EMBO/sd-character-level-ner huggingface dataset:
x stabilizes the expression of the phosphorylated form of BimEL and reduces Bim-ubiquitination in PMA-stimulated 293FT cells.Figure 2A Usp27x inhibits the PMA-induced degradation of BimEL in 293FT cells. 293FT-TetR-3xFlagUsp27x or TetR-3xFlag-Usp27xC87A cells were treated with dox to induce 3xFlag-Usp27x or Usp27xC87A, or with PMA [16.2 nM] (to induce BimEL degradation), or with the combination of dox, PMA and QVD [10µM] (to inhibit apoptosis) as indicated for 24 h (left) or 48 h (right). Cell lysates were analysed by Western blotting. The blots are representative of n=4 similar experiments. See also Appendix Fig. S5B. * Probably modified form of wild-type 3xFlag-Usp27x (such as dimer, ubiquitinated Usp27x or a stable complex of Usp27x with another interacting partner); this form is only seen if high expression levels of catalytically active Usp27x are reached (see Appendix Fig. S5A where Usp27x was transfected into 293FT cells) or if wt Usp27x is enriched during immunoprecipitation.** Probably degradation product of 3xFlag-Usp27x. These forms of Usp27x were not seen in every experiment. B 293FT-TetR-3xFlagUsp27x or TetR-3xFlag-Usp27xC87A cells treated as in A were analysed using conditions of higher resolution. A band very likely corresponding to phosphorylated BimEL is detectable. Similar results were obtained in n=2 separate experiments. C Usp27x stabilizes expression of p-BimEL (Ser69) in Bim-degrading conditions without reducing β-TrCP levels293FT-TetR-3xFlagUsp27x were treated with dox, PMA [16.2 nM], UO126 [10µM] or QVD [10µM] as indicated for 24h, and levels of phosphorylated BimEL at serine69 were analysed by Western blotting using a phospho-Bim (Ser69) specific antibody. Scans are from the same membrane and exposure times. Similar results were obtained in n=2 separate experiments. D UO126 reduces phosphorylation-associated shift of BimEL293FT-TetR-3xFlagUsp27x were treated for 24h with the indicated drug combinations and Bim was detected by Western blotting. UO126 was added 30 minutes prior other stimulation. Similar results were obtained in n=3 separate experiments (see Appendix Fig. S5D). E Usp27x-expression reduces ubiquitination of Bim293FT-TetR-3xFlagUsp27x cells were transfected with a vector coding for 6His-ubiquitin (left) or GFP (right). After 24h cells were treated with PMA and QVD, and 3xFlag-Usp27x was induced by dox at the same time. After additional 20h cells were treated for 4h with MG132 (40 µM) to block proteasomal degradation of ubiquitinated proteins. Cells were lysed under denaturing conditions and His-ubiquitin-labelled proteins or proteins from only GFP expressing cells were purified by Ni2+-NTAaffinity chromatography. Ubiquitinated Bim (Bim-Ubn) was detected by Western blot using anti-Bim antibodies (left). Similar results were obtained in n=3 separate experiments (another experiment including a Coomassie stain is shown in Appendix Fig. S3E). *Indicates non-His-ubiquitinated BimEL that was bound unspecifically to the Ni2+-agarose beads
Figure 4Schematic of ribophagy detection with Keima-based reporter system; FP: fluorescent protein. Validation of STX16/STX17 DKO by Western blot analysis in HEK293 cells stably expressing RPS3-Keima (left panel) and HCT116 cells stably expressing RPL28-Keima (right panel). WT or STX16/STX17DKO HEK293 RPS3-Keima cells were cultured in full medium or starved for 8 hours, and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm. Masks: white, cells identified based on nuclei; red, Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm, respectively; lower panel shows Keima puncta without masks. Scale bar: 20 µm.   Quantifications of autolysosomal Keima puncta in WT or STX16 /STX17DKO HEK293 RPS3-Keima cells treated as in (C). Data shown as means ± SEM of Keima puncta per cell, minimum 1000 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).  WT or STX16/STX17DKO HCT116 RPL28-Keima cells were cultured in full medium or starved for 8 hours, and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm. Masks: white, cells identified based on nuclei; red, Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm, respectively; lower panel shows Keima puncta without masks. Scale bar: 20 µm.   Quantifications of autolysosomal Keima puncta in WT or STX16/STX17DKO HCT116 RPL28-Keima cells treated as in (E). Data shown as means ± SEM of Keima puncta per cell, minimum 1000 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant; **, p < 0.01 (two-way ANOVA).
Figure 1(A) Schematic overview of cloning, expression and purification of 143 carbohydrate recognition domain (CRD) - mouse IgG2a-Fc fusion proteins, from 168 annotated murine CRD containing proteins. The constructs were expressed in HEK293F cells and secreted Fc-fusion proteins were purified using protein A columns. (B) Exemplified SDS-PAGE of purified Clec7a and Mgl2 stained with Coomassie blue. (C) Glycosylation map of the SARS-CoV-2 Spike and RBD. The most prominent glycan structures are represented for each site, with at least 15% relative abundance. * marks highly variable glycosylation sites in which no single glycan structure accounted for >15% relative abundance. The different monosaccharides are indicated using standardized nomenclature. NTD, n-terminal domain; RBD, receptor binding domain; S1/S2 and S2', proteolytic cleavage sites; HR1 and HR2, α-helical heptad repeat domains 1 and 2; GlcNAc, N-acetylglucosamine.
Figure 5(A) siRNA-based knockdown of endogenous Tom70, Tom20, Tom40, and Sam50. HeLa cells treated with the corresponding siRNAs and immunoblotted with the indicated antibodies. AIF, Apoptosis-inducing factor. (B) HeLa cells were immuno-stained with anti-Flag and anti-catalase antibodies after treatment of each siRNA. Tom20- and Tom40-siRNA treatment inhibited import of Su9-GFP (the targeting sequence of the FoF1 ATPase subunit 9) into the mitochondria, leading to an accumulation of Su9-GFP precursor proteins in the cytosol and cell nucleus. 3Flag-MITOL still localized to mitochondria in Tom20, Tom40, and Sam50 siRNA-treated cells. In contrast, 3Flag-MITOL dispersed into the cytosol when Tom70 was knocked down. Scale bars, 10 µm. (C) Statistical analysis of the MITOL subcellular localization following 15 µM CCCP treatment for 3 hours in cells treated with control, Tom70, Tom20, Tom40, or Sam50 siRNAs. Su9-GFP was not imported into the mitochondria, but rather localized to the cytosol and nucleus following Tom20 and Tom40 knockdown. The number of HeLa cells with cytosol-localized Su9-GFP or 3Flag-MITOL in each siRNA experiment were determined. Data represent the mean ± s.e.m from > 100 cells in three biological replicates. (In case of Tom40, and Sam50 siRNAs, data represent the mean ± s.e.m from > 60 cells in three biological replicates.) Statistical significance was calculated using a one-tailed Welch's t-test. (D) HeLa cells treated with siControl or siTom70 were fractionated into cytosolic and mitochondrial fractions and then immunoblotted with anti-lactate dehydrogenase (LDH), anti-Tom20, and anti-Flag antibodies. LDH was used as a cytosolic marker. Although some MITOL detached from mitochondria during fractionation, sufficient amounts of MITOL were collected in the mitochondria-enriched fraction in control cells. In contrast, almost all of the MITOL was collected in the cytosolic fraction in siTom70-treated cells.
Figure 3A CTV-labeled splenocytes (20 x 106 cells) from either Arhgap45-/-or WT mice were mixed with CMTPX-labeled WT splenocytes (20 x 106) and injected intravenously in WT recipient mice. After 4 hours, single cell suspensions were prepared from the spleen and mesenteric LNs (mLN) and the percentages of CTV- and CMTPX-labelled B and T cells determined by flow cytometry. B The ratio of CTV/CMTPX-labeled cells present in each organ (Ro) was determined and normalized by dividing it with Ri, the ratio of CTV/CMTPX-labeled cells present in the cell mixtures prior to injection. Ro/Ri ratio corresponding to each of the specified mice are shown. Data shown for Arhgap45-/-mice correspond to 4 independent experiments (E1 to E4) involving a total of 15 individual mice whereas data shown for WT mice correspond to 2 independent experiments (E1 and E2) involving a total of 5 individual mice. The Arhgap45-/- mice analyzed on the same day as the control mice corresponded to the E1 and E2 experiments. Mean and SD are shown. ns, non-significant, **** P ≤ 0.0001; multiple unpaired T tests. C Expression of CCR7, and LFA-1 on naïve CD4+ and CD8+ T cells found in LNs of WT and Arhgap45-/- mice, analyzed by flow cytometry. Gray shaded curves, isotype control staining. D Quantification of data shown in (C). The MFI corresponding to each analyzed mouse is shown together with mean and SD. Each dot represents an individual mouse and CD4+ T cells from at least 4 mice have been analyzed for each condition. Mice analyzed on the same day are represented by similar dots (see key in bottom right corner). ns, non-significant, ** P ≤ 0.002; multiple unpaired T tests.
a) Myosin VI and Tom1/Tom1L2 co-immunoprecipitate from LNCAP cell lysates.figf6(a) Myosin VI and Tom1/Tom1L2 co-immunoprecipitate from LNCAP cell lysates. Immunoprecipitation of myosin VI with three separate affinity-purified rabbit polyclonal antibodies (B4, B7, 2401) was performed alongside an IgG control immunoprecipitation. The immunoprecipitates were analysed by western blotting with antibodies against Tom1/Tom1L2. (b) Binding of myosin VI to Tom1 requires the WWY motif. Bottom, the mammalian two-hybrid assay was used to test binding of full-length Tom1 and Tom1L2 or various truncated versions of Tom1, annotated by amino acid numbers, against wild-type myosin VI tail or the myosin VI ΔWWY or ΔRRL tail. The graph shows the mean values from n = 2 independent experiments. Top, Tom1 domain structure and truncation mutants. VHS, Vps-27, Hrs and STAM; GAT, GGA and Tom1. (c) RPE cells stably expressing GFP-myosin VI were processed for immunofluorescence microscopy to evaluate GFP-myosin VI and endogenous Tom1/Tom1L2 or Rab5 co-localization. The insets show higher magnifications of the areas outlined in the main panels. The arrows indicate areas of co-localization. (d) RPE cells with stable expression of GFP-myosin VI tail were subjected to mock or Tom1 siRNA transfection followed by western blot analysis (left) and immunofluorescence microscopy (middle) to evaluate GFP-myosin VI tail localization. Cells were either fixed directly or saponin-extracted before fixation. Quantification (right) was performed on RPE cells stably expressing GFP-myosin VI tail and transfected with mock, Tom1 or TNO siRNA. Cells were processed for immunocytochemistry, immunolabelled for GFP and nuclei labelled with Hoechst followed by quantification of GFP-myosin VI tail punctae per cell using an automated Cellomics VTi microscope. More than 600 cells per group from n = 3 independent experiments were analysed and are represented as the mean number of GFP-myosin VI tail punctae per cell (±s.d.). Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.
Statistical modeling of the traveling ratio reveals the independent dynamics between population and transcriptional regulation in regenerating skeletal muscle tissues.Figure 4(A) Immunofluorescent images of the mouse tibialis anterior muscle on the indicated days after CTX treatment. The images of anti-mouse ChIL probe for PolII-S5P (red) and anti-rabbit IgG for laminin (green) are shown. Scale bar: 20 µm. (B) PolII S5P ChIL-seq signal of the marker genes of mature skeletal muscle (Acta1) and macrophages (Cd68). (C) Proportion of sequenced reads (%UMI) occupied by the representative cell types in muscle regeneration. The single cell data (GSE143437) by De Micheli et al.(De Micheli et al) was re-analyzed. (D) Extraction of independent dynamics of the population and transcriptional regulation. Change in PolII distribution at the gene loci: a gene (blue) was transcriptionally activated (red nuclei) following the stimuli, while population size was unchanged. Change in the height of PolII distribution: a type of cells (yellow) was grown after the stimuli, while the transcriptional activity was maintained. (E) Estimated mean (95% confidence interval) of TR and the CPM of PolII S5P ChIL-seq at TSS. Representative genes of mature skeletal muscle cells and immune cells are shown. (F) Bulk-tissue expression levels (TPM) of the representative genes. (G) Different activities of two major cell-types in muscle regeneration. Scatter plots of log2FC of day-3 vs. day-0 of TR (x-axis) and the TSS-level (y-axis) are shown: immune cell marker genes (left); myogenic genes: right. Colors indicate significance in TR and TSS-level based on |log2FC| > 1 (two-fold) and FDR < 0.1. (H) Activities of major cell types in muscle regeneration. The colors of the heatmap show the log2FC to day 0 (uninjured) of TR and TSS levels. Representative genes with significant changes in TR are shown. (I) The dynamics of the biological process in muscle regeneration and the participating cell types. Genes were assigned to five groups (C1-5) based on highest time point of TR. OR indicates the specificity of participation to the biological processes.
a,b) Midguts dissected from either early third instar larvae (a) or at puparium formation (b) that express GFP-labelled mitochondria in enterocytes (larger nuclei) and contain Uba1H33 loss-of-function mutant cell clones (lacking RFP).figf8(a,b) Midguts dissected from either early third instar larvae (a) or at puparium formation (b) that express GFP-labelled mitochondria in enterocytes (larger nuclei) and contain Uba1H33 loss-of-function mutant cell clones (lacking RFP). Wild-type control cells possess stronger RFP and heterozygous cells have weaker RFP. Representative images are shown. (c) Midguts dissected at puparium formation that express Uba1IR (GFP in nucleus and cytoplasm) and stained with ATP synthase complex V (ATPV) to detect mitochondria in all cells. Representative images are shown. (d-f) Representative immuno-TEM images of Uba1IRclone cells at puparium formation. Uba1IR-expressing cells possess gold particles; control cells lack gold particles (d). Control cells possess numerous autolysosomes in the cytoplasm (arrowheads) and few mitochondria (asterisks); Uba1IR-expressing cells possess numerous mitochondria and few autophagic structures. Scale bars, 20 μm (a-c) and 1 μm (d-f).
Open bars, mice treated with a placebo; filled bars, mice treated with CCI-779.figf6Open bars, mice treated with a placebo; filled bars, mice treated with CCI-779. (a-c) Behavioral tests in mice expressing mutant huntingtin after treatment at 16 weeks (CCI-779, n = 14; placebo, n = 14), 18 weeks (CCI-779, n = 10; placebo, n = 9), 20 weeks (CCI-779, n = 9; placebo, n = 7) and 22 weeks (CCI-779, n = 5; placebo, n = 6) of age. Scores are explained in Methods. Data for all time points are given in Supplementary Figure 13 online. (a) Tremors. Overall effect from all treated time points: P = 0.0006, odds ratio (OR) = 0.21, 95% confidence interval (95% c.i.) = 0.098-0.45. 16 weeks, P = 0.01; 18 weeks, P = 0.08; 20 weeks, P = 0.14; 22 weeks, P = 0.07. (b) Wire maneuver. Overall effect from all treated time points: P = 0.02, OR = 0.51, 95% c.i. = 0.29-0.92. 16 weeks, P = 0.29; 18 weeks, P = 0.40; 20 weeks, P = 0.003; 22 weeks, P = 0.008. (c) Grip strength. Overall effect from all treated time points: P 0.0001 OR = 12.73, 95% c.i. = 4.35-37.2. 16 weeks, P = 0.1827; 18 weeks, P = 0.0412; 20 weeks, P = 0.0021; 22 weeks, P = 0.0285. (d) Accelerating rotarod tests in mice expressing mutant huntingtin after treatment at 4 weeks, (CCI-779, n = 16; placebo, n = 17), 14 weeks (CCI-779, n = 11; placebo, n = 10), 16 weeks (CCI-779, n = 14; placebo, n = 14) and 18 weeks (CCI-779, n = 10; placebo, n = 9) of age. Overall effect from all treated time points: P = 0.035. 4 weeks, P = 0.227; 14 weeks, P = 0.012; 16 weeks, P = 0.0025; 18 weeks, P = 0.057. CCI-779 had no discernable effects on the performance of nontransgenic mice on any of these behavioral tests (data not shown). There was no difference in the male:female ratios of the groups treated with placebo or with CCI-779.
The Tfcp2l1-Cpt1-FAO axis promotes the survival of 2i-ESCs in response to mTOR inhibition.Figure 6A. Pharmacological (INK-128) and physiological (glutamine deprivation) inhibition of the mTOR signaling. B. Immunoblotting of phosphorylated and total S6K (a substrate of mTOR) showing the inhibitory effect of INK-128 (200 nM) and glutamine deprivation on mTOR activity. C. Percentage of cell death of WT, Tfcp2l1_KO, and Tfcp2l1_KO 2i-ESCs with Cpt1a overexpression (OE) treated with 200 nM INK-128 for 24 hours. n=3 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test. D. 0.5 million of 2i-ESCs with indicated genotypes were seeded and treated with 200 nM INK-128 on day0. Cell numbers were counted on day2, 4, and 6. n=4 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test. E. Immunoblotting of WT and Tfcp2l1_KO 2i-ESCs untreated or treated with 200 nM INK-128. F. Real-time PCR showing Cpt1a mRNA levels in WT and Tfcp2l1_KO 2i-ESCs in the presence and absence of 200 nM INK-128. n=3 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test. G, H. Percentage of cell death in 2i-ESCs treated with 50 µM Eto in the presence or absence of 200 nM INK-128 or 100 µM of hexanoate (G) or octanoate (H) as indicated for 24 hours. n=4 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.
Overexpression of ASCL1 and NEUROD1 promotes the expression of their responsive genes.Figure 1(A) Western blot showing the protein expression of ASCL1 and/or NEUROD1 in NCI-H69, NCI-H82, NCI-H146, NCI-H510A, NCI-H526, SHP-77 and DMS-53 SCLC cells (lanes 4-10); A549 and NCI-H460 NSCLC cells and a IMR-90 HFL cell lines (lanes 1-3). Tubulin (α-Tub) is shown as loading control. Each blot is representative from at least three independent experiments. (B, C) Venn diagrams showing the overlap between DMS-53 RNA-seq (Blue circle) and either ASCL1 ChIP-seq (Orange circle) (B) or NEUROD1 (Green circle) (C) ChIP-seq promoter annotated peaks in untreated conditions (t=0). Top panels. Tables showing the common and unique features from Venn diagram. According to RNA-seq data, a gene has been considered transcribed, if it has a coverage (number of reads per base of the gene) of more than 1 and a cut off value of TPM (transcript per million reads) of 4. DMS53 RNA-seq: genes being transcribed; ASCL1- and NEUROD1-Chip-seq: genes targeted byASCL1 or NEUROD1; DMS53 RNA-seq and either ASCL1- or NEUROD1-seq: genes bound by either ASCL1 or NEUROD1 and being transcribed; DMS-RNA-seq only: transcribed genes not targeted by ASCL1 or NEUROD1; ASCL1- and NEUROD1-Chip-seq only: non-transcribed genes in our experimental conditions. Lower panel : Venn diagram showing the overlap between ASCL1 and NEUROD1 (Dark green Circle) targeted and downregulated genes (3470 genes) in untreated conditions. (D) Picture of ASCL1 (above) and NEUROD1 (below) ChIP-seq associated motifs around the genome (P=1e-6800 and P=1e-60, respectively). 13254 RNAseq filtered genes by mean coverage >1 and mean tpm>= 4. 10657 had 1 or more ASCL1 motif in their custom promoter region. 8925 Genes had 1 or more NEUROD1 motif in their custom promoter region.
Figure 3A. In vitro methylation reactions were performed using recombinant His14-MBP-NSUN3 (NSUN3) or the catalytically inactive mutant His14-MBP-NSUN3-C265A (C265A), 3H-labelled S-adenosyl methionine as a methyl group donor and in vitro transcribed mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. The RNA was then separated on a denaturing polyacrylamide gel, stained with ethidium bromide (EtBr) to indicate inputs and exposed to an X-ray film to analyse methylation (3H-Me). B. The distribution of Illumina sequence reads along the mt-tRNAMet sequence obtained from CRAC experiments with NSUN3 after UV (light grey) or 5-AzaC crosslinking (dark grey) is given as reads per million mapped reads. The position of the anticodon is indicated by a bar. C. Cloverleaf scheme of the mt-tRNAMet sequence. Nucleosides that were exchanged in the mutational analysis shown in the following panels are marked with arrows and the nucleotide positions in the tRNA are given. D. In vitro methylation assays were performed as described in (A) with His14-MBP-NSUN3 and in vitro transcribed wildtype mt-tRNAMet and cytidine mutants of the anticodon stem and loop region indicated in (C). Two exposure times of X-ray films are shown: 16 hours (short), 3 days (long). E. In vitro methylation assay of in vitro transcribed mt-tRNAMet and chemically synthesized mt-tRNAMet containing an m5C modification at the wobble position. The experiment and analysis were performed as described in (A).
Figure 4Rathippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA (shA3) or a control shRNA (shCtrl) and tag RFP. Three days after transduction (DIV3+3), neurons were transfected with (G4C2)80HIGH(+0) and analyzed at (DIV3+7). (A) Neurons were fixed, immunostained and imaged by confocal microscopy. Double immunofluorescence for poly-GA aggregates (green) and tag RFP (red). Nuclei were labeled with DAPI. Scale bar represent 20 μm. Rat hippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA (shA3) or a control shRNA (shCtrl) and tag RFP. Three days after transduction (DIV3+3), neurons were transfected with (G4C2)80HIGH(+0) and analyzed at (DIV3+7). (B) Efficient knockdown of endogenous rat hnRNPA3 with its targeting shRNA. Rat hippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA (shA3) or a control shRNA (shCtrl) and tag RFP. Three days after transduction (DIV3+3), neurons were transfected with (G4C2)80HIGH(+0) and analyzed at (DIV3+7). (C) Poly-GA aggregates were detected in a filter trap assay using an anti-Flag antibody. (D) The amounts of poly-GA aggregates were quantified and are presented as the fold change of signals from neurons treated with the control shRNA or the repeat construct. Means ± SD of three independent experiments are shown. * p<0.05 by a Student's t-test. n= 3 replicates.
Figure 5A Time lapse images of B. subtilis Δbkd cells stained with FM 5-95 and grown in PF medium. See Movie EV1 for full time lapse. Appendix Fig S6 shows the changes observed in relation to natural homeoviscous adaptation. B Images of B. subtilis Δbkd cells grown with IB or without precursor (IBPF). Cells were stained with membrane fluidity-sensitive dye Laurdan and imaged at 460 nm, 520 nm and as the corresponding colour-coded Laurdan GP map. C Images of E. coli fabA(Ts) cells grown at 30°C or shifted to non-permissive 37°C for 120 min. Cells were stained and imaged as described in panel B. For a more pronounced view on domain formation associated with local differences in membrane fluidity, see Appendix Fig S7, showing E. coli fabA(Ts) grown in LB instead of M9 minimal medium with glucose/casamino acids. D Images of B. subtilis Δbkd cells grown and stained with Laurdan (imaged at 460 nm) as in panel B, but additionally expressing WALP23-mCherry. For corresponding fluorescence intensity correlation between images, see Appendix Fig S8A. Co-localisation of membrane dye FM 5-95 and transmembrane peptide WALP23 is shown in Appendix Figs S9 and S8C. E Images of E. coli fabA(Ts) cells grown and stained as in panel C, but additionally expressing WALP23-mScarlet-I. For corresponding fluorescence intensity correlation between images, see Appendix Fig S8B. Dat