EMBO/sd-character-level-ner huggingface dataset:
SH-BC-893 acutely restores a tubular mitochondrial network in the liver and brain of HFD-fed mice.Figure 3Body weights of mice that had consumed a SD for 22 weeks or a HFD for 26 weeks; n=4-5. Mean ± SD. Mice are grouped to reflect their randomization to vehicle or SH-BC-893 groups, weight measured prior to treatment. NAD(P) H autofluorescence evaluated by confocal microscopy in freshly resected livers from mice in (A) 4-8 h after treatment with vehicle (n=5) or 120 mg/kg SH-BC-893 (n=4) by gavage at ZT8.5. Mice were sacrificed and imaged in pairs (SD and HFD). Aspect ratio (C) and roundness (D) of mitochondria in the livers shown in (B) calculated per field as described in Fig. EV3; 40 fields examined from each treatment group. Body weights of mice that had consumed a chow or HFD diet for 22 weeks; n=3. Mean ± SD. Mice are grouped to reflect their randomization to vehicle or SH-BC-893 groups, weight measured prior to treatment. Coronal brain section showing the areas where mitochondria were imaged in G-L. Citrate synthase staining in the arcuate nucleus of mice in (E) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=3. Aspect ratio (H) or roundness (I) of the mitochondria in (G); 18 fields examined from each treatment group. imaging was performed in the areas of the cortex As in G-I but imaging was performed in the areas of the cortex outlined in (F). 18 fields examined from each treatment group.
Figure 5A. Fluorescent microscopy images showing that CRY2, CIB1 and CO co-localized to the nucleus. Scale bars: 2 μ B. Co-IP assays showing that CRY2, CIB1 and CO form a protein complex. Samples were prepared from 10-day-old WT or 35S:Myc-CIB1/35S:CO-Flag or 35S:Myc-CIB1/35S:CO-Flag /cry2-1 seedlings grown in LDs (16L/8D), and samples were collected at ZT16. C. Co-IP assays of samples prepared from 10-day-old 35S:Myc-CIB1/35S:CO-Flag 35S:Myc-CIB1/35S:CO-Flag /cry2-1 seedlings grown in LDs (16L/8D). Samples were collected at the times indicated over the course of one day. D. Diagram depicting the putative promoter and genomic region of FT. The diamond symbol indicates the position of CORE. The white circles indicate positions of E boxes (CANNTG) located at proximal promoter of FT. E. Representative results of the ChIP-qPCR assays. Chromatin fragments (~500 bp) were prepared from 10-day-old 35S:Myc-CIB1/35S:CO-Flag seedlings, immunoprecipitated by the anti-CRY2, anti-Flag (CO-Flag) , anti-Myc (Myc-CIB1) antibody or anti-preimmune IgG, and the precipitated DNA was analyzed by qPCR using the primer pairs indicated. The IP/input ratio of anti-preimmune is set to 1.0. Data information: the relative IP/input ratios are shown with their standard deviation (n = 3). Two biological replicates were performed. F. Box plots (the minimum, first quartile, median, third quartile, and maximum are shown, with data >1.5 interquartile ranges denoted with circles) for relative reporter activity (LUC/REN) in plants expressing different reportors and effectors. The LUC/REN in plants expressing ProFT alone is set to 1.0. Immunoblots were done to show GFP-CRY2W374A, Myc-CIB1 and Myc-CO protein levels in the transient Dual-LUC assay. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, and with the anti-GFP antibody. Data information: the relative LUC activities normalized to the REN activity are shown (LUC/REN) with their standard deviation (n =6). The letters "a" to "f" indicate statistically significant differences determined by Tukey's HSD test (P≤0.05). Two biological replicates were performed. G. Model representing the action of CIB1 and CO on FT expression under long day condition.
Figure 5Cell fractionation upon inhibition of DPP9 activity. HeLa cells were incubated for 4 days with the DPP9 inhibitor 1G244 or as control with DMSO. Cells were subjected to fractionation into 'cytosol' (supernatant) and 'mitochondria' (pellet) by differential digitonin lysis and centrifugation. The IMS marker proteins CPOX and cytochrome c were exclusively localized to the pellet fraction at one specific digitonin concentration (lanes 2,5,8,11) irrespective of 1G244 treatment. Likewise, the cytosolic marker protein LDH localized under all conditions to the same fractions. Conversely, endogenous AK2 appeared at this digitonin concentration in the 'cytosol' fraction but only upon 1G244 treatment (compare lanes 2 and 5). Thus, inhibition of DPP9 results in appearance of AK2 in the cytosol. Quantification shows loading-control adjusted AK2-signals of 1G244-treated 'cytosol' fraction (lane 5) over DMSO-treated 'cytosol' fraction (lane 2). White arrow head indicates the endogenous AK2 appearing in the cytosolic fraction upon 1G244 treatment. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. Localization of DPP9-insensitive AK2-HA variants. Experiment was performed as described in Figure 2C. Compared to the wild type, DPP9-insensitive and import-competent AK2-HA variants co-localized with cytosolic markers indicating partial cytosolic localization. The colocalization of AK2-HA variants with Mitotracker was quantified using Fiji (Pearson's correlation coefficient). A one-way ANOVA with post-hoc Tukey test was performed. Numbers of analysed cells are indicated in the plot. ** represents p<0.01. Data shown in Figures 2C, 5B, S2 and S5 are derived from the same experiments. The black horizontal line represents the median. Data information: Scale bars: 10 μm. Localization of AK2C40S-HA upon proteasomal inhibition. Experiment was performed as described in Figure 2C. Upon MG132 treatment, colocalization of AK2C40S-HA with the cytosolic marker increased. A one-way ANOVA with post-hoc Tukey test was performed. Numbers of analysed cells are indicated in the plot. *** represents p<0.001.The black horizontal line represents the median. Data information: Scale bars: 10 μm.
Anaerobic growth of the five HC ATPase strain variants.Figure 7A) Time course of biomass concentration. B) Time course of glucose concentration. C Data information: The means for n = 3 biologically independent samples are shown. The error bars represent ± SD.  C) Specific glucose (Glc) uptake rate and specific productivity for ethanol (Eth), acetate (Ace), formate (For), lactate (Lac), and succinate (Suc). D) Yield of Eth, Ace, For, Lac, and Suc. Data information: The reaction rates in C were calculated for the exponential phase under assumption of quasi-steady state. The means ( and the means and individual data for n = 3 biologically independent samples are shown. The error bars represent ± SD.
Colocalization of HCV Proteins with organelle-specific markers in HCV infected cells.Figure 1Huh7 cells were infected with HCV (strain Jc1) using 30 TCID50/cell and 48 h later cells were fixed and processed for fluorescence microscopy. In case of samples shown in panels B-D, cells were first transfected with expression constructs specified in the left of each panel and 24 h later cells were infected as described above. Samples were analyzed with a Nikon TE2000-E inverted confocal microscope at 60× magnification. (A)-(D) Colocalization of HCV proteins specified in the top of each panel with protein disulphide isomerase (PDI; an ER marker), GFP-Rab21 (marker for early endosomes), GFP-Rab7 (marker for late endosomes) or βCOP-YFP (marker for COP I vesicles). The upper panels represent a low magnification overview; boxed areas are shown as enlargement in the corresponding panel below. The nucleus was stained with DAPI (blue). Scale bars represent 10 µm (top panels) and 2 µm (lower panels). The quantification of the degree of colocalization (Pearson's correlation coefficient) is given at the top of the enlarged pictures.
Figure 6A. Myc-TRAPPC9 directly interacted with FLAG-γ-COP. The presence of FLAG-γ-COP in immunoprecipitations of Myc-TRAPPC9 and Myc-TRAPPC10 from cell lysates of the indicated genetic backgrounds was investigated by immunoblotting. B,C. Co-immunoprecipitation between FLAG-γ-COP and HA-Rab18 in HEK293T cells with indicated genetic backgrounds. The presence of FLAG-γ-COP in immunoprecipitates of HA-Rab18 (and reciprocal IP in C) was investigated by immunoblotting. D. The interaction between endogenous COPI and Rab18 was TRAPPII dependent. Endogenous Rab18 was immunoprecipitated by anti-Rab18 antibody from lysates of indicated genetic background. The presence of γ-COP and various TRAPP subunits were detected by their specific antibodies as indicated. E. Co-immunoprecipitation between TRAPPII and HA-Rab18 in control (siFFL) and γ-COP (si-γ-COP) siRNA depleted cells. TRAPPII was precipitated with transfected Myc-TRAPPC10 and the presence of HA-Rab18 was determined by immunoblotting. F. Co-immunoprecipitation between FLAG-γ-COP and TRAPPII in control (siFFL) and Rab18 (si-Rab18) siRNA depleted cells. TRAPPII was precipitated with Myc-TRAPPC10 and the presence of FLAG-γ-COP was determined by immunoblotting. The molecular weight of endogenous Rab18 overlaps with IgG light chain, and therefore, the amount of endogenous Rab18 precipitated with TRAPPII cannot be determined in this experiment.
SNX18 interacts with LC3 family members.Figure 6.(A) GFP, GFP-LC3, or GFP-LC3 G120A were immunoprecipitated from transfected HeLa cells. The immunoprecipitates and 2% of the lysate were analyzed by immunoblotting against SNX18. The GFP proteins were detected by Ponceau staining. (B) Lysates from fed or starved MDBK cells were incubated with the indicated glutathione-Sepharose-bound GST-tagged proteins. SNX18 was detected by immunoblotting. Ponceau staining shows the GST proteins. (C) In vitro translated GFP-p62 or GFP-SNX18 were incubated with the indicated GST-tagged Atg8 family proteins. Bound proteins were detected by autoradiography and GST proteins by Coomassie blue staining.
The DNA damage response is largely driven by extrinsic signals.Figure 3Spearman's correlation between green (A-D) or yellow (E) fluorescence range and red fluorescence range over the lifetime of the cell (105 < n < 153). The data shown are from 2 independent experiments. On the x-axes P2P1recA-GFPdes (A-D) or a C-term translational reporter of SSBa fused to mCitrinewt (E). On the y-axes, a C-term translational reporter of RecA fused to mCherrywt (A and E), transcriptional reporters of ruvC (B) and radA (C), and mCherrydes expressed from a constitutive control promoter (D). Colors (A-D) indicate subpopulations segregated by VMR of P2P1recA-GFPdes: gray (0<VMR≤1), dark green (1<VMR≤10), and light green (VMR>10). Colors (E) indicate subpopulations segregated by VMR of RecA-mCherrywt: gray (0<VMR≤1), plum (1<VMR≤10), and magenta (VMR>10). Pearson's correlation between green (F-I) or yellow (J) fluorescence and red fluorescence, measured 3 hours after MMC exposure (203 < n < 314). The data shown are from 2 independent experiments. On the x-axes P2P1recA-GFPdes (F-I) or a C-term translational reporter of SSBa fused to mCitrinewt (J). On the y-axes, a C-term translational reporter of RecA fused to mCherrywt (F and J), transcriptional reporters of ruvC (G) and radA (H), and mCherrydes expressed from a constitutive control promoter (I). Positive correlations between recA and other DNA-damage markers both under normal growth conditions, where spontaneous DNA-damage events occur at the subpopulation level, and in the presence of an alkylating agent that induces double-strand breaks in the entire population, validate recA as a reliable proxy for DNA damage in single cells.
Figure 3A) Fragment size distribution, determined by sWGS, in CSF (blue line), plasma (red dashed line) and urine (black dashed line) from patient G1. The 3 samples were collected simultaneously before initiation of treatment. B) Fragment size distribution of cfDNA, determined by sWGS, in CSF from 13 glioma patients. The samples contained a high fraction of reads from DNA fragments with lengths corresponding to ~145 bp and ~167 bp. The fragment size profile of cfDNA from the plasma sample from patient G1 is shown in red. All samples were collected simultaneously before initiation of treatment C) Cumulative frequency analysis of the average density of fragment size in CSF (blue) and plasma (red). The vertical dashed line represents 167 bp Mean fragment size density in patients with detected SCNAs in the CSF (yellow), and those with no detected SCNAs (dark blue) Log2 ratio of the difference in cfDNA fragment sizes between CSF samples with detected SCNAs and those samples in which no SCNAs were detected The amplitude of the 10 bp periodic peaks in fragment size is a signature for non-tumor DNA in the CSF. The amplitude of the 10 bp periodic peaks in fragment length distribution (in the range between 75 and 150 bp, see Methods) when compared to the mean copy number alteration calculated from sWGS revealed a significant negative correlation (Pearson -0.85; p=0.0002)
Figure 2A. Overview of the synaptic organization of the retina in RBEWT/WT and RBEKO/KOmice. Cryostat sections from littermate mice were labeled by double immunofluorescence for the RIBEYE B-domain (CtBP2; green) and SV2. Scale bar: 20 µm; abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. B. Double immunofluorescence staining of retinas from RBEWT/WT and RBEKO/KO mice for PSD95 (green, to label postsynaptic specializations) and mGluR6 (red, to label specifically postsynaptic sites formed by bipolar neurons in photoreceptorsynapses). Abbreviations are as in A. Scale bars: 20 µm (left and right overview), 5 µm (left panel magnification), 2 µm (right panel magnification).C. Same as B, except that retinas were stained for PKCα (green, to label bipolar neurons) and PSD95 (red, to label amacrine cells).
Figure 4A. Theoretical distribution of lengths of mature cells when NEZ correlates with 1/ln(rEZ) (as in panel C) and when it does not (as in panel D). In the correlated case, the values extracted for real wild type plants for l0EZ , rEZ and NEZ were used (n=122, all data in panel C). For the non-correlated case, the real values of rEZ and NEZ were randomly coupled in pairs and the l0EZ value for the associated rEZ was used. B. Theoretical distribution of root growth computed when NEZ correlates with 1/ln(rEZ) (as in panel C) and when it does not (as in panel D). In the correlated case, the values extracted for real wild type plants for Rprod, l0EZ , rEZ and NEZ were used (n=24, white symbols in panel C). For the non-correlated case, the real values of rEZ and NEZ were randomly coupled in pairs and the Rprod ,l0EZ values for the associated rEZ were used. C-D: NEZ versus 1/lnrEZ in the presence or absence of the meristem-elongation correlation, when Rprod is changed two-fold. E-H: Theoretical distribution of mature cells length (E,F) and of root growth (G,H) in the presence (E,G), or absence (F,H) of the correlation, for different values of the meristematic activity (Rprod) (n=35). In all cases data was computed using the real experimental values for 1/ln(rEZ), 1/(2ln(rEZ)) and 2/(ln(rEZ)) and hypothetical values for NEZ chosen to either preserve or not the correlation. p<0.005 ***, p<0.01 **, p<0.05 * Boxplot representation as in Fig. 2.
Figure 7A. LPS (5mg/Kg for 2 hrs) induced higher expression levels of cytokine genes in wa3 hearts than in littermate WT hearts. The results are presented as fold induction of untreated controls. The mRNA expression levels of Pln and Ryr2 was reduced in both wa3 and WT hearts, while of Mybpc3 and Sgcg was unchanged. For the full list see Dataset EV3 and for functional clustering in Appendix Figure S7. Differences between knockdown and control with p values =0.04998 (Student's t test) are indicated with an asterisk. B shows that LPS induced a higher mortality rate of wa3 mice than of littermate controls following two weekly subsequent injections of LPS (1mg/Kg).
Figure 1p53 ChIPseq in MEFs of the indicated genotype treated with or without 10 µM nutlin-3a (Nutlin) for 16 hours. Shown are 2 kb regions surrounding the summit of the 468 p53 binding peaks called in Nutlin-treated p53+/+, but not p53-/- or p53EE/EE MEFs. For p53EE/EE MEFs three independent replicates are shown. De novo motif search using MEME-Chip was performed on the 468 p53 binding peaks (as in A). The best hit motif is reported with corresponding E-value and logo (upper part). Graphs depict a CentriMo enrichment analysis for the best MEME motif (middle) and for known transcription-factor binding sites (bottom). RNAseq was performed with MEFs of the indicated genotype untreated or treated with 10 µM Nutlin for 16 hours. Shown are all Nutlin-regulated genes from the MSigDB gene set Hallmarks_P53_Pathway with a mean log2FC≥1 in p53+/+ cells. Shown are the z-transformed RNA expression values (FPKM). RNAseq data were subjected to gene set enrichment analysis (GSEA). Shown are enrichment plots for the indicated set of p53 downstream genes in pairwise comparisons of Nutlin-treated MEFs with the indicated p53 genotypes. Summary of GSEA results for p53-related gene sets. NES, normalized enrichment score; nom P, nominal P value. Reverse transcription quantitative PCR (RTqPCR) analysis of p53 target genes in MEFs treated for 24 hours with 1 µg/ml doxorubicin (Doxo). Shown are expression values normalized to β-actin as mean ± SD (n=6). Western blots of protein lysates prepared from MEFs treated for 24 hours with (G) 10 µM Nutlin Western blots of protein lysates prepared from MEFs treated for 24 hours with 0.4 µg/ml Doxo.
Impact of Ub phosphorylation on E1 and E2 enzymesPhosphoUb is abbreviated to pUb in this Figure.Figure 5A E1‐mediated charging of E2 enzymes by Ub and phosphoUb in a time course analysis. Non‐reducing Coomassie‐stained 4-12% SDS-PAGE gradient gels are shown, and bands are labeled. "˜Ub" refers to generation of a thioester, while "Ub‐"refers to additional covalent ubiquitination of the E2 enzyme. * contaminant from E2 purification. B SAXS‐derived ensemble of UBE2D3 (Pruneda et al, ), where the E2 is shown under a blue surface and the Ub is shown as a green ribbon. Ser65 is shown in sphere representation. Ser65 does not contact the E2 enzyme in any orientation of the Ub at the E2 enzyme. PDB files were downloaded from the Klevit‐lab website (http://depts.washington.edu/klvtlab/). C Quantitative mass spectrometry analysis for UBE2T, UBE2E1 and UBE2R1 with Ub and phosphoUb using UbAQUA peptides reveals impaired chain formation with phosphoUb (see also Supplementary Fig S10). D Charging of UBE2N, and UBE2N/UBE2V1‐mediated Lys63 chain assembly with Ub and phosphoUb. Charging of UBE2N as in (A) proceeds identically with both Ub and phosphoUb (left 6 lanes), while UBE2V1‐mediated Lys63 chain assembly only proceeds with Ub but not phosphoUb (right 6 lanes and Western blot). E Structure of Saccharomyces cerevisiae Ubc13‐Mms2 (blue/red, Mms2 is a homolog of UBE2V1) with the acceptor Ub (cyan) bound non‐covalently to Mms2 [pdb‐id 2gmi (Eddins et al, )]. Ser65 of the acceptor Ub interacts with Mms2, and Ser65 phosphorylation would prevent binding due to Mms2 Glu18. F PhosphoUb competition assays for UBE2N/UBE2V1‐mediated Lys63 diUb formation. Time course with K63R Ub (donor) and Ub ΔGG (acceptor) generates Lys63 diUb and is not inhibited upon increasing concentrations of phosphoUb, revealing phosphoUb does not compete with Ub for UBE2N/UBE2V1‐mediated Lys63 diUb formation.
Figure 7(A) Cell cycle distribution of immortalized Cdca5 Δ/Δ sor-LAP-AID fibroblasts after 24-hour incubation with aphidicolin and release. (B) Western blot showing cell extracts after incubation with aphidicolin and release. (C) Still images of a fluorescence recovery after photobleaching experiment. Size bar, 5 μm. Bleach area radius, 2 µm.(D) Graph depicting the normalized intensity after photobleaching to quantify turnover of sororin (n = 15).(E) Quantification of the relative abundance of sororin-LAP-AID on chromatin. Error bars denote s.e.m.(F) Quantification of the chromatin residence time of sor-LAP-AID at different times. Error bars denote s.e.m.
Presence of the STAT1Y701F mutant reduces IFNβ-stimulated binding of STAT2 to nuclear ISRE sequences.Figure 3A IFNβ-stimulated binding of STAT2 to ISRE sequences of Mx2 and IRF7 promoters. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with STAT2-specific antibody. The amount of precipitated DNA was measured by Q-PCR. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (**, p≤ 0.01); the p-values were calculated using paired ratio t-test. B Impact of STAT2 deficiency on IFNβ -stimulated STAT1 association with the Mx2 ISRE. BMDMs of wild type (WT), Stat1-/- (S1) and Stat2-/- (S2) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with STAT1-specific antibody. The amount of precipitated DNA was measured by Q-PCR. Bars represent mean values of three independent experiments; error bars represent standard error of mean (SEM). C Simultaneous association of STAT1 and STAT2 with the Mx2 ISRE analyzed by ChIP-reChIP. BMDMs of wild type (WT) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with either STAT1-specific antibody and re-immunoprecipitated with STAT2-specific antibody or vice versa. The amount of precipitated DNA was measured by Q-PCR. Bars represent mean values of three independent experiments; error bars represent standard deviation (SD). D Impact of STAT1Y701F on delayed, STAT2-mediated expression of IFN-induced genes. Stat1-/- fibroblasts were transfected with plasmids driving expression of the indicated proteins. 24 h after transfection, 250 IU/ml of IFNβ was added to the transfected cells and ISG expression was determined by Q-PCR after 48h of cytokine treatment. Gene expression was measured by Q-PCR and normalized to Gapdh. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM) and asterisks denote the level of statistical significance (*, p≤ 0.05); the p-values were calculated using paired ratio t-test.
Figure 5(A) Stereo view of the superposition of the ADP-MjRad50 (3AUX) complex (black) onto the ATPγS-MjMR-DNA complex (green and blue) by aligning their lobe II domains. The α1-α2 loop that could collide with parts of the DNA molecule is indicated. (B) Aligning the lobe II domains of two structures in Fig 5A shows that rotation of the lobe I opens the gate for Mre11, which could allow the access of the unwound DNA. Color schemes are same as those in Fig 5A. (C) DNA-unwinding activities of TmMR. Lane 2, no protein; lanes 3 and 4, no ATP; lanes 5 and 6, with ATPγS; lanes 7 and 8, with ATP. The DNA: protein ratio was 1:2 for lanes 3, 5, and 7, and 1:5 for lanes 4, 6, and 8. Plotted data represent the mean of three experiments, with error bars indicating one standard deviation. A graph of the quantified data is shown in the lower panel. (D) Comparison of the ATP-dependent DNA unwinding activities of WT and mutant TmMR proteins. Lane 2, no protein; lane 3 and 4, WT TmMR; lane 5 and 6, Rad50 (Δ54-56) mutant; lane 7 and 8, R87E mutant; lane 9 and 10, K95E mutant; lane 11 and 12, R94E/K95E mutant; lane 13 and 14, K115E mutant. For each TmMR, reaction contains 1:2 or 1:5 ratio of DNA: protein.