EMBO/sd-character-level-ner huggingface dataset:
TFEB stimulates lysosomal–autophagosomal fusion and clearance.Figure 5Confocal microscopy images of live fibres from GFP-LC3:GAA−/− mice.Muscle was transfected with mCherry-LAMP1 only. The image (a single frame from the time-lapse series presented in Movie 5) shows lysosomes (red), LC3-positive autophagosomes (green) and a number of autolysosomes (yellow).Muscle was transfected with Flag-TFEB and mCherry-LAMP1. Massive formation of autolysosomes is indicated by yellow structures; the three lower panels provide a snapshot of the process of exocytosis. The structures at the plasma membrane are labelled with both LC3 and LAMP1, indicating that they represent amphisomes (a product of fusion between autophagic vesicles and late endosomes) or autolysosomes (a product of fusion between autophagic vesicles and lysosomes). Bar: 10 µm for all panels.
Centriolar satellites regulate AURKA localization, abundance and activity in quiescent cells A, B.Figure 4A, B. Representative images and quantification of basal body levels of (A) AURKA and (B) phospho-AURKA (p-AURKA) in control and PCM1-depleted cells. RPE1 cells were transfected with control or PCM1 siRNA #1 or siRNA #2 for 48 h. Following 24 h serum starvation, cells were fixed and stained with the indicated antibodies. Centrosomal AURKA and p-AURKA fluorescence intensities were measured from maximum projections, and average means of the levels in control cells were normalized to 1. n>500 cells per experiment. Data for (A) AURKA and (B) p-AURKA represent mean value from three experiments per condition ± SEM (****p < 0.0001, Unpaired Student's t-test.) Scale bar, 10 μm C. Total AURKA and p-AURKA levels in control and PCM1-depleted cells. RPE1 cells were transfected with control or PCM1 siRNA#1 for 48 h. Following 24 h serum starvation, cell lysates were prepared and run on an SDS-PAGE gel. Proteins were detected by immunoblotting with antibodies against AURKA, p-AURKA and GAPDH (loading control). D. Cycloheximide chase experiment for quantification of AURKA half-life. Cells were transfected with control or PCM1 siRNA#1 for 48 h, then treated with 200 nM cycloheximide along with serum starvation for indicated time points. AURKA intensities were quantified by immunoblotting for AURKA and vinculin (loading control) and AURKA levels were normalized to vinculin levels. Data represent mean value from three experiments per condition ± SEM (**p < 0.01, Unpaired Student's t-test)
Figure 5(A) A summary of altered metabolic fluxes in liver by integrating metabolomics data accounting individual variations. Solid arrows indicate one or more metabolic reactions. Plasma metabolites increased due to metabolic cofactor supplementation are highlighted in rounded rectangle frames. The red/blue color of arrows and metabolite names indicate increased/decreased flux/pool with metabolic cofactor supplementation compared to control study. (B-Q) Changes in fluxes of key metabolic reactions highlighted in (A) compared to baseline fluxes (dashed line) based on personalized modelling, where 'SX' indicating flux obtained by the modeling of subject number 'X' as shown in Table EV13.
Accumulation of Ub in autophagy-deficient brain.Figure 1.Accumulation of Ub in autophagy-deficient brain. Ub profile from the 1% Triton X-100-soluble and -insoluble fraction of autophagy-deficient brain homogenates captured with P2UBA resin. (A) Age-matched control Atg7 littermates (Atg7flox/+;nestin-Cre:p62+/−; denoted +) at 6 wk (open circles; n = 4) or Atg7 KO (Atg7flox/flox;nestin-Cre:p62+/−; denoted −) at 6 wk (closed diamonds; n = 3) are shown. Accumulation of Ub in autophagy-deficient brain. Ub profile from the 1% Triton X-100-soluble and -insoluble fraction of autophagy-deficient brain homogenates captured with P2UBA resin. (B) Control Atg5 littermates (Atg5flox/+;nestin-Cre; denoted +) at 16 (open circles; n = 4) and 26 wk (open diamonds; n = 3) or Atg5 KO (Atg5flox/flox;nestin-Cre; denoted −) at 16 (closed circles; n = 4) and 26 wk (closed diamonds; n = 3) are shown. Each symbol represents one animal. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005.
a) Cell-cycle distribution and cell number count of cells transfected with control or VHR siRNA and observed for 4–5 days.figf3(a) Cell-cycle distribution and cell number count of cells transfected with control or VHR siRNA and observed for 4-5 days. The cells were stained with propidium iodide for DNA content and analysed by FACS after the indicated number of days in culture. (b) Confocal microscopy of the same HeLa cells stained for VHR (green) and BrdU (red) to indicate S-phase entry, and DNA (DAPI, blue). Control siRNA-treated cells and cells treated with VHR are shown. The bottom panels represent merges of the 3 images above (c) Quantification of the BrdU positive cells in b. The indicated numbers of cells were counted and the data are given as percentage of BrdU positive cells of this total number. d) Quantification of cells staining for phosphoH3. The indicated number of cells were counted and the data are given as percentage of phosphoH3-positive cells of this total number. (e) Confocal microscopy of control or VHR siRNA-transfected HeLa cells stained for VHR (green) and phospho H3 (red) to indicate M-phase entry, and DNA (DAPI, blue). The bottom panels represent merges of the images above. (f) Immunoblots of total lysates of control siRNA transfected cells and VHR siRNA cells with the indicated antibodies. Note that VHR was downregulated by approximately 80%, wheras actin levels were unchanged. (g) Immunoblot with anti-p21Cip1-WAF1 of total lysates of cells transfected with control siRNA (lanes 1-2), two different VHR siRNAs cells (lanes 3-6) or another control siRNA (lanes 7-8). The scale bars represent 10 μm in b and e.
Figure 4Representative FRET time traces (black, FRET signal; yellow, fit) of McjD NBD C547 labeled with Alexa 555 and Alexa 647-maleimide under apo conditions at 10 ms time resolution. Approximately ~85% of all apo-McjD traces show no significant fluctuations beyond shot-noise. Apo-McjD is predominantly in the low FRET and thus open state with E* values <0.3 Within the complete data set of apo-McjD (N = 80 traces) comprising donor and acceptor, ~15%, show infrequent fluctuations to a higher FRET efficiency state with a lifetime of (C) 82 ± 25 ms where E* values > 0.5 are observed during short dwells. The data set represents a total recording time of 4.6 mins with a temporal resolution of 10 ms. To exclude unwanted influence on McjD due to surface-immobilization, experimental verification is provided that ATP-induced NBD switching occurs similarly with detergent solubilized McjD on the surface in Figure EV4
Figure 4(Ai) Confocal microscopy images of LLO-treated HeLa cells immunolabelled for the indicated proteins. Insets show colocalization between NMHCIIA and WRAMP components at cortical bundles. (Aii) Quantification of Pearson's correlation coefficient (PCC) between NMHCIIA and the indicated proteins within entire LLO-treated cells, cortical bundles or equivalent size control cellular ROI (Ctrl ROI). For Calpain 2 cortical bundles were defined by actin staining. Data are representative of one experiment repeated 3 independent times with similar results. p-values were calculated using one-way-ANOVA with Tukey post hoc analyses ** p<0.01; *** p<0.001, ns - non significant (B) Immunoblots of NMHCIIA, Gp96 and Filamin A levels from WCL and Gp96 IP of HeLa cells left untreated (U) or treated with LLO (0.5 nM, 15 min) (LLO). (C) Immunoblots of NMHCIIA and Gp96 levels from WCL and Gp96 IP of HeLa cells left untreated (U) or treated with 25 μg/ml of Wnt5a for the indicated time. (D) Confocal microscopy images of HeLa cells untreated (Mock) or treated with 25 μg/ml of Wnt5a for 30 min. Cells were immunolabelled for ER-KDEL (blue), Gp96 (red) and NMHCIIA (green). Arrows indicate polarised localization of NMHCIIA, ER-KDEL and Gp96 in Wnt5a-treated cells. (E-F) Confocal microscopy images of shCtlr, shGp96 or shNMHCIIA HeLa cells stained for actin (red) and DAPI (blue) and immunolabelled for (E) NMHCIIA (green) or (F) focal adhesion kinase (FAK) (green). (A, D-F) Scale bar - 10 μm. Insets show sites with focal adhesion points and arrows indicate stress fibers or distinct focal adhesion points. Quantification of the % of cells with (G) stress fibers or (H) focal adhesion points labelled by FAK. (I-J) Wound closure assay. (I) Sequential frames of time-lapse microscopy of HeLa cells grown to confluence separated by a stopper. Stopper was removed at t0 and cellular migration was imaged for the indicated times. (J) Quantification of the % of wound area occupied by migrating cells over time stopper removal (upper panel) and respective rate of closure (lower panel).
a) Whole-cell lysates of HL-1 cells treated with 0.f3(a) Whole-cell lysates of HL-1 cells treated with 0.02 μM DOX and transfected with FLAG-tagged Parkin or the FLAG-empty vector were immunoprecipitated with the anti-Flag-M2 antibody and blotted with anti-p53 and Flag antibodies. (b) Whole-cell lysates of rat neonatal cardiomyocytes treated with the indicated concentrations of H2O2 and DOX for 24 h were immunoprecipitated with the anti-Parkin antibody. (c) Cytosolic heart lysates of adult and aged mice were immunoprecipitated with anti-Parkin, p53 and control IgG antibodies. (d) Schematic representation of N-terminally FLAG-tagged WT Parkin and various mutants of Parkin. (e) The association of endogenous p53 with various Parkin mutants in DOX-treated HeLa cells. The immunoprecipitation assay revealed that the RING0 domain was essential for the interaction with p53. (f) Schematic representation of the GST-p53 fusion proteins. (g) GST pull-down assays using GST-p53 fusion proteins and in vitro transcribed/translated FLAG-Parkin. Residues 81-160 of p53 were sufficient to bind Parkin.
Figure 1A Detection of the activation of IRE1α, PERK and ATF6 UPR branches by immunoblotting in control (shCtrl) and Fam20C stable knockdown (shFAM20C) HepG2 cells treated with or without 5 μM Tg for 30 min. ATF6-FL, full-length ATF6; ATF6-N, N-terminal cleavage product of ATF6; Asterisk, unspecific background bands. Fam20C knockdown was verified by protein immunoblotting of Concanavalin A-Sepharose (Con A)-enriched culture medium. Ponceau staining was shown as a loading control. B (Left) Detection of spliced XBP1 (S) and unspliced XBP1 (U) mRNA in shCtrl and shFAM20C HepG2 cells treated with or without 5 μM Tg for 1 h. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test). C (Left) Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing RNAi-resistant codon-altered Fam20C wild-type (WT) or its inactive D478A mutant (DA) treated with or without 5 μM Tg for 1 h. Fam20C expression levels were shown by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test). (Left) Detection of XBP1 mRNA splicing in shCtrl and shFAM20C HepG2 cells (D) treated with or without 2 μg/ml Tm for 8 h. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test). Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing Fam20C WT or DA (E) treated with or without 2 μg/ml Tm for 8 h. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test). F Volcano plot depicting the log2 of fold change versus - log10 (p-value) for Fam20C-interacting proteins in Tg-treated HepG2 cells. The red dots represent proteins showing statistically significant increase of binding with Fam20C (log2 fold change ≥ 1; - log10 (p-value) ≥ 1.3); the blue dots represent proteins showing statistically significant decrease of binding with Fam20C (log2 fold change ≤ - 1; - log10 (p-value) ≥ 1.3); the green dot is Fam20C. G HepG2 cells expressing FLAG-tagged Fam20C were treated with or without 5 μM Tg for 30 min. FLAG-immunoprecipitates were analyzed by PDI immunoblotting.
Summary schematic of novel CDKL5 substrates and functions in microtubule dynamics.Figure 8 CDKL5 phosphorylates microtubule-associated proteins EB2, MAP1S and ARHGEF2 at their RPXS motifs. Double phosphorylation of the MAP1S LC microtubule-binding region decreases MAP1S affinity for microtubules. Loss of these MAP1S phosphorylations leads to impaired microtubule dynamic instability in CDKL5 KO neurons. Altered microtubule stability in CDKL5 KO neurons is also likely to be responsible for reduced cargo trafficking
Figure 8(A) UBQLN4 interacts with non-glycosylated (and thus defective) IL-2Ra WT in SRP54-knockdown cells. After treatment of HeLa cells with SRP54 siRNA (10 nM) for 48 h, Flag-tagged IL-2RaWT and T7-tagged UBQLN4 proteins were expressed, and the cells were treated with (+) or without (-) 10 µM MG-132 at 4 h before harvesting. UBQLN4 was affinity-purified from cell extracts and probed with an anti-Flag antibody. Note that the levels of non-glycosylated IL-2RaWT (indicated as Defective) increased, while the glycosylated form of this protein (indicated as Assembled) decreased in SRP54 knockdown cells. (B, C) SRP54 knockdown stimulates polyubiquitinated proteins co-precipitation of UBQLN4. Flag-tagged UBQLN4 and T7-tagged ubiquitin were expressed in SRP54 siRNA-treated cells, and Flag precipitates were probed with an anti-T7 antibody to detect polyubiquitinated clients co-precipitation with UBQLN4 under no addition of MG-132 (B). Note that the interaction of UBQLN4 with polyubiquitinated substrates was further increased in SRP54 knockdown cells treated with MG-132 for 4 h before harvesting (C).
APEX2:RAB expression lead to endosomal biotinylation.Figure 1(A) Diagram representing APEX2:RAB-mediated endosomal biotinylation of endogenous proteins. (B) Illustrative representation of APEX2:RAB endosomal microdomains. APEX2 only or APEX2:RAB were found to biotinylate endogenous proteins. (C) Streptavidin Western blotting of total biotinylated proteins in APEX2:RAB or APEX2 only Flp-In/T-REx HeLa cells. (D) APEX2:RAB biotinylated proteins (streptavidin) are partially colocalized with EEA1 in HeLa cells, n=2 independent experiments. Scale bars: 10µm or 5µm in enlarged views.
Figure 3A). Schematic representation of interactions between PTP-MEG2 and the NSF-pY83 site. (B). Relative values of the decreased phosphatase activities of different PTP-MEG2 mutants towards pNPP (p-Nitrophenyl phosphate) compared with wild-type PTP-MEG2. Data information: *** P<0.001: PTP-MEG2 mutants compared with the control group. Data were obtained from 3 independent experiments. N.S. means no significant difference. All the data were analyzed using one-way ANOVA, and displayed as the mean ± s.e.m. In the red column indicates that the mutation sites have greater effects on the enzyme activities toward phospho-NSF segement than the pNPP. The green column indicates that the mutation sites only have significant effects on the enzyme activity toward NSF phospho-segement but not pNPP. The white column stands for the mutants that have similar effects on pNPP and NSF phospho-segement. (C). Fold-change decreases in the phosphatase activities of different PTP-MEG2 mutants towards the NSF-pY83 phospho-segment compared with wild-type PTP-MEG2. Data information: *** P<0.001: PTP-MEG2 mutants compared with the control group. Data were obtained from 3 independent experiments. N.S. means no significant difference. All the data were analyzed using one-way ANOVA, and displayed as the mean ± s.e.m. In the red column indicates that the mutation sites have greater effects on the enzyme activities toward phospho-NSF segement than the pNPP. The green column indicates that the mutation sites only have significant effects on the enzyme activity toward NSF phospho-segement but not pNPP. The white column stands for the mutants that have similar effects on pNPP and NSF phospho-segement. D). Sequence alignment of PTP-MEG2 from different species and with other PTP members. Important structural motifs participating in phosphatase catalysis or the recognition of the NSF-pY83 site are shown, and key residues contributing to the substrate specificity are highlighted.
Figure 6(A) Rescue of a delayed cortical migration in CAMDI-KO mice by Tubastatin A. Immunohistochemical analysis of Cux1 expression in the somatosensory cortex at P2 after intraperitoneal injections of Tubastatin A into pregnant CAMDI-KO mice from E12.5-17.5. Scale bar, 100 μm.(B) Quantification of the number of Cux1-positive neurons. Note the abnormal distribution of neurons in deep cortical layers of CAMDI-KO mice. n = 3 mice/genotype (WT (vehicle) = 3,628 cells, KO (vehicle) = 2,816 cells, WT (Tubastatin A) = 1,847 cells, KO (Tubastatin A) = 2,364 cells). *, p<0.05, **, p<0.01, Two-way ANOVA followed by Tukey HSD test. Data are presented as mean ± SEM. (C) Enhanced α-tubulin acetylation at P0 by Tubastatin A. Ac-tubulin level was assessed by immunoblot analysis.(D) Quantification of the α-tubulin acetylation. n=3 independent experiments. *, p<0.05, Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.
affects anti-EV71 restriction of SAMHD1.Figure 3TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. Illustration of gRNA-guided TRIM21 knockout in cells. TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. Negative control or RD-CRISPR-Cas9-TRIM21 RD cells were infected with EV71 at a MOI of 0.05 for the indicated time and harvested for SAMHD1, TRIM21 and EV71-VP1 detection by IB. Tubulin served as a loading control. TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. EV71 RNA levels in (B) were detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P < 0.01, paired t-test). TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. Viral titres in the supernatant were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (**P < 0.01). Overexpression of TRIM21 in HEK293T cells increased EV71 replication by degrading SAMHD1. HEK293T cells were transfected with VR1012 or TRIM21-HA for 24 h and then infected with EV71 at a MOI of 0.05. Cells were harvested at the indicated time points and subjected to IB with tubulin as a loading control. Overexpression of TRIM21 in HEK293T cells increased EV71 replication by degrading SAMHD1. HEK293T cells were transfected with VR1012 or TRIM21-HA for 24 h and then infected with EV71 at a MOI of 0.05. EV71 viral RNA in cell lysates was detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P < 0.01, paired t-test)). TRIM21 itself has no effect on EV71 replication. VR1012 or TRIM21 was transfected into pLKO.1 or sh-SAMHD1 HEK293T cells for 24 h and infected with EV71. Cells were harvested and subjected to IB analysis. TRIM21 itself has no effect on EV71 replication. VR1012 or TRIM21 was transfected into pLKO.1 or sh-SAMHD1 HEK293T cells for 24 h and infected with EV71. Viral titers in the supernatants were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (*P < 0.05).
Figure 8A) As depicted in the cartoon and shown by representative confocal images, patch-clamp recordings were performed in the dorsal striatum, in the area of HSV-LacZ (blue, left) and HSV-RGS9-2 (red, right) viral infection, the latter showing an increased RGS9-2 immunolabelling with respect to the sham contralateral striatum of the same mouse. Representative confocal images at higher magnification show colocalization of RGS9-2 immunolabeling with Lucifer Yellow (LY) fluorescence in two ChIs loaded intracellularly with LY during whole-cell recordings in the infected area B) Representative patch-clamp recordings of ChIs from hMT (top, perforated configuration) and Tor1aΔgag/+ (bottom, cell-attached configuration) mice, showing that viral-mediated delivery of RGS9-2 rescues the physiological response to the DRD2 agonist quinpirole (10 µM, 2 min). The dot plot reports the firing frequency changes induced by quinpirole in hMT striata (N=6, HSV-RGS9-2 n=9, HSV-LacZ n=14, t test P<0.0001) C) Representative traces show a typical response of Tor1a+/+ ChIs to quinpirole, characterized by a reduction in the frequency of the ongoing firing activity of the cell, as opposed to an aberrant increase in firing frequency in Tor1a+/- ChIs, quantified in the plot (right, Tor1a+/+ N=6, n=14; Tor1a+/- N=10, n=27; Tor1a+/+ vs. Tor1a+/- in quinpirole t test ***P=0.0005; Tor1a+/- basal vs. quinpirole paired t test ***P=0.0009) D) LV-mediated delivery of RGS9-2 rescues the physiological response to quinpirole in Tor1a+/- slices. Dot plot reporting firing frequency changes induced by quinpirole in Tor1a+/+ (N=3) and Tor1a+/- (N=6) striatal ChIs infected with LV/HSV-RGS9-2 or control LV-GFP and HSV-LacZ ChIs (Tor1a+/+ RGS9-2 n=7; Tor1a+/- RGS9-2 n=9; Tor1a+/- LacZ/GFP n=8; Tor1a+/-: LacZ/GFP vs. RGS9-2 t test *P=0.0136)
Figure 3(A) ROS accumulation in Gmlmm1-1 and Gmlmm1-2 mutant plants. Soybean leaves (aged 3 weeks) were subjected to ROS accumulation examination by DAB staining. Scale bar, 1 cm. Data information: The experiments were performed three times as biological replicates, with similar results. The exact number (n) and p values are shown in the source data. (B) ROS accumulation upon P. sojae treatment. The leaves (aged 3 weeks) were inoculated with P. sojae isolate P7076, and DAB staining was performed at 16 hpi. The left panel was photographed under bright field (scale bar, 1 cm). The middle and right panels were photographed under a microscope (scale bar, 50 μm). Data information: The experiments were performed three times as biological replicates, with similar results. The exact number (n) and p values are shown in the source data. (C) Enrichment of defense-related genes in the Gmlmm1-1 mutant. The Gmlmm1-1 and WT plants (aged 2 weeks) were subjected to RNA-seq analysis. The genes that were highly expressed in Gmlmm1-1 compared with the WT (2-fold changes) are presented. The enrichment (%) of the GO terms indicated is presented, and the p-values are calculated by Fisher's tests. Detailed data are shown in the source data. (D The increased resistance of the mutants to Psp (D) infection. The indicated plants (aged 2 weeks) were infected with the indicated pathogens (Mean ± SD, n ≥ 8, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The lesions are shown by trypan blue staining. Scale bar, 1 cm. Data information: The experiments were performed three times as biological replicates, with similar results. The exact number (n) and p values are shown in the source data. E) The increased resistance of the mutants P. sojae (E) infection. The indicated plants (aged 2 weeks) were infected with the indicated pathogens (Mean ± SD, n ≥ 8, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The lesions are shown by trypan blue staining. Scale bar, 1 cm. Data information: The experiments were performed three times , as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.