of surrounding introns (left panels), or over entire gene bodies including introns (pre-mRNA; middle panels), or as metagenes over exons only (Metagene mRNA; right panels). F. Box plot showing the distribution of fold enrichment values on series of introns of comparable size (4kb in average, ranging from 3 to 5kb) sorted by number of sense CACACA motifs per intron (2+, two or more motifs), n= 5782, 899, 960, and 3234 introns for all introns, 0, 1, and 2+ CACACA categories, respectively. R2, Linear regression coefficient between 0, 1, and 2+. Dashed line represents an IP/Input ratio of 1, taken as a reference. Data information: All box boundaries in Fig 4 represent 25th and 75th percentiles; center line represents median; whiskers indicate ±1.5×IQR; points are values of outliers. All P-values are * P < 0.01, ** P < 0.005, ***P < 0.001; two-tailed Student's t-test. G, H. Average read profiles on two groups of RefSeq genes, in the size-range 4 to 15kb, containing the highest (G) or lowest (H) CACACA motif density.
Transport granule component TOG D1 interacts weakly with hnRNPA2 LC.Figure 2A) AlexaFluor488-tagged (green) hnRNPA2 LC undergoes LLPS, while AlexaFluor555-tagged (red) TOG D1 does not. However, TOG D1 partitions into hnRNPA2 LC droplets when mixed at a 1:1 ratio. Conditions: 20 µM indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. hnRNPA2 LC control duplicated from Figure 1A as hnRNPF PLD and TOG D1 samples were made concurrently. B) Similar to hnRNPF PLD, AlexaFluor555-tagged TOG D1 does not undergo LLPS at 300 µM or partition into AlexaFluor488-tagged FUS LC droplets with both proteins at 300 µM. Conditions: 300 µM proteins (~1% fluorescently tagged), 20 mM MES pH 5.5 150 mM NaCl, 150 mM urea. Scale bar: 10 µm. C-D) TOG D1 homology structure with Γ2 values from PRE experiments for hnRNPA2 LC (c) S285C and (d) S329C. Amino acids are colored based on Γ2 value: red corresponds to Γ2 > 2, orange to 2 > Γ2 > 1, yellow to 1 > Γ2 > 0.5. E) AlexaFluor555-tagged hnRNPF PLD or FL and AlexaFluor405-tagged TOG D1 partition simultaneously into AlexaFluor488-tagged hnRNPA2 LC droplets. Conditions: 20 µM of each indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 20 µm.
Figure 4The Bd1971 cNMP dimer (light/dark blue) situates its output domain (EAL) on the opposite side to that of CRP (DNA-binding subdomain, dimer coloured pink/red). Both Bd1971 and CRP dimerize via a central C-helix:C-helix interface. Close-up of hinge helix to C-helix crossover (circled). Tilt of the hinge helix relative to the C-helix allows Bd1971 to bridge from the cNMP domain body into the C-helix of the opposing monomer; the arrangement in CRP differs by pairing the C-helix with the cNMP body of the same monomer. Despite this difference, the respective C-helix dimers remain in the same orientation, as observed in panel (A). Overlay of the cAMP-binding pocket of Bd1971 and CRP (aligned on cAMP ligand, Bd1971 residue numbering given first), ligand-contacting sidechains shown in stick form, residues from the opposing monomer labeled by a '.
Figure 2A. HeLa cell stably expressing GFP-LGN and transiently expressing RFP-H2B was treated with noscapine to induce chromosome misalignments. Cells were imaged every 5 minutes. The time is relative to NEB. The red arrows indicate cortical regions where the chromosomes are in close proximity to the cell boundaries, accompanied by the displacement of LGN from the cortex. B. Similar assay as described in supplementary figure 2D was carried out in the presence of a small molecule inhibitor against Aurora-B (ZM447439) in combination with the proteasome inhibitor MG-132 to keep cells arrested in mitosis and in B. with an inhibitor against Plk1 (BI2536). Drugs were added at the same timing as Hoechst, 30 minutes prior to imaging.
Analysis of functional, binding and regulatory moieties of the endocytic coat protein architecture.Figure 7A Topology of functional, binding and regulatory motifs and domains in the proposed endocytic coat architecture. Known motifs and domains of indicated coat proteins were classified according to their main function and color-coded as shown on the right. Ark1/Prk1 phosphorylation sites were taken from (Huang et al., 2003). B Comparison of the FRET-based protein proximity mapping with previously described interactions of studied proteins. Physical interactions of indicated proteins (observed in more than one study) were taken from Saccharomyces Genome Database as per February 2019. FRET proximities mirroring previously detected protein interactions are shown as violet lines and numbers. Proximities not found as interactions and vice versa are shown as blue and gray lines/numbers, respectively.
A negative effect of HOXA4 on VSMC phenotypic switching is mediated by YAP/TEAD.Figure 6Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding shRNAs of HOXA4 and TEAD1/3/4. Quantification of alpha smooth muscle actin and calponin protein levels is expressed as the means ± SEM of three independent experiments. *P<0.05, **P<0.01, by ANOVA with Sidak's correction. Proliferation assay in VSMCs transduced with indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cell viability was analyzed by Cell Counting Kit-8 at indicated days at each time point as indicated. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments. Representative immunoblotting images and quantification of the interaction between endogenous TEAD1 and HOXA4 or YAP using co-IP experiments in human VSMCs with knockdown of HOXA4. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments. ChIP assays of CTGF, CYR61 and α-SMA promoters using anti-FLAG-tag, anti-Myc-tag, anti-TEAD1 antibodies in human VSMCs transfected with plasmids expressing Myc-tagged YAP-5SA and/or FLAG-tagged HOXA4. *P<0.05, **P<0.01 vs FLAG-GFP, by unpaired two-tailed Student's t-test. Data are expressed as the means ± SEM of three independent experiments.
MIR376 family miRNAs in the DLKI/GTL2 gene cluster.Figure 1 MIR376 family miRNAs in the DLKI/GTL2 gene cluster. (A) Individual miRNA genes were indicated. Sizes of spacer sequences between miRNA genes were marked as nucleotides (nt). (B) Comparison of mature miRNA sequences with identical "seed sequences" (bold, underlined). Identical nucleotides were indicated by asterix (*). ClustalW tool was used for the analysis. (C) ClustalW comparison of the stem-loop sequences of MIR376A and MIR376B. Seed sequences were marked as bold and underlined.
Figure 2Relative levels of c-di-GMP in matched cell biomass host-independent (HI) cultures of bd1971::kan compared with fliC1 merodiploid KnR HI control strain (Wild-Type for bd1971). The bd1971 mutant strain contains significantly more c-di-GMP than the control. Data are from 3 independent experiments. Relative levels of c-di-GMP in matched cell biomass wild-type predatory cultures of HD100 strain, silent Bd1971 deletion strain (Δ1971), Bd1971D306307A strain (bd1971DD) and Bd1971R67D strain (bd1971R). All mutant strains had higher levels of c-di-GMP compared to the wild type strain. Error bars show SEM. Data are from six independent experiments for HD100 and ΔBd1971 and from four independent experiments for Bd1971DD and Bd1971R mutants.
JARID2 exists as a LMW form (~80kDa) in many cell types.Figure 1A schematic diagram showing functional domains of JARID2: UIM, ubiquitin interaction motif; transcription repressive domain consisting of sub-domains 1-3; JmjN, JumonjiN; ARID, AT-rich DNA binding domain; GSGFP which is a SUZ12 binding domain; JmjC, JumonjiC; and ZF, zinc finger domain. Domains 1-3 constitute PRC2 associated domains. Domain 1 is required for PRC2 stimulation, domain 2 is needed for binding to EZH2 and domain 3 is needed for nucleosomal binding. The RNA binding domain has been indicated with dashed box. Immunoblot of whole cell lysates from multiple human lineage-committed cell-lines using a C-terminal anti-JARID2 antibody. The size of canonical form is 140kDa but an additional strong band was detected at around ~80kDa. The blot is divided according to cell-types, which are indicated in the figure. qPCR measurements of JARID2 mRNA levels in different cell lines. The RNA levels were measured relative to 18S rRNA. Means and SEs are calculated over at least 2 independent experiments and 3 technical replicates. The data is represented as mean ± SE. Immunoblot showing that the ~80kDa band disappears upon treatment of HEK293T and HaCaT cells with siRNAs against exon 3 and exon 15 of JARID2 RNA. Non-silencing control (NSC) was used as a transfection control.
Figure 5(A) Molecular docking assay showing the interaction of Palbociclib with STING. Key amino acids responsible for the interaction were indicated. (B) Alignment of the protein sequence of mouse and human STING were performed and the evolutionarily conserved amino acids were indicated. (C) Immunoblotting of the streptavidin precipitates of the mixture of biotin-Palbociclib (100 μM) and the lysates of HEK293T cells transfected with corresponding Flag-STING mutants. The data shown are representative of n=3 biological replicates. (D) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-STING and Flag-STING or Flag-STING Y167A in the presence of DMSO and Palbociclib (10 μM). The data shown are representative of n=3 biological replicates. Representative immunofluorescent assay of STING and ERGIC in HeLa cells stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) (E). Scale bars, 25 μm. STING and ERGIC in HeLa cells stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) The quantification data is shown in (F). The data shown are mean ± SD from indicated number of cells from 1 representative of n=3 biological replicates. Statistical significance was determined by two-tailed Student's t-test. ***, P<0.001; ****, P<0.0001; ns, not significant. (G) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-TBK1 and Flag-STING or Flag-STING Y167A. The data shown are representative of n=3 biological replicates. (H) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-STING and Flag-STING C91A in the presence of DMSO and Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.
Oxidative stress observed in absence of TP53INP1 is at the origin of presence of lipid droplets (LD) in MEFi.Figure 7MEFi deficient (−/−) or not (+/+) for TP53INP1 were seeded in media containing or not 10 mM NAC. Forty-eight hours later, MEFi were treated or not (NT) with 100 μM hydrogen peroxide (H2O2) during 1 h in serum free media. Cells were left to recover for 4 h in normal media containing or not NAC (10 mM), GW-9662 (PPARγ inhibitor, 10 μM) or PNU-74654 (Wnt/β-catenin pathway inhibitor, 50 μM) before being harvested. Cells were observed by TEM.Number of LD normalized by cytoplasmic surface area is shown in both histograms at the bottom of the figure. Scale bar represents 0.5 μm. Results are expressed as the mean ± SEM and are representative of three independent experiments. *P (−/− versus +/+; NT) = 0.00086; #P (H2O2 versus NT; +/+) = 0.013. Cells were also observed by fluorescence microscopy after Bodipy493/503 staining of LD (green) and DAPI staining of nucleus (blue). L = lipid droplets. Scale bar represents 100 μm. Results are representative of three independent experiments. TCLs from MEFi were analyzed by immunoblotting for β-catenin, PPARγ, ATGL, MGLL and β-tubulin. Results are representative of three independent experiments.
Figure.Figure 5(A) qPCR analysis of FSCN1, miR-143 and miR-145 expression in a 1205Lu xenograft nude mice model treated with the BRAFi Vemurafenib compared to control mice (n = 6). Paired Student t-test was used for statistical analysis. ***P≤0.001, ****P≤0.0001. (B) FSCN1 immunofluorescent staining and quantification of fluorescence intensity in M238P cells treated or not with BRAFi (Vemurafenib, 3 μM) in the presence or the absence of LNA-based anti-miR-143 (LNA-143) or anti-miR-145 (LNA-145) (50 nM) or a combination of the two. Data are represented as scatter plots with mean ± SD from 10 independent fields representative of 3 independent experiments, Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm. M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (C) Western Blot analysis of cell cycle markers on cell lysates from M238P cells cultured for 72 h following transfection with the indicated siRNAs. M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (D) Migration assay performed in Boyden chambers. Representative images showing migration of M238P cells treated with the indicated siRNAs. The bar graph represents the quantitative determination of data obtained of mean ± SD from 5 independent fields representative of 3 independent experiments, using ImageJ software. Paired Student t-test was used for statistical analysis. *P≤0.05, ***P≤0.001 M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (E) Western Blot analysis of phenotype-switch markers on cell lysates from M238P cells transfected with the indicated siRNAs. BRAFi-resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct. M238R transduced with a Ctrl lentivirus were used as control. (F) Effect of FSCN1 overexpression on cell proliferation assessed by time-lapse analysis using the IncuCyte system. Graph shows quantification of cell confluence. Two-way ANOVA analysis was used for statistical analysis. ****P≤0.0001. BRAFi-resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct. M238R transduced with a Ctrl lentivirus were used as control. (G) Western Blot analysis of FSCN1, phenotype switch markers and ECM remodeling markers on cell lysates from control and FSCN1 overexpressing cells. BRAFi-resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct. M238R transduced with a Ctrl lentivirus were used as control. (H) Crystal violet viability assay of M238R cells stably overexpressing FSCN1 during 6 days with the indicated doses of the BRAFi Vemurafenib. Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. ****P≤0.0001.
Figure 1A Gene enrichments analysis on differentially expressed genes between sorafenib-sensitive and sorafenib-resistant human liver tumors. B Venn diagrams show overlapped genes in signaling pathways response to sorafenib. Heatmap of overlapped expressed genes in response to sorafenib-resistance. D The mRNA expression level of METTL3 in sorafenib-sensitive (n=3) and sorafenib-resistant (n=3) human liver tumors. Data information: In all relevant panels, ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments. E Expression of METTL3 was significantly down-regulated in advanced stage of liver cancer from TCGA. F METTL3 RNA levels between naïve HepG-2 cells (n=3) and sorafenib-resistant HepG-2 cells (n=10). Data information: In all relevant panels, ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments. G The IC50 of sorafenib-resistant HepG-2 cells treated with sorafenib under hypoxia condition (1%O2). Data information: In all relevant panels, ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments. H The protein level of METTL3 in sorafenib-resistant HepG-2 cells treated with sorafenib under hypoxia condition (1%O2). I The global RNA m6A level in naïve HepG-2 and sorafenib-resistant HepG-2 determined by dot-blotting assay under hypoxia condition (1%O2).
Figure 4(A) The central channel in the PfFNT-MMV007839 protomer viewed from the parasite's membrane plane. The channel, calculated by HOLE (Smart et al., 1996), is indicated by blue dots. There is one constriction site (colored red) formed in the central channel. Residues forming the constriction are in green sticks. Residues that line the rest of the channel are in cyan sticks. (B) Representative figure of the calculated diameters of a PfFNT-MMV007839 protomer. (C) A cartoon of the central channel formed within a PfFNT protomer. The channel contains one constriction site in this conformational state. Residues forming the constriction and the K35-D103-N108 and K177-E229-N234 triads are illustrated as sticks. Residues F94, I97 and L104, which form the first constriction site in the apo-PfFNT structure, is also included in the figure. (D) Surface representation of the inside of the channel formed by a PfFNT protomer (red, negative -15 kT/e; blue, positive +15 kT/e). The five residues K35, F90, F94, K177 and H230, which are subjected for mutagenesis studies, are in green sticks.
Figure 7A. 3D7 ring stage parasites were treated with DMSO or UCT943 or MMV048 for 20-24 h, washed with HBSS before incubation with BODIPY-ceramide and DAPI followed by live imaging. TVN formation seemed to be impaired upon inhibitor treatment (Scale bar-1 μm). B. Ring-infected erythrocytes were treated with MMV048 for 24h and then incubated with FM4-64 and DAPI to assess lipid uptake. Subsequently, FM4-64 associated fluorescence was detected by fluorescence microscopy (upper panel) or quantitated by FACS (lower panel) and % change in FM4-64 uptake in inhibitor-treated parasites was determined. (mean± SEM, n=3; biological replicates, *p<0.05, t-test; Scale bar-1 μm). C. Synchronized PfCDPK7-KO or 3D7 ring/trophozoite stage parasites were labelled with BODIPY-ceramide and DAPI followed by live imaging as described for panel A. While tubular vesicular networks/structures (TVNs), vesicles were predominantly labeled in 3D7 parasites, most PfCDPK7-KO parasites did not exhibit TVN; instead, small punctate spots (white arrowheads) were seen in the iRBC cytoplasm (Scale bar-1 μm). D. Trophozoite stage infected erythrocytes were incubated with FM4-64 and DAPI to assess lipid uptake by 3D7 or PfCDPK7-KO parasites. Subsequently, FM4-64 associated fluorescence was visualized by live microscopy (top panel) and also analyzed by FACS and % change in FM4-64 uptake in KO parasites was determined (bottom panel) (mean± SEM, n=3; biological replicates, *p<0.05, t-test; Scale bar-1 μm).
Figure 1Relationship between carbonylation site distribution, protein vulnerability to reactive oxygen species, and stress phenotypes.   Structural systems biology workflow for proteome-wide carbonyl site prediction. Red circles = carbonyl sites (CS); black circles = non-oxidized RKPT residues; gray protein regions = non-RKPT residues