EMBO/sd-character-level-ner huggingface dataset:
111 phosphorylation Upon mitochondrial depolarisation, PINK1 activation leads either to activation of a kinase or to inhibition of a protein phosphatase that targets Ser111 phosphorylation of Rab8A.Figure 10Schematic representation of PINK1 regulation of Rab8A Ser111 phosphorylationUpon mitochondrial depolarisation, PINK1 activation leads either to activation of a kinase or to inhibition of a protein phosphatase that targets Ser111 phosphorylation of Rab8A. Phosphorylation of Rab8A at Ser111 impairs Rabin8 (GEF)‐mediated GDP exchange leading to GDP‐bound inactive Rab8A.
Figure 4A. RT-PCR products with divergent and convergent primers showing circularization of has_circ_0079480 and has_circ_0087391. cDNA, complementary DNA; gDNA, genomic DNA. B. qRT-PCR evaluated the knockdown efficiency of has_circ_0079480 and and has_circ_0087319 in SW620 and HCT116 cells transfected with two unique shRNAs (#1, #2). **P < 0.01, Student's t-test, mean ± SD. C. Representative images of the migration phenotype in HCT116 cells with knockdown of candidate circRNAs. D. The relative fold-change of the transwell migration for indicated knockdown cells over those of control cells. **P < 0.01, Student's t-test, mean ± SD. E-F. Representative hematoxylin and eosin (H&E) staining and statistical results of the micro-metastatic nodules in the liver from mice injected with the indicated cells into the spleen for 45 days. N=8 per group. **P < 0.01, Student's t-test, mean ± SD. G-H. Representative H&E staining and statistical results of metastatic lung nodules from mice injected with the indicated cells via the tail vein for 60 days. (Five sections evaluated per lung). N=8 per group. **P < 0.01, Student's t-test, mean ± SD.
ER protein reflux is constitutive in cancer cells.Figure 3Subcellular protein fractionation of several ER resident proteins in (A) GL261, (B) U87 and (C) A549 cells treated with the indicated concentrations of Tm or Tg using Digitonin. (NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer) Quantification of the subcellular protein fractionation of several ER endogenous proteins in GL261, U87 and A549 cells as in (A-C) respectively. Biological triplicates, mean ± SD calculated using Prism 9 (GraphPad). Subcellular protein fractionation of several ER resident proteins in A549 cells treated with the indicated concentrations of Tm or Tg using differential centrifugation protocol. Quantification of the subcellular protein fractionation of several ER endogenous proteins in A549 as in (G). Biological triplicates, mean ± SD calculated using Prism 9 (GraphPad).
Figure 7In early prophase I, ASY1 is expressed and imported into nucleus, facilitated by PCH2. Concomitantly, CDKA;1 becomes enrichted in the nucleus, localizes on chromosomes, and phosphorylates ASY1. The phosphorylation enhances the binding affinity of ASY1 to ASY3 and the self-assembly and thus, in turn antagonizes the releasing force of PCH2. At the same time, high CDKA;1 activity in the nucleus may block other axis disassembling factors that will be activated later in synapsed regions where CDKA;1 is not present.
Figure 7A. Immunoblots showing the levels of MCM3 and PrimPol proteins in PRIMPOL WT and KO cells. MEK2 and Ponceau-S are shown as loading controls. B. Schematic of experimental design and examples of replicative structures (fork/ first-label origin/ terminations) in stretched DNA fibers. Scale bar, 10 µm. C. Percentage of first-label origins in each condition (average and SD of three assays). Circle dots in each column represent the values of individual replicates. Statistical analysis: one-way ANOVA followed by Bonferroni post-test. p-values are indicated. D. Histograms show the percentage of the different replication patterns observed in each experimental condition (average and SD of three assays). Circle dots in each column represent the values of individual replicates. Statistical analysis: two-way ANOVA followed by Bonferroni post-test. p-values of the indicated comparisons are shown. E. IF detection of FANCD2 foci in WT and PRIMPOL KO cells 48 h after TMP-UVA (2 µM TMP, 2 h followed by 5 s irradiation) with or without MCM3 downregulation. Nuclear DNA is counterstained with DAPI. Scale bar, 25 µm. F. Top, dot plot shows the distribution and mean value (horizontal red line) of the number of FANCD2 foci per cell in each condition. The pool of three replicates is represented (n ≥100 cells per condition). Statistical analysis was conducted with Kruskal-Wallis test and Dunn's post-test. p-values are indicated. Bottom, histograms show the average foci number and SD of three assays. Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of the indicated comparisons are shown. G. Cell survival assays of WT and PRIMPOL KO cells with or without MCM3 downregulation in the presence of increasing amounts of MMC or TMP (followed by 90 sec UVA irradiation) for 72 h. The same control curves without MCM3 downregulation are also shown as part of Figure EV6D. Each point in the curve represents the percentage of surviving cells (average and SD of three assays for MMC and two assays for TMP). Statistical analysis was conducted with two-way ANOVA followed by Bonferroni post-test. p-values of comparisons between KO and KO-siMCM3 are indicated. H. Model for the participation of PrimPol in the ICL traverse reaction (left branch), and the alternative fork convergence mechanism (right branch), both of which generate a suitable template for FA-mediated ICL repair. In the absence of PrimPol, ICL traverse is much less efficient and cells rely on dormant origin activation to promote fork convergence at the ICL. When dormant origins are not available, a fraction of the ICLs remain unrepaired, generating chromosomal instability. See text for details.
Figure 5(A) Enrichment of the ZEB1, AP-1 and TEAD4 DNA-binding motifs is shown, as identified by HOMER known motif analysis on 200 bp regions centred on different subsets of ZEB1 peak summits. (B) Hypergeometric testing showed that while there is a significant association between the AP-1 and TEAD4 motif within ZEB1 peaks, both motifs are associated with the absence of the ZEB1 consensus motif (Z-box). Odds ratio (OR): measure of association between categorical variables with OR>1 indicating a positive association. (C) The upper panel shows a schematic representation of the ANKRD1 luciferase reporter constructs. The lower panel shows luciferase reporter assays with these different ANKRD1 promoter constructs in MCF7 cells upon overexpression of ZEB1, YAP and JUN/FOSL1 or different combinations of the factors. n = 4-5; shown is mean ± s.e.m.; *P ≤ 0.05; **P ≤ 0.01; ratio paired t-test. Firefly luciferase activity was normalized to co-transfected renilla luciferase. (D) Left panel: EMSA using nuclear extracts from MDA-MB-231 and labelled TEAD binding side as probe (black arrow). Competition of specific binding complex (read arrow) with cold probe (c), anti-JUN, -panTEAD, and -ZEB1 antisera. Right panel: recombinant GST-ZEB1, but not GST-Ctrl, only binds to Z-box.
Figure 2(A) Volcano plot shows genes where RNA reads are statistically significant under- or over-expressed in 5 RIRCD affected individuals versus 6 controls resulted from DESeq2 analysis. Decreased genes are represented in red, the up-regulated ones are in green while blue dots highlight most outlying genes (defined by absolute (log2(fold change) + log10(padj))>10).Genes that were not statistically significant are represented in black. Grey and black gene names indicate top 30% of the most dysregulated genes with a highlight on ones involved in metabolism. (B) Representation of the KEGG terms associated with differentially expressed RNAs and their corresponding proteins in 5 affected patients versus 6 controls showing major pathway changes in patient muscle (C) Abnormal transcription of genes involved in various biological metabolic pathways derived from our RNAseq (RNAseq read counts/ person/ gene for n=6 RIRCD affected individuals, n=5 controls and n=1 (biological replicate analysed in 4 technical replicates) recovered) data showing that their expression returned close to normal levels in patient muscle after recovery. The central band represents the median, the lower and upper hinges correspond to the first and third quartiles (25% and 75%) while the whiskers extend to the highest and lowest points within the data (1.5x the inter-quartile range). (D) Relevant functional changes in gene expression showing the major genes that return to baseline or contribute to the recovery in RIRCD patients. The data depicted is between the affected and recovered biopsies of the same patient (F6/1M). (E) Comparative proteome profiling of proteins that are significantly under- or over-expressed in RIRCD affected versus control individuals (p-Anova <0.05-statistically significant - horizontal line). Proteins which are decreased are represented in red while the up-regulated ones are in green while proteins that did not reach statistical significance threshold are marked in black. Vertical lines are delimiting the regulated proteins resulted from the proteomics analysis (the cut-off values were determined based on the 2× standard deviation and the normal distribution from all identified protein's log2 ratio in which the bell curve is symmetric around the mean, therefore, an average log2 ratio of a protein which <−2.2 or > 0.98 was considered as regulated). (F) Proteomap representation of the major altered pathways in RIRCD affected muscle compared to control individuals where the size of each circle or hexagon represents the fold change.
Figure 2A, CRISPR-modified cells (Brightfield, left) that express GFP-nesprin-2 (middle) and mCh-LAC (right) show nesprin (orange arrow) but not lamin accumulation at the front of the nucleus as it is deformed through a narrow constriction. B, Quantification of intensity of GFP-nesprin-2 and mCh-LAC, normalized to the intensity at the sides. Error bars correspond to the standard deviation. Constriction: n=19 cells over 5 biological replicates, 15 µm: n= 27 over 6 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars (*P < 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.
CENATAC is a novel component of the minor spliceosome.Figure 3Phylogenetic profiles (presences (blue) and absences (white)) of the top 15 genes co-occurring with CENATAC in 90 eukaryotic species. Top: phylogenetic tree of the eukaryotic species (see Table EV1) with colored areas for the eukaryotic supergroups. 1 for NMT1 no human ortholog was found and instead the Arabidopsis thaliana ortholog is depicted. Genes associated with the minor (U12-dependent) spliceosome are depicted in orange. See also Table EV2. GO term analysis of the genes co-occurring with CENATAC as in (A) with a correlation score of >0.5. Note: the amount of hits and fold enrichment score were manually changed to accomodate the recently discovered SCNM1 (Bai et al, 2021) Graph of fold-changes of proteins enriched (P-value < 0.05) in proteomics analysis of CENATAC vs. control co-immunoprecipitations of HeLaEGFP-CENATAC cells (three biological replicates). Splicing factors are depicted in orange. See also Appendix Figure S7. Examples (D) and quantification (E) of Northern blot analyses of minor (U6atac, U4atac, U11, and U12) and major (U2, U1, U4, U5, and U6) spliceosome snRNAs in HeLaEGFP-AID-CENATAC cells (three biological replicates, normalized to the control). See also Appendix Figure S8. Glycerol gradient (10-30%) analysis of HeLa S3 nuclear extracts. snRNAs were detected by Northern blot analysis, proteins (CENATAC and PRPF4) by western blot. Locations of the U6atac mono-snRNP, U4atac/U6atac di-snRNP and U4atac/U6atac.U5 tri-snRNP are indicated. Schematics showing key assembly stages in minor intron splicing and minor tri-snRNP assembly: intron recognition (A-complex) and the catalytic spliceosome (C-complex). For simplicity, several stages of spliceosome assembly are omitted, such as the pre-B-complex, which consists of the intron recognition complex together with the tri-snRNP before architectural changes lead to the exclusion of U11 to give rise to the B complex, after which subsequent architectural changes lead to the exclusion of U4atac to give rise to the BACT complex, which is a precursor stage for the catalytically active C complex depicted in this figure (Turunen et al, 2013b).
Figure 6RGCC was co-stained with PAX6 on proliferating rosettes of hCOs at day 30. RGCC exhibits specifically higher expression level on NSCs around apical surface (marked by arrow head) adjust to ventricle-like region (marked by white dash line). Scale bar: 50 μm. Representative image of one M-phase NSC with higher RGCC expression around nucleus were show in right panel and marked by yellow arrow head. Scale bar: 5 μm. RGCC was co-stained with PCNT on 3D hCO slices. RGCC was with stronger expression in NSCs during M-phase. prometaphase (B'), metaphase (B'') anaphase (B''') and telophase (B''''). RGCC was expressed around nucleus in interphase (B''''') . NSCs undergoing prometaphase, metaphase, anaphase and telophase were pointed out by yellow arrow. Interphase were pointed out by white arrow (B'''''). Immunostaining of 2D-differentiated NSCs in metaphase showed partial colocalization of RGCC with α-TUBULIN in the spindle (B'''''', yellow arrow). Scale bar: 5 μm. The Western blots results. Proteins were collected from WT hCOs and RGCC KO hCOs #6 at day 45. B-ACTIN were used as references. PCNT was co-stained with β -TUBULIN on WT NSCs and RGCC KO NSCs differentiated by 2D cultured dual-SMAD inhibition method. Scale bar: 5μm. Representative images of measuring fluorencense intensity of β -TUBULIN and PCNT. Step-by-step details can be found in the Materials and Methods section. Scale bar: 5μm. Quantification of fluorescence intensity of β -TUBULIN positive spindle fibers. Quantification of mean fluorescence intensity of β -TUBULIN positive spindle. Quantification of fluorescence intensity of PCNT positive centrosomes. Data were presented as mean ± SEM. Each plot represents the average intensity of two PCNT positive region in one dividing cell (n=11 for H9 cells; n=12 for RGCC-KO cells).Statistical significance was tested by one-way ANOVA. ****, p<0.0001.
a) The Hiw-associated complex was purified by TAP (see Online Methods) and analyzed by one-dimensional SDS-PAGE gel followed by Sypro Ruby staining.figf1(a) The Hiw-associated complex was purified by TAP (see Online Methods) and analyzed by one-dimensional SDS-PAGE gel followed by Sypro Ruby staining. Mass spectrometry identified the full-length Hiw, β-tubulin, Rae1 and Fsn, indicated by arrows. (b) The Rae1-associated complex purified from fly brains by TAP was analyzed by one-dimensional SDS-PAGE gel followed by Coomassie blue G-250 staining. Mass spectrometry identified Hiw, Nup98 and Rae1, indicated by arrows. (c) Larval brain lysate of wild-type (WT), hiw null mutant (hiwΔN) and Fsn mutant (Fsnf06595/Df(2R) 7872) flies was subject to co-immunoprecipitation with antibody to Hiw (Hiw2b). Both the input and the immunoprecipitated complexes were analyzed by western blots with antibodies to Hiw or Rae1. The membrane was also blotted with antibody to β-tubulin to ensure equal amount of total proteins in the input samples. Full-length blots are presented in Supplementary Figure 8.
Jumpy R336Q mutant associated with centronuclear myopathy is defective in inhibiting autophagy.Figure 7(A-K) C2C12 cells were transfected for 48 h with YFP, YFP‐Jumpy WT (Jumpy WT), YFP‐Jumpy C330S (Jumpy CS), YFP‐Jumpy Y462C (Jumpy YC) or YFP‐Jumpy R336Q (Jumpy RQ), fixed and immunostained with anti‐p62 antibody (red). p62 puncta were quantitated by confocal fluorescence microscopy. Representative confocal images of YFP (A), YFP‐Jumpy WT (B), YFP‐Jumpy C330S (C), YFP‐Jumpy Y462C (D) and YFP‐Jumpy R336Q (E) transfected cells, immunostained for p62 (F), (G), (H), (I) and (J), respectively. Scale bars, 5 μm. Quantitation of p62 puncta per cell (K). Bars, s.e.m. (n=3 independent experiments, 30 cells per experiments). *P0.05 (t‐test), ns: nonsignificant. (L) HeLa cells were transfected for 24 h with YFP, YFP‐Jumpy WT (Jumpy WT) or YFP‐Jumpy R336Q (Jumpy RQ), incubated with or without 50 ng/μl rapamycin (BafA1 (100 nM) was present in both control and rapamycin treated cells) for 2 h, lysed and analysed for LC3, YFP and actin by immunoblotting. Densitometric LC3‐II/actin ratios are shown underneath the blot.
Figure 6A, B The period (A) and power (B) of DD locomotor rhythms of flies with not knocked down and controls. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down. C Plots of relative mRNA abundance vs CT for clock genes determined by qRT-PCR in whole head extracts of not RNAi (timG4/+;Udcr2/UnotRNAi) and control (timG4/+;Udcr2/+) collected on DD1 (n=5). D ChIP assays to detect ub-H2B binding at E-box, TSS and gene body of tim and Pdp1ε in not RNAi (timG4/+;Udcr2/UnotRNAi) and control (timG4/+;Udcr2/+) (n=3). Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Statistical difference is measured using two-way ANOVA, P<0.001, significant effect of genotypes were found for tim E-box (P<0.01), Pdp1ε E-box (P<0.05), tim TSS (P<0.05) and Pdp1ε TSS (P<0.001) and tim gene body (P<0.01). G4, GAL4; U, UAS. E, F The period of DD locomotor rhythm of flies when knocking down Nipped-A and over-expressing not. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down. G The period of DD locomotor rhythm of flies co-expressing Nipped-A RNAi and not RNAi. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down.
Figure 4A. Huh7 cells were co-transfected with the HBV expression plasmid pCH-HBV1.3 and the four principal constructs examined in this study, or with an empty AAV expression plasmid that served as negative control. Another negative control were mock-transfected cells. HBeAg and HBsAg were quantified in the supernatant48 h later. B. Measurement of HBV DNA in cell culture supernatants four days after co-transfection of Huh7 cells with the plasmid encoding the 1.1-fold over-length HBV sequence (pCH-9-3091) and the same RNAi expression plasmids as in panel A. PCR was performed using primers that discriminate between HBV rcDNA and the 1.1-fold over-length HBV sequence contained in the plasmid. C. RT-qPCR results using primers specific for the 3.5 kb HBV transcript (including pregenomic RNA and the precore transcript; left), or primers that detect all HBV transcripts (right). D. Southern blot analysis of lysates obtained 72 h post-transfection of HepG2 cells with the same plasmids as described in panel A.
The LuTHy procedure.Figure 1A Schematic representation of the workflow of the LuTHy method. Expression vectors encoding NL and PA-mCit-tagged hybrid proteins are cloned and co-transfected into mammalian cells. Binary interactions are detected with a double-readout; first, in vivo with in-cell bioluminescence resonance energy transfer (BRET) and second, ex vivo with a luminescence-based co-precipitation (LuC). B Schematic representation of control proteins and fusion proteins of interest for investigating the interaction between PA-mCit-BAD and NL-BCL2L1 in proof-of-principle LuTHy experiments. C Calculated BRET ratios for the indicated protein pairs. The positive control protein PA-mCit-NL and the interacting fusion proteins NL-BCL2L1 and PA-mCit-BAD show high BRET ratios. D Calculated LuC ratios for the indicated protein pairs. The positive control proteins PA-NL and PA-mCit-NL and the interacting proteins NL-BCL2L1 and PA-mCit-BAD show high LuC ratios.
Characterization of RAB21 knockout HeLa cells.Figure 4(A) Low RAB21 expression levels in two independent RAB21 KO cell populations. Immunoblot analysis of endogenous RAB21, RAB5, RAB7 and GAPDH. (B) Endo-lysosomal compartments are unaffected by the loss of RAB21. Immunofluorescences of APPL1, TfR, RAB7 and LAMP1 in parental HeLa cells and in the two RAB21 knockout cell populations. Scale bars :10µm, n=3 independent experiments.
'-phosphopantetheine.Figure 8Our current model predicts that defects in pantothenate kinase 2 impair the phosphopantetheinylation of mtACP and lead to impaired complex I activity (Fig. 2E), iron-sulfur biogenesis (Fig. 2C), and mitochondrial fatty acid synthesis. Lipoic acid modification is required at the E2 domain of PDH complex. Decreased production of lipoic acid in mitochondria would diminish activity of this enzyme (Fig. 2D). The clinical features of PKAN, including abnormal iron accumulation and parkinsonism, can be explained by these metabolic defects. Their rescue by exogenous 4'-phosphopantetheine suggests that the CoA pool critical for mtACP phosphopantetheinylation is at least partially dependent on pantothenate kinase 2.