EMBO/sd-character-level-ner huggingface dataset:
Figure 3(A) Representative confocal microscopy images of F-actin and 3D reconstruction of z-stacks from nuclear region with IMARIS software in Phalloidin-stained HCC cells (A) Red: Phalloidin Blue: DAPI. shCtrl, control shRNA; shTFAM, shRNA against TFAM; EV, empty vector; TFAM, expression vector encoding TFAM. Scale bars, 20μm for microscopy images, 40μm for 3D reconstruction. Representative confocal microscopy images of F-actin and 3D reconstruction of z-stacks from nuclear region with LifeAct-mCherry Venus expressing cells with treatment as indicated (B) (n = 3 independent experiments). Red: LifeAct, Blue: DAPI. shCtrl, control shRNA; shTFAM, shRNA against TFAM; EV, empty vector; TFAM, expression vector encoding TFAM. Scale bars, 20μm for microscopy images, 40μm for 3D reconstruction. (C) Immunofluorescence analysis of TFAM and Phalloidin staining in human HCC cells from paired clinical samples (n=100). The 3D reconstruction of z-stacks from nuclear region is showed in the right side. Red: LifeAct, Blue: DAPI, Green: TFAM. Scale bars, 20μm for microscopy images, 40μm for 3D reconstruction. (D) Statistical analysis for fluorescent intensity of nuclear F-actin in (A), (B) and (C). Fluorescence intensity are quantified from at least 30 cells for each group in (A) and (B). One-way ANOVA. *P < 0.05; **P < 0.01.
Figure 8Average ratios of A, C, G, and T nucleotides in the human internal exons, which are evenly divided into 50 groups according to their lengths. The mean and standard error (SE, semi-transparent shade) are plotted. Average ratios of amino acids in all human internal exons, which are evenly divided as in (A). The numbers in parentheses indicate the ratio of C-nucleotides in the codons considering codon usage in humans. The mean and SE (semi-transparent shade) are indicated. Note that proline and serine are frequently used in IDRs and are rich in C-nucleotides in their codons.   Violin plots showing the fractions of disordered residues in all human internal exons, which are evenly divided as in (A). The mean (green) with standard error (SE, semi-transparent shade) and the median (red) of the fractions of disordered residues are indicated.   Average fractions of disordered residues in large internal exons in different species. All internal exons are divided into 50 groups as in (A) based on their lengths. The last 10 groups (> 173 nt) are shown. The mean and SE are indicated. The phylogenetic tree of analyzed species with distance from humans is shown at the bottom. Clustering analysis and heatmap showing P-values of GO terms enriched in genes containing large (> 180 nt) and C-enriched (> 30%) internal exons in each species. The top 30 GO terms enriched in the human genes carrying large and C-enriched exons were selected. GO terms associated with transcription are indicated in red. Clustering and heatmap showing the relationship between the codon ratios and exon lengths in large exons (> 180 nt) in 265 species. The codon enrichment was estimated by linear regression between the codon ratios and exon lengths in large exons (> 180 nt). A regression coefficient with p < 0.01 is indicated by a heatmap, whereas a regression coefficient without statistical significance is indicated in gray. Codons preferentially used in large exons are color-coded in blue. Enlarged view with species names is shown in Appendix Fig S5. Pie charts showing the number of codons with amino acids in Clusters 1 and 2 in (F). Sequence logos created by the codons in each cluster are also shown. Schematic of C-nucleotide enrichment in exons and splicing regulation of large exons in vertebrates. The C-nucleotide content is biphasically increased with increasing exon length. The first part is accounted for by an increase in C-nucleotide number at the third position of a codon (NNC, N stands for any nucleotide), which has marginal effects on amino acid compositions. The second part is accounted for by an increase in C-nucleotide number at the second position of a codon, which increases the IDR-constituting proline and serine content to secure the phase separation of transcription factors. The splicing-suppressive effect by hnRNP K is masked by SRSF3 so that C-rich large exons are constitutively spliced.
Figure 5A) The GCaMP5 reporter was used to visualize activation of the SRD-1 receptor by the sex pheromone in AWA neurons. To facilitate real-time imaging of the process, the heads of the transgenic worms were immobilized on soft-agar pads permeable to sex pheromone. B) Excitation of the GCaMP5 reporter in AWA neurons could be observed through both lateral and top views but the top view was clearer and more suitable for quantification. Arrowheads: AWA neurons. Scale bar = 50 μm. C) Relative fluorescence-intensity change was quantified through calcium imaging. Wild-type males showed receptor excitation by sex pheromones from C. elegans hermaphrodites or C. remanei females. This response was lost in srd-1-mutant-strain males but was restored following AWA-specific rescue of srd-1. Red triangles: time points of sex pheromone addition. Three males were assayed from each strain.
Figure 1(A) Summary of the metabolic cofactor supplementation and control study as well as the dosage of the metabolic cofactors before (left regimen) and after (right regimen) dosage adjustment based on this study. (B) Changes in plasma level of each cocktail substances in both supplementation and control studies (NR is detected in control study) compared to time baseline based on untargeted metabolomics measurement. The grey shaded area represents the 95% confidence level interval. For boxplots limits, the middle line represents the median. The upper and lower box limits represent the 25% quantiles. The upper and lower error bars correspond to 75% quantiles. The p-values are derived from one-way ANOVA (FDR< 0.05).
Figure 3Profiles of nucleosome midpoint positions by MNase-seq experiments. Samples were digested with the indicated MNase concentrations in both raffinose (raf) and galactose (gal) containing media. Midpoints of nucleosomes are smoothed by 31 bp. In galactose the stable nucleosomes move away from the Gal4UAS, creating space for an additional fragile nucleosome (indicated by arrow).
Fig.Figure 6(A) In vitro translation of a luciferase mRNA with or without of 0.5 μM (PR)20 and in the presence of increasing amounts of a 646 nt non-coding RNA (n=3). Data represent mean values ± SEM. (B) HTM-mediated quantification of OPP levels per cell (PR)20 (10 µM, 16h) alone or together with a 38 nt ssDNA oligonucleotide at the indicated doses. Black lines indicate median values. (C) Percentage of viable cells as evaluated with a CellTiter-Glo luminescent assay in U2OS cells treated with increasing doses of (PR)20 alone or together with 2 μM of ssDNA oligonucleotides (19 nt or 38 nt) (n=3). Data represent mean values ± SEM. (D) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 2 μM of a 19 nt ssDNA oligonucleotide for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test. (E) Immunofluorescence of HA-(PR)20 (green) and Cy5 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 4 μM of Cy5-labeled 19 nt ssDNA or ssRNA oligonucleotides for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test. (F) Clonogenic survival assay of U2OS cells exposed to 7.5 μM with the indicated peptides or protamine (PROT.) alone or together with 2 μM of a 38 nt ssDNA oligonucleotide or 0.5 μM Heparin. (G) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with heparin (0.5 μM). Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.
No obvious developmental defect in HoipK778R/K778R knockin mice is observed while TNF-responses are suppressed in HoipK778R/K778R cells.Figure 3Numbers of weaned mice of the indicated genotypes from Hoip+/K778R crosses. A gross appearance image of Hoip+/+ and HoipK778R/K778R male mice at 6-week old. Immunoblotting to detect TNF-induced degradation and phosphorylation of IκB-α, or phosphorylation of IKK in immortalized Hoip+/+ and HoipK778R/K778R MEFs treated with human TNF (20ng/ml) for the indicated times. Immunoblots of anti-Vinculin antibody and anti-α-Tubulin antibody shown for monitoring loading amount. Representative data from three independent experiments Induction of TNF-dependent NF-κB target genes, ICAM, VCAM and IκB-α in Hoip+/+ or HoipK778R/K778R immortalized MEFs determined by qRT-PCR. RNA extraction and cDNA synthesis from MEFs treated with hTNF (20ng/ml) for the indicated time subjected to examine transcripts of ICAM, VCAM and IκB-α. Normalization to β-actin. Representative data from three independent experiments, n=3. TNF-dependent induction of cleavage of PARP and Caspase 3 in primary Hoip+/+ or HoipK778R/K778R MEFs determined by immunoblotting. Total cell extracts of MEFs treated with hTNF (100ng/ml) and CHX (1µg/ml) for the indicated times subjected to SDS-PAGE followed by immunoblotting using antibodies as indicated. Anti-α-Tubulin antibody used to monitor loading amount. Representative data of two independent experiments. TNF-dependent induction of Caspase 3 activation in immortalized Hoip+/+ or HoipK778R/K778R MEFs measured by using DEVD-AFC. MEFs treated with hTNF (100ng/ml) and CHX (1µg/ml) for 4 hours subjected to the Caspase 3 activity assays. TNF-induced Caspase 8 activity in Hoip+/+ or HoipK778R/K778R immortalized MEFs treated with hTNF (100ng/ml) with or without Cycloheximide (CHX) (1µg/ml) or z-VAD (20µM). Representative data from three independent experiments, n=4. TNF-receptor complex II formation in Hoip+/+ or HoipK778R/K778R immortalized MEFs. Total cell extracts of MEFs treated with hTNF (100ng/ml), CHX (1µg/ml) and zVAD (25µM) for the indicated time immunoprecipitated using an anti-FADD antibody. Recruitment of RIPK1, HOIP and SHARPIN monitored by immunoblotting. The anti-Vinculin antibody blot shown for monitoring loading amount. The black arrows indicating HOIP and SHARPIN in the IP samples and unmodified RIPK in the input samples, respectively. Representative data of two independent experiments.
Figure 1A-F Immunostaining of primary HL with H2A.B (brown, indicated by black arrows) with a nuclear counterstain (blue). Human testis (positive control) (A), Human tonsils (negative control) (B), Nodular sclerosis classical cHL, double staining with CD15 (pink) to identify HRS cells and H2A.B (brown) (C), Mixed cellularity cHL (D), Lymphocyte rich cHL (E), Nodular sclerosis HL (F). Scale bar for panels A, B, D-F is 50 µm; scale bar for panel C is 15 µm. G Percentage of H2A.B-positive HRS cells in all 81 tumours. Lymphocyte Rich cHL (LRCHL), Mixed Cellularity cHL (MCCHL), Nodular sclerosis cHL (NSCHL), Nodular Lymphocyte Predominant HL (NLPHL), unclassified cases (U/C). Error bars are standard deviations, and the black line represents the average.
Sample Processing, the Loop-Mediated Isothermal (LAMP) Reaction and Synthetic RNA Validation.Figure 1.(A) Clinical and environmental samples collected with Nasopharyngeal (NP) and isohelix swabs respectively, were tested with RNA-sequencing, qRT-PCR, and LAMP. (B) The test samples were prepared using an optimized LAMP protocol from NEB, with a reaction time of 30 minutes. (C) Reaction progress was measured for the Twist COVID-19 synthetic RNA (MT007544.1) from 1 million molecules of virus (106), then titrated down by log10 dilutions. The colorimetric findings of the LAMP assay are based on a yellow to pink gradient with higher copies of SARS-CoV-2 RNA corresponding to a yellow color. The limit of detection (LoD) range is shown with a gradient after 30 minutes between 10 and 100 viral copies (lower right). (D) Replicates of the titrated viral copies using LAMP, as measured by Quantifluor fluorescence. (E) The sensitivity and specificity of the LAMP assay from 201 patients (132 negative and 69 positive for SARS-CoV-2, as measured by qRT-PCR). Thresholds are DNA quantified by the Quantifluor.
a) HCT116 cells were treated with serum starvation for 24 h (upper panels) or 0.figf4(a) HCT116 cells were treated with serum starvation for 24 h (upper panels) or 0.5 mM H2O2 for 12 h (lower panels); cytosolic proteins were extracted for co-immunoprecipitation with anti-FoxO1 and probed with anti-acetylated lysine (Ace-lyn). Both bottom panels display cytosolic FoxO1. IP, immunoprecipitation. (b) HCT116 cells were serum-starved for 24 h and the cytosolic proteins were extracted for co-immunoprecipitation with anti-FoxO1 or anti-SIRT2, followed by probing with anti-SIRT2 or anti-FoxO1, respectively (top and middle panels). FoxO1(WT) was also transfected into H1299 cells and the cytosolic protein was extracted, followed by co-immunoprecipitation with anti-FoxO1 and probing with anti-SIRT2 (bottom panels). (c) HCT116 cells were transfected with SIRT2 RNAi plasmid or an empty control plasmid. At 48 h after transfection, the cytosolic lysate was extracted for probing with SIRT2 or FoxO1 (upper panels), or for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-acetylated lysine (lower panels). IB, immunoblot. (d) HCT116 cells were treated with AGK2 for 24 h at different doses, as indicated. The cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-acetylated lysine.
Figure 76-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (A) Serum AST and ALT levels. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA 6-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (B) Hepatic TG contents. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA 6-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (C) Body weights and liver/body weight ratio. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA 6-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (D) H&E and Sirius red staining of liver sections (left), and quantitation of Sirius red staining (right). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA 6-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (E) Immunofluorescence staining of CD36 and β-catenin. (F) Quantitation of CD36 positive area. (G) Co-localization rate of CD36 and β-catenin. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA 6-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (H) Protein levels of palmitoylated CD36 in mouse livers (left), and its quantitative results (right). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA 6-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (I) CD36 protein expression in the plasma membrane fractions, and its normalization to ATP1a1. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA
Figure 3CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-Flex-EGFP-P2A-mNachBac or control GFP vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. Weekly body weight gain after viral delivery (A) at 11 weeks after viral delivery. CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-Flex-EGFP-P2A-mNachBac or control GFP vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. measurements of fat mass (B) and lean mass (C) at 11 weeks after viral delivery. Measurements of energy expenditure during the 7th week after viral delivery. Comparison of real time O2 consumption traces during the 4-day measurement period (D), of average O2 consumption between the 2 groups (E), and of the difference between day and night periods between the 2 groups (F). (G-I) Measurements of feeding during the 7th week after viral delivery. Comparison of real time feeding c traces during the 4-day measurement period (G), of average food intake between the 2 groups (H), and of the difference in food intake between day and night periods between the 2 groups (I).
Figure 6(A-B) Quantification of tRFTyrGUA induction in response to oxidative stress as a function of time in MCF10A (A) (n=4) and in HBEC30 (B) (n=6) with mean ± s.e.m shown for each cell line. (C) Volcano plot of mass spectrometry results from a synthetic 5'-biotinylated tRFTyrGUA co-precipitation experiment with cell lysate. Log2 fold enrichment values of proteins identified from tRFTyrGUA relative to scrambled tRF control samples. (D) Western blot validation of mass spectrometry results for three of the top hits in (A), showing co-precipitation of endogenous proteins with transfected tRFTyrGUA relative to a scrambled control sequence (Scr).
IRF2BP proteins are SUMOylated at their highly conserved C-terminus.Figure 2(A) Schematic representation of the domain structure of IRF2BP1 and IRF2BP2. The primary sequence suggests two putative SUMO-sites that are conserved among different species. (B) Identification of the endogenous SUMO-sites in IRF2BP1 (left panel) and IRF2BP2 (right panel). HeLa cells were transfected with HA-tagged wildtype (WT) or mutant (KR) proteins, endogenous SUMO-IPs were performed and the HA-signal was analyzed by immunoblotting. Mutation of the C-terminal SUMO-site abolished SUMOylation of IRF2BP1 and IRF2BP2 in HeLa cells. (C) Clustal omega analysis of IRF2BP1 and IRF2BP2 from H.sapiens, M.musculus, G.gallus, X.laevis, D.rerio, D.melanogaster and C.elegans shows high conservation of the C-terminal region including the SUMO-site. (D) Schematic representation of the creation of stable, untagged and siRNA resistant IRF2BP1 WT and K579R HeLa cells. Constructs expressing IRF2BP1 variants in an pIRES-hrGFP II ("pIRES") vector were transfected, selected with antibiotics and FACS sorted for low GFP expression. (E) Stable HeLa cells expressing pIRES-empty vector, IRF2BP1 WT or IRF2BP1 K579R were treated with siRNA against endogenous IRF2BP1 or non-targeting (nt) siRNA. Exogenous siRNA-resistant IRF2BP1 was expressed at low levels similar to endogenous IRF2BP1. * refers to an unspecific band. (F) Wt and mutant IRF2BP1 localizes in the nucleus. After knockdown of endogenous IRF2BP1, stable IRF2BP1 (WT or K579R) cell lines were immunostained for IRF2BP1. Exogenous IRF2BP1 variants show a similar nuclear localization. Scale bar = 10 µm. (G) IRF2BP1 wt and mutant associate with chromatin to a similar extent. HeLa cells were lysed in 0.075 % NP40 (Input). After centrifugation, the nuclei were incubated and vortexed with a nuclear extract (NE) buffer containing 170 mM NaCl. The eluates were collected and the procedure was repeated using a NE buffer with higher salt concentrations, first 290 mM, then 420 mM. Wildtype IRF2BP1, the SUMO-deficient K579R mutant and the SUMOylated wildtype form (*) all behave similarly.
Figure 5(A) Graphical representation of XPO1 protein. C528 to S amino acid mutation confers XPO1 resistance to both KPT-185 and KPT-330 chemical inhibitors [24]. S1055 amino acid correspond to the phosphorylation target of the STK38. Finally, the ΔCter region is also highlighted corresponding to a XPO1 construct lacking the 39 C-terminal residues (B-C) XPO1_S1055 phosphorylation is required and sufficient for STK38 nuclear exit upon nutrient starvation. (B) HeLa cells were transiently transfected with myc-STK38(wt) in addition to indicated Flag-XPO1 mutant plasmids. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT-185 (final concentration = 1 µM) in order to inhibit endogenous XPO1 activity. Cells were then fixed and stained for Flag and myc tags. Only cells positives for both Flag-XPO1 and myc-STK38(wt) were captured. Representative images are shown and scale bars are 40 µm. (C) Graphical representation of myc-STK38(wt) nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Mann-Whitney test). Expression of wt XPO1 failed to induce cytoplasmic localization of STK38 upon nutrient starvation in presence of KPT-185 where C528S mutant recapitulates results described in Figure 2B-C. Expression of phosphonegative XPO1 (S1055A) failed to induces STK38 cytoplasmic localization upon EBSS treatment while phosphomimetics XPO1 (S1055D and S1055E) were sufficient to promote STK38 cytoplasmic localization without autophagic stimuli. Finally, expression of XPO1 lacking in its 39 C-terminal residues mimicked phosphomimetics variants. (D) HAP1 cells carrying genomic XPO1 mutations of S1055 were subjected for IMDM (nutrient rich medium) or EBSS (starvation) incubation for 4 hours followed by western-blot analysis for p62 level measurement. This result indicates that p62 degradation in starvation conditions was inhibited in XPO1_S1055A HAP1 cells whereas p62 degradation was potentiated in both phosphomimetics variant (S1055D & S1055E) (n = 4 independent experiments, mean ± SEM; *p<0.05, ns, not significant, Mann-Whitney test (E) Hek-HT-iRFP-LC3 cells were transiently transfected with siRNA targeting endogenous STK38 (or with non-targeting siRNA (siNT)). Two days after, cells were transiently transfected with indicated Flag-XPO1 mutant plasmids. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT-185 (final concentration = 1 µM) in order to inhibit endogenous XPO1 activity and with chloroquine (final concentration = 10 µM). Cells were then fixed and the number of iRFP-LC3 dots per cell was recorded only in cells positive for Flag-XPO1 mutants As expected, silencing of endogenous STK38 prevented the formation of LC3 dots upon starvation where introduction of phosphonegative XPO1 (S1055A) failed also to increase LC3 dots upon starvation. Interestingly, phosphomimetic XPO1 (S1055D) was sufficient to increase the number of LC3 dots even in complete and also STK38-depleted conditions.
Figure 6A) Schematic of the Fft2 protein showing the location of the catalytic function-abrogating point mutation K581R. Error bars represent standard deviation of reverse transcriptions of biological duplicates. B) Bar graph representing RNA levels as in Fig. 1D, relative to a control locus (act1) and to WT levels, as measured by qPCR. Error bars represent standard deviation of nucleosome occupancy of biological duplicates. C) TSS shift in Fft deletion and catalytic mutants is accompanied by reduced LTR nucleosome occupancy, measured by MNase-qPCR as in Fig. 5B.