EMBO/sd-character-level-ner huggingface dataset:
Figure 2A. Growth curves of the library (black) compared to two different controls under increasing selective pressures. DSB (double-stranded break) Negative Control is a cassette designed to introduce a stop codon at the unrelated gene galK. n=3 for each curve B. Plasmid-barcode based mapping of enriched variants across all targeted genes under AEC selection. The innermost circle represents the ORFs in the E. coli genome, with the green bars highlighting regions that were zoomed 50X. Positive log2 enrichment scores of variants under increasing selective pressures are plotted as orange bars facing outward. Not enriched variants are plotted in gray bars facing inward. Two distinct biological replicates are combined in this plot, using a weighted enrichment score (described in the methods section) C. Mapping the number of enriched mutations in genes that were classified under the different categories. The classification of each gene is the same as shown in Fig. 1A. D. Log2 enrichment scores for each gene under each category
Mitochondrial localization of Parkin is etiologically important.Figure 1.(A) HeLa cells expressing HA-Parkin were treated with CCCP or DMSO (control) and then immunostained with the indicated antibodies. (B) HeLa cells stably expressing HA-Parkin or intact SH-SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments. I, C, and M indicate input, cytosol-rich supernatant, and mitochondria-rich membrane pellet, respectively. (C) Schematic diagram of disease-relevant mutants of Parkin used in this study. IBR, in between RING; Ubl, ubiquitin like. (D) Polarized mitochondria stained with MitoTracker red (red arrowheads) were not labeled by Parkin. In contrast, damaged mitochondria marked by Parkin (green arrowheads) were not stained with MitoTracker red. (E) HeLa cells expressing HA-Parkin with various pathogenic mutations were treated with CCCP, followed by immunocytochemistry. (A, D, and E) Higher magnification views of the boxed areas are shown in the insets. (F) Parkin colocalization with mitochondria was analyzed in >100 cells per mutation. Example figures indicative of robust colocalization (counted as 1), weak colocalization (counted as 0.5), and no colocalization (counted as 0) are shown. Error bars represent the mean ± SD values of at least three experiments.
Precursor API is trapped within a vesicle in cvt17 mutants.Figure 4Precursor API is trapped within a vesicle in cvt17 mutants. (A) Precursor API is in a protease-protected compartment in cvt17 mutants. THY32 (cvt17) spheroplasts were subjected to differential lysis and protease treatment in PS200 buffer as in Fig 3 B) Precursor API is not free in the vacuolar lumen in cvt17 mutants. Differential fractionation was continued by resuspending the P200 fraction in PS0 buffer and separating the supernatant (S0) and pellet (P0) fractions by centrifugation at 10,000 g. The S0 and P0 fractions were treated with proteinase K in the presence and absence of Triton X-100 as indicated. All fractions were precipitated with 10% TCA and resolved by SDS PAGE; proteins of interest were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated.
XBP‐1 loss‐of‐function enhances autophagy during ER stress in Mfn2‐ablated cells.Figure 6(A) Immunodetection of XBP‐1s in WT and Mfn2 KO cells stably expressing a scrambled shRNA (Scr) or a shRNA directed to XBP‐1 (XBP‐1 KD), and treated with Tg for 6 h. (B) Caspase activity was detected by measurement of DEVD‐AFC substrate processing. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. #P0.05 versus Scr group. (C, D) Total and cleaved caspase 3 levels were detected by western blot. WT and Mfn2 KO cells subjected or not to XBP‐1 silencing (Scr, and KD) and treated with 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. (E, F) Immunodetection of LC3b‐I and ‐II in Scr and XBP‐1 KD WT or Mfn2 KO cells treated as indicated (1 μM Tg; 100 nM Baf) for 6 h. Densitometric quantification is shown in F. Data are mean±s.e.m. (n=3). #P0.05 versus Scr group. (G) Mfn2 KO cells stably expressing mCherry‐GFP‐LC3b and subjected or not to XBP‐1 silencing (Scr or KD) were treated with 1 μM Tg, 100 ng/ml Bref, or 0.5 μg/ml Tm for 24 h and examined by confocal microscopy. Scale bar: 10 μm. (H) Quantification of mCherry+/GFP‐ (mCherry‐LC3) red puncta per cell (a measure of acidic autophagosomes) is shown. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). #P0.05 versus Scr group.Source data for this figure is available on the online supplementary information page.
Figure 5A qRT-PCR analysis of G-rich and polyA+ tiTERRA in cells carrying one tiTEL and expressing Trt1-myc (CAF610) cultured in EMM medium with the indicated combinations of THI and TSA for 24 h. Values are normalized to ACT1 mRNA and expressed as fold increase over THI+ TSA- samples. Bars and error bars are averages and SD from 4 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, (relative to THI+ TSA-; two-tailed Student's t test). B Telo-PCR analysis of tiTELs using genomic DNA extracted from cells as in (A). Marker (m) molecular weights are on the right in base pairs. C RIP experiments performed using anti-myc antibodies and extracts from cells as in (A) followed by qRT-PCR analysis of G-rich and polyA+ tiTERRA. Values correspond to fraction of input RNA expressed as fold increase over an untagged (unt) control tiTEL strain (CAF545). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01 (relative to unt; two-tailed Student's t test). D ChIP experiments performed using anti-myc antibodies and extracts from cells as in (A) followed by qPCR using tiTEL specific oligonucleotides. Values correspond to fraction of input DNA expressed as fold increase over THI+ TSA-. Unt: untagged strain (CAF545). Bars and error bars are averages and SD from at least three independent experiments. * P < 0.05, ** P < 0.01 (relative to THI+ TSA-; two-tailed Student's t test).
Figure 7A Biosynthesis pathway of Moco with all stable intermediates. Gephyrin G- and E-domain catalyze the last two steps as depicted.B In vitro Moco-synthesis assay using 150 pmol purified 6His-tagged gephyrins. D580A is an activity-deficient gephyrin mutation previously identified in Moco-deficient patients (Reiss et al., 2011). Assays without the addition of gephyrin (-gephyrin) or molybdenum (-Mo) served as internal controls of the assay (see also (Belaidi Schwarz, 2013)).C Experiment as in B but with pre-incubation of 6His-gephyrin/G375D in a 1:1 ratio to simulate the heterozygous mutation. n = at least 3 with two independently purified protein batches. Results are expressed as mean ± SEM.
Fig.Figure 5A Representative CT images (upper panel) and H&E staining (lower panel) of lungs from EML4-ALK tumor-bearing mice are shown. The complete set of CT images refers to Appendix Figure S10A. Mice were treated with 30 mg/kg XMU-MP-5 twice daily for 3 weeks. H: heart; arrow indicated tumor. Scale bars, 200 μm. B IHC analyses of lungs from EML4-ALK transgenic mice. pALK and Cl-caspase 3 were probed. Scale bars, 100 μm. C Representative CT images (upper panel) and H&E staining (lower panel) of lungs from EML4-ALK(L1196M) tumor-bearing mice are shown. The complete set of CT images refers to Appendix Figure S10B. Mice were treated with 40 mg/kg XMU-MP-5 twice daily for 1 week. H: heart; arrow indicated tumor. Scale bars, 200 μm. D IHC analyses of lungs from EML4-ALK(L1196M) transgenic mice. pALK and Cl-caspase 3 were probed. Scale bars, 100 μm.
TIP60 S90 phosphorylation affects its interaction with H3 (A) FLAG- immunoprecipitations were performed from nuclear extracts of U2OS cells stably expressing FLAG-tagged TIP60WT, TIP60S90A, TIP60S86A or empty vector (vec).Figure 3 (A) FLAG- immunoprecipitations were performed from nuclear extracts of U2OS cells stably expressing FLAG-tagged TIP60WT, TIP60S90A, TIP60S86A or empty vector (vec). Immunoprecipitated material and input lysate were subjected to Western blot analysis (B) The experiment as shown in (A) was repeated three times. The graphs and error bars represent mean and standard deviation of these three individual experiments (one-way ANOVA, Fisher"s LSD test, WT vs. S86A p=0,5992, WT vs. S90A p=0,0429, S90A vs. S86A p=0,0176 (*p < 0,05; n.s. not significant, p > 0,05))  (C) HEK293 cells were transfected with pcDNA3.1 encoding FLAG-TIP60WT, FLAG-TIP60S90A or empty vector. FLAG-immunoprecipitations were performed from nuclear extracts. Immunoprecipitated material and input lysate were subjected to Western blot analysis  (D) U2OS cells stably expressing FLAG-TIP60WT or empty vector were treated with 2µM SNS-032 (SNS) for 2 hours as indicated. FLAG-immunoprecipitations were performed from nuclear extracts. This material and input lysate were subjected to Western blot analysis  (E) U2OS cells stably expressing FLAG-TIP60WT, FLAG-TIP60S90A or empty vector (vec) were subjected to ChIP using anti-FLAG antibody. Immunoprecipitated DNA and input were subjected to quantitative real-time PCR using primers annealing in the myc locus about 50bp upstream (myc+50) or about 500 bp downstream (myc-500) of the transcription start site. Promoter occupancy of FLAG-TIP60WT or FLAG-TIP60S90A was determined as percent input in relation to FLAG -TIP60WT. The graphs and error bars represent mean and standard deviation of three individual experiments (one-way ANOVA, Bonferroni"s multiple comparison test, myc+50 WT vs. S90A p=0,0289, myc-500 WT vs. S90A p=0,0155 (*p < 0,05))
Figure 10A-F. Schematic representation of capped (m7G-capped) and A-capped (non-functional capped) monocistronic mRNAs with poly(A)-tail-encoded firefly luciferase (Fluc) and fused upstream with (A) c-myc IRES, (B) no insertion, (C) non-specific 105-nt luc RNA (Non105), (D) huORFchop-1-105-nt, (E) huORFchop-69-105-nt, and (F) huORFchop-81-105-nt as shown above each bar graph. The RNA transfection assay of each transcript equipped with m7G-capped or A-capped monocistronic mRNA in HEK 293T cells was compared. To normalize the luc activities of capped and A-capped Fluc transcripts, the m7G-capped Renilla luc (Rluc) mRNA was used as an internal control. The luc activity of cells transfected with Cap-monocistronic mRNA in each graph was set as 100%. G. The luc activity driven by the indicated A-capped monocistronic mRNA in the presence of Endouc or EndoucK242A in HEK 293T cells. The m7G-capped Rluc mRNA was used as an internal control to normalize the activities of A-cap Fluc transcripts. The egfp mRNA was used as a negative control, and the luc activity of cells transfected with egfp mRNA and A-cap Fluc mRNA containing full-length huORFchop-1-105-nt was set to 1. The data were averaged from three independent experiments and presented as mean ± S.D. (n = 3). Student's t test was used to determine significant differences between each group (**: P<0.005, ***: P <0.001).
a–c) P-mTOR levels in wild-type retinae.figf5(a-c) P-mTOR levels in wild-type retinae. Dorsal (top) enrichment of P-mTOR (a). Higher magnifications of the dorsal and ventral regions are shown to the right, with P-mTOR in red and cone segments in green, as detected by PNA. Dorsal retinal sections stained for P-mTOR (red) and PNA (b) or antibody to β-galactosidase (green, c). Higher magnification (insets) images suggest that the P-mTOR signal is located in the lower part of the outer segment (OS). IS, inner segment. (d-g) Rapamycin treatment of wild-type mice led to downregulation of red/green opsin ventrally (e) but not dorsally (red, d). Ventral blue opsin (red, f) remained unaffected, as did PNA (d-g) (green) and P-mTOR itself (red, g). (h-m) Reduced levels of dorsal P-mTOR during photoreceptor degeneration (red). The wild-type control is shown in h and the Pde6b mutant is shown in i and j. Reduction started during rod death at P15 (i) as the outer segments (green, PNA) detached from the retinal pigmented epithelium. By P30, only a few cones (green signal: α-β-galactosidase) showed high levels of P-mTOR (red) (j). (k-m) Similar reduction was seen in dorsal cones of the other three mutants (cones marked in green by PNA; Pde6g−/− is shown at P35 in k; Rho−/− is shown at PW20 in l; P23H is shown at PW70 in m). All panels show immunofluorescence microscopy on retinal flat mounts (photoreceptor side up) with the exception of b, c and g, which show retinal sections. Blue indicates DAPI.
Figure 5Chemical structure of the BMPS hetero-bifunctional (amine- and thiol-reactive groups) cross-linker. Introduction of Cys at position 241 in NCT allows chemical cross-linking of NCT and APPC99 with BMPS. Representative SDS-PAGE/western blot show cross-linked NCT-APPC99, NCT and APPC99 bands in detergent-extracted membrane proteins from BMPS cross-linker-treated membranes prepared from MEF cell lines expressing the indicated WT or mutant NCT GSEC complexes together with APPC99 or in the absence of the substrate. Orange, black, and white arrows indicate the NCT-APPC99/Aβ crosslinked, mature NCT, and immature NCT bands, respectively. Arrowheads indicate the position of molecular weight markers. Analysis of the western blot density profiles in panel (B) shows that co-migration of NCT (orange arrow) with APPC99 shifts the cross-linked band towards a higher molecular weight than the observed for mature NCT (black arrow). Immature NCT is indicated with an open (white) arrow. Full-range molecular weight density profiles are shown in Figure EV3C Schematic representation of the GSEC-APPC99 complex modified at NCT- I241 and APP-K28 by the introduction of Cys substitutions. Representative SDS-PAGE/western blot presents NCT-APPC99, NCT and APPC99 expression in detergent-extracted membranes prepared from MEF cell lines co-expressing the mutant NCT- I241C GSEC complex with the APPC99-K28C substrate. As negative control, the same sample was supplemented with β-mercaptoethanol (BME) to reduce all disulfide bridges. A full-range molecular weight western blot is shown in Figure EV3B. Orange, black, and white arrows indicate the NCT
Figure 1Freezing phenotype (A) of Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA for non-acclimation, −5°C, 30 min; CA for cold acclimation, −8°C, 40 min). Representative pictures are shown in (A). survival rate (B), and ion leakage (C) of Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA for non-acclimation, −5°C, 30 min; CA for cold acclimation, −8°C, 40 min). Expression of CBFs (D) in Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were placed at 4°C for 3 h (D) and total RNA was extracted and subjected to qRT-PCR analysis. Relative transcript levels in untreated Col-0 plants were set to 1. Expression of CBFs targets (E) in Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were placed at 4°C for 24 h (E), and total RNA was extracted and subjected to qRT-PCR analysis. Relative transcript levels in untreated Col-0 plants were set to 1.
Conserved seed sequences control envelope homeostasis in V.Figure 7Outer membrane damage misfolded OMPs activate an intra-membrane proteolysis cascade resulting in the release of σE from its anti-σ factor RseA. Free σE activates expression of at least 73 transcripts in V. cholerae, including the rpoE-rseABC operon and the MicV and VrrA sRNAs. MicV and VrrA employ the conserved base-pairing region R1 to downregulate omp mRNAs, restoring membrane homeostasis, and the rpoE-rseABC operon. VrrA specifically downregulates pal and lpp, encoding two major lipoproteins, via the base-pairing region R2.
Figure 2IFA of ISP1 and ISP3 expression from zygote to ookinete of the isp1::6HA and isp3::6HA parasites, respectively. Scale bar = 5 μm. B Co-localization of ISP1 and ISP3 from zygote to ookinete in the doubly-tagged strain (DTS, isp1::3V5;isp3::6HA). Scale bar = 5 μm. C Western blot of ISP1 and ISP3 expression in zygotes of isp1::6HA and isp3::6HA parasites. The numbers are the relative intensities of band in blot. Right panel: the quantification of band intensity. Values are means ± SEM (n=4 biological replicates), two-tailed t test, ****, P<0.0001. D Western blot of ISP1 and ISP3 expression in zygotes of DTS, DTS/Δisp1, and DTS/Δisp3 parasites. The numbers are the relative intensities of band in blot. Three replicates performed. E RT-qPCR analysis of isp1 and isp3 transcripts in zygotes of the WT, Δisp1, Δisp3, and Δisp1/3 parasites. The expression is normalized to the 18s rRNA. Values are means ± SEM (n=4 biological replicates). F IFA analysis of ISP1 and ISP3 expression dynamic of DTS/Δisp1 and DTS/Δisp3 parasites. Scale bar = 5 μm. G Proposed model of polarized co-localization and expression compensation between ISP1 and ISP3.
Figure 7(A) Western blot analysis of total and pS776 wild-type and mutant Atxn1 in the cerebella of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SD, **P<0.01, ***P<0.001, one-way ANOVA, n = 7, 6, 4, 7 following the order of genotypes in the western blot, respectively. (B) Rotarod motor performance test result of 11-12 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SEM, *P<0.05, two-way ANOVA, n = 18, 12, 13, 11, 19, 13, 15, 13 following the order of genotypes in the legend on top of the figure, respectively. (C) Representative immunofluorescent (IF) images of cerebellar molecular layer between the cerebellum lobule V and VI across different genotypes at 36 weeks old, stained with anti-Calbindin 1 antibody. Scale bar = 100 μm. (D) Quantification of the thickness of top and bottom molecular layers shown in (C). Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from three sections per mouse.
Figure 8A. Volcano plot of differential expressed genes analysis results for RNA-seq data on TCGA comparing hTrmt13 expression low group patients (first quartile) versus high group patients (fourth quartile). Significant differentially expressed genes (FDR corrected p-value < 0.05) were colored by red, interesting genes with oncogenic implications are labelled in blue. Pseudo values 1e-15 were added to FDR correlated p-value for better visualization. B. Stacked bar plot of patients number diagnosed as various breast cancer subtype in TCGA group by the expression level of CDH1 or MMP1 or hTrmt13 (low as the first quartile, high as the fourth quartile). C. GSEA plot showing an interesting gene set from MSigDB were enriched for genes expressed higher in hTrmt13 low group for SOTIRIOU_BREAST_CANCER_GRADE_1_VS_3_UP (upper) and enriched for genes expressed higher in hTrmt13 high group for TURASHVILI_BREAST_LOBULAR_CARCINOMA_VS_LOBULAR_NORMAL_UP (down). D. Lung metastasis assay in mice model. MDA-MB-231-Luc-D3H2LN cells infected with lentivirus carrying control (shSCR) or shRNA against hTrmt13 (sh-1) were injected through the tail vein of 6-week-old female SCID mice (n=8). Lung metastasis was monitored by bioluminescent imaging after three weeks of injection. Representative in vivo bioluminescent images are shown (left); Bioluminescent quantitation of lung metastasis (right). Statistical analysis was performed using t-tests. E. Immunohistochemical staining of tissue arrays containing 24 breast carcinoma samples paired with adjacent normal mammary tissues for hTrmt13 expression. Representative images are shown (left), and the mean staining intensity was calculate using Image-Pro Plus software (right). Statistical analysis was performed using t-tests. F. Kaplan-Meier survival analysis of Kaplan-Meier plotter (http://kmplot.com/analysis/) for the relationship between the relapse-free survival (RFS) or overall survival time (OS) of breast cancer patients (BRCA, n=349 (low)/205 (high)), liver cancer patients (LIHC, n=270 (low)/100 (high)), patients with kidney renal clear cell carcinoma (KRIC, n=156 (low)/374 (high)), and patients with kidney renal papillary cell carcinoma (KIRP, n=208 (low)/79 (high)) and their expression of hTrmt13.
Fig.Figure 3(A) Primary sequence of Mrp17 highlighting charged amino-acids, Tom20-binding motifs (TBM1, TBM2) and secondary structure (from PDB:5MRC); (B) In vitro mitochondria translocation capacity of WT Mrp17, Mrp17K-R with all lysines (K) substituted with arginines (R), and Mrp17K-A with all lysines (K) substituted with alanines (A) fused to DHFRmut. Import was performed as described in Fig. 2 legend. (C) In vivo mitochondrial targeting capability of WT Mrp17, Mrp17K-R, and Mrp17K-A fused to GFP. Scale bar is 10 µm. (D) Substitution of lysines with alanines in the regions 1-60 or 30-60 of Mrp17 abolishes mitochondrial import capacity: (top) schematics of substitution positions, TBMs are in green, targeting signal outlined in grey, substitutions are denoted by arrows; (bottom) in vitro translocation of the mutants. (E) Mrp17 variants rescue ∆mrp17 strain growth defect on respiratory media: the indicated variants or empty vector (EV) were introduced in WT yeast and then the genomic MRP17 was disrupted by knock out, the resulting mutants were serially diluted 10× and spotted on media containing glucose or glycerol as a sole carbon source.
Germ plasm aggregation was disrupted in sinhcaf mutant embryos.Figure 3(A) Whole-mount in situ hybridization was performed in 4-cell stage WT, MZsinhcaf -/- zebrafish embryos with germ plasm marker vasa. Red arrowheads point to cleavage furrows in the 4-cell embryos. Scale bars, 100 μm (B) Relative mRNA levels of germ plasm genes vasa, dnd, nanos3, dazl and buc in WT and MZsinhcaf -/- mutant eggs, respectively, as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using 2-way ANOVA followed by the Holm-Sidak post-hoc test; NS, no significant difference. (C) Maximal intensity projection of a confocal z-stack of 4-cell stage WT and MZsinhcaf -/- zebrafish embryos injected with GFP-Buc and RFP mRNA. White arrowheads point to cleavage furrows of 4-cell embryos. Scale bars, 100 μm. (D) Relative GFP-Buc density at the cleavage furrow of 4-cell stage WT and MZsinhcaf -/- zebrafish embryos injected with GFP-Buc and RFP mRNA. Statistical analysis was performed using unpaired two-tailed Student's t-test. Data shown are mean ± SEM (n = 12, the number of embryos analyzed). (E) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. (F) Western blots for GFP-Buc expression in 4-cell stage WT and MZsinhcaf -/- zebrafish embryos injected with GFP-Buc and RFP mRNA
Association between gene expression levels of transcription factors (TFs) and DNA methylation levels at PD‐hypermethylated enhancers from PD iPSC‐derived DAn.Figure 5Relative enrichment of TF‐binding sites (TFBSs) overlapping with the 2,087 DMCpGs detected in PD iPSC‐derived DAn (n = 10). This analysis shows an enrichment of binding sites for 23 TFs in regions hypermethylated in PD.Bar plot showing the results of the Spearman correlation analysis between levels of TF gene expression and of average DNA methylation at the 376 enhancer sites hypermethylated in PD iPSC‐derived DAn (n = 10). Coef, coefficient. Graphical representation of average methylation at PD‐associated enhancers and gene expression of the key TFs FOXA1,NR3C1,HNF4A, and FOSL2 in iPSC‐derived DAn (n = 14). Scatter plots of key TF gene expression and DNA methylation levels at enhancers in iPSC‐derived DAn (n = 14).