EMBO/sd-character-level-ner huggingface dataset:
Figure 8A) Projection images of three confocal sections (1 μm) at the basal side of wing discs expressing the Bac constructs for Hh-GFP and Ptc-cherry, used to analyse co-localization between the ligand and the receptor in wild type conditions and after inhibition of vesicle fusion (Syb RNAi), vesicle trafficking (Hrs RNAi) or Ptc protein degradation (Dor RNAi). Boxplot shows co-localization rates using the Manders coefficient for proportion of red covered by green in projected images for wing discs temporally expressing RNAi against ESCRT proteins Hrs and Vps4, SNAREs Syb and α-Snap, and Dor as control for effects after Ptc degradation-blocking, as well as temporally over-expressing the Tsp96F-HA. Central horizontal lines show median values of N=8-12 box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding. Note the significant reduction in co-localization rates (1= total coverage of red by green) after RNAi expression for both ESCRT and SNARE proteins as well as after Tsp96F-HA over-expression, while RNAi expression for Dor presents greater co-localization. B) Projection images of three confocal sections (1 μm) from the apical side of the wing discs. Quantification of co-localization rates using apical projection images; central horizontal lines show median values of N=8-12 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note that at the apical side no clear significant reduction in co-localization rates (1= total coverage of red by green) is observed after RNAi expression or in Tsp96F-HA over-expression. C) Correlation coefficients between the maximum value of quantified integrated density (integrating both fluorescence intensity and signal area) for Ptc-GFP values (Yac construct) and distance from the basal side (-1=basal, +1=apical). Box plot shows quantification data of discs after RNAi expression for the vacuole sorting protein Dor (necessary for Ptc degradation), the ESCRT Hrs, the SNARE Syb as well as after over-expression of the Tsp96F-HA construct and wild type condition. Central horizontal lines show median values of N=8-12, box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note the apical localization of Ptc-GFP highest values after degradation impairment (Dor RNAi) in contrast to the basal shift resulting from exocytosis deficiency (Syb RNAi).
Figure 6A Overlay of ScV1 and the cryo-EM structure of the V1Vo holoenzyme in "state 2" (Zhao et al, 2015). The structures of ScV1 and ScV1Vo were overlaid by aligning the A3B3 catalytic hexamers, thus highlighting structural differences in the central rotor and peripheral stator subunits. Coloring of ScV1Vo (shown as semi-transparent ribbons) is as used throughout for ScV1 for all but the A3B3F (tan), subunit H (dark green), subunit C and Vo domain (gray). The EG1-HNT interaction is boxed. Note the shift in position of HNT and EG1 between ScV1 and ScV1Vo. B Comparison of peripheral stalk EG1 and HNT in ScV1 versus ScV1Vo. The boxed view in A is shown enlarged. The N-terminal α helices of peripheral stalk EG1 and HNT change in conformation, likely induced by the movement of HCT when going from the holo enzyme to the autoinhibited conformation. C View as in A rotated 150º around the vertical. Comparisons of peripheral stalk EG3 and HCT are boxed.D Comparison of peripheral stalk EG3 in ScV1 versus ScV1Vo. In ScV1Vo, EG3 binds to the C subunit head domain (Chead; gray) whereas in ScV1, EG3 is bent towards the C-terminal domain of a catalytic A subunit (enlarged view of box labeled EG3 in panel C).E Comparison of HCT's conformation in ScV1 versus ScV1Vo. Enlarged view of box labeled HCT in panel C highlighting the conformational changes and binding interactions of HCT; for clarity only aNT, HCT and the central rotor (DF) are shown. In ScV1Vo, the most C-terminal α helix (dark green) of HCT (green) binds to aNT (light gray), with the inhibitory loop (green spheres) rotated away and inaccessible for binding the B subunit and the central stalk (transparent red). In ScV1, the HCT (yellow) inhibitory loop (yellow spheres) binds to the central stalk subunit D (red) and the B subunit of the open (AB) 1 catalytic site. The difference in conformation between ScV1Vo and ScV1 involves a ~150° rotation of HCT. Note that the HCT loop (spheres) is rotated away from the central stalk in ScV1Vo whereas in ScV1, the C-terminal α helix (orange) involved in binding aNT is rotated away and unavailable for rebinding to the membrane. F HCT conformational changes. Available subunit H structures as obtained for isolated H (Sagermann et al, 2001) (purple), in ScV1Vo (Zhao et al, 2015) (green) and in ScV1 (yellow) are shown aligned by their N-terminal domains (HNT) illustrating the conformational flexibility of HCT. The loop involved in inhibition of ScV1 is shown as red spheres, highlighting the 180º rotation of HCT when going from the conformation of isolated H (HCT 1ho8) to the inhibitory conformation in ScV1(HCT ScV1).
Figure 4PDGF-AA stimulation enhances localization of active Akt (phospho-S473Akt) at the basal body of primary cilia (right side panels show higher magnification). Quantification of co-localization of phosphorylated Akt (green) and INVS (red) at the indicated time points after PDGF-AA stimulation revealed a time-dependent increase. Colocalization was measured by counting yellow pixels (colocalized area) using Imaris software (Bitplane AG). Results presented are means ±SE (n=31 at time 0, n=35 at 1 min., and n=33 at 3 min, respectively). Three independent experiments were analyzed with similar results. Statistical significance was determined by student's t test. Co-immunoprecipitation assays using HEK293T cells show that INVS preferentially binds to WT-Akt (second panel, lane1) or phospho-mimetic Akt (T308D/S473D, lane2), but fails to interact with phosphorylation-defective forms of Akt (T308A/S473A, lane 3).PDGF-AA stimulation of HEK293T cells results in augmented interaction of endogenous INVS with Akt (top panel, comparison between lane 1 and lane 2, untreated and PDGF-AA-stimulated conditions, respectively). Both WT and 3A-INVS, which are localized at the INVS compartment of primary cilia under serum-starved condition (left side panels, lower panels show higher magnification), translocate to the basal body of the cilium after PDGF-AA stimulation (right side panels, lower panels show higher magnification). Results presented are means ± SE of yellow pixels demonstrating colocalization of EGFP-INVS (green) with γ-tubulin (red) in hTERT-RPE1 cells (n=28). Three independent experiments showed similar results. Statistical significance was determined by student's t test. Fluorescence intensities of INVS (green), acetylated-α-tubulin (blue), and γ-tubulin (red) along the line (a-b) are plotted underneath.
Figure 4(A) Expression of cGAS, STING, NF-κB, ASC, IL-1β (FL), caspase-1 (FL), and GSDMD in primary microglia lysates was detected using western blot. (B) Quantitative analysis for Western blot analysis of cGAS, STING, NF-κB, ASC, IL-1β (FL), caspase-1 and GSDMD. (C) The protein levels of caspase-1 p20 and IL-1β (p17) in the supernatant of cultured primary microglia were determined by western blot. (D) Quantitative analysis for Western blot analysis of caspapse-1 and IL-1β. *P < 0.05, **P<0.01 compared with the untreated group, #P < 0.05, ##P < 0.01 compared with the LPS+poly(dA:dT) group. (E-F) Immunofluorescence was conducted to detect the expression levels of cGAS, NF-κB, and pyroptosis associated molecules (GSDMD, caspase-1, and IL-1β) in BV2 cells. n = 3 in each group. Scale bars, 50 μm.
Analysis of intersubunit rearrangements and movement of the HCV IRES Domain II.Figure 2(A) Comparison of 40S subunit positions in Rolled, Rotated, Classical/no head tilt (orange) and Classical 80S*HCVIRES complex (yellow) in common 60S alignment. The cryo-EM map of the Classical 80S*HCVIRES was filtered to 6 Å. Arrows indicate the direction of movement during transition between two different states. The distance changes in the 40S subunit positions resulting from the rigid body transformation are color-coded in Å units.(B) Comparison of the HCVIRES in Classical 80S*HCVIRES complex (gray) and upon P-tRNA binding (Classical, no head tilt; purple) in a common 40S body alignment. The distance of the movement of domain II was indicated with an arrow and Å units
Figure 3(A) Nuclear extracts of murine intestinal crypts were immunoprecipitated with anti-SRCAP antibody or rabbit IgG followed by ADASDS-PAGE, sliver staining and mass spectrometry. (B) SRCAP and REST were stained in murine intestinal crypts. Scale bar, 10 μm. (C) A schematic diagram of full-length and truncated fragments of REST protein was shown. (D) Flag-tagged REST fragments were expressed in 293T and purified by immunoprecipitation with anti-Flag antibody. REST proteins were incubated with crypt lysates for pulldown followed by immunoblotting. (E) C-terminal domain of REST was sufficient and essential for SRCAP association by pulldown assay. (F) Intestinal crypt lysates were incubated with anti-REST antibody, followed by immunoblotting with indicated antibodies. (G) Intestinal crypt lysates were incubated with anti-REST antibody and protein A/G beads in 4℃ overnight. Recombinant SRCAP protein was added with different concentrations, followed by immunoblotting.
EGFR signaling is required for endo-lysosomal trafficking.Fig 9(A-C) repots>GFP-LAMP1. (D-F) repots>GFP-LAMP1+Shits1. (G-I) repots>GFP-LAMP1+DERDN. Adults were incubated at 28°C for 12 h (A, D, G), 24 h (B, E, H), and 48 h (C, F, I), respectively. The GFP-LAMP1 signal (green) was induced at 12 h and progressively enhanced in (D-F) and (G-I). (J) repots>Rab7-mCherry+H2B-RFP. (K) repots>Rab7-mCherry+Shits1. (L) repots>Rab7-mCherry+DERDN. The adults were shifted to 28°C for 2 days. Rab7-mCherry puncta (red) were increased in (K, L). DAPI: nuclei (white in J'-L'). Scale bar: 20 μm.
New roles for Ser65‐phosphoUbPhosphorylation of Ub by PINK1 has multiple consequences.Figure 9Phosphorylation of Ub by PINK1 has multiple consequences. Structurally, it alters the electrostatic potential of Ub, but also generates a new UbretraCT conformation with a retracted C‐terminus. The UbretraCT conformation is in slow exchange with the common Ub conformation. Functionally, Ub phosphorylation leads to a gain‐of‐function of Ub, as it becomes an allosteric activator of Parkin. We here show that phosphoUb also has loss‐of‐function effects, as it inhibits some assembly and disassembly systems. Additionally, it is possible that phosphoUb is recognized specifically by Ub receptors.
SNX18 tubules are positive for TfR.Figure 8.SNX18 tubules are positive for TfR. HeLa GFP-LC3 cells were transfected with myc-SNX18 or a control myc plasmid and then immunostained for myc and TfR (A) or Rab11 (B). Bars: (A) 10 µm; (B) 5 µm. Insets show enlarged views of the boxed regions. (C, i) Clusters of tubulo-vesicular structures together with small multivesicular body-like vesicles were observed upon plastic embedding of HeLa GFP-LC3 cells overexpressing myc-SNX18. (ii-vi) Immuno-EM showed that similar clusters labeled strongly for GFP-LC3 (ii, and enlarged in iii from the boxed region), myc-SNX18 (v), and TfR (vi). Strong GFP-LC3 labeling in structures resembling more typical autophagosomal vesicles was also detected (iv). See also Fig. S4.
Figure 2(A) Mean telomere length (kb) of whole blood cells from patients and their parents estimated with the TRF method. (B) Representative picture of telomeric signals used for Q-FISH analysis on metaphase spreads from SV40T-transfomed fibroblasts and Muntjac cells used to normalize the signals. (C) Individual and mean values of Q-FISH analyses for control and patients. Mean fluorescence intensity of Muntjac cells was set to 1. Error bars indicate s.d.. (D) Graphical representation of (C) showing the average of fluorescence ratios (relative to Muntjac). Error bars indicate s.e.m.. The non-parametric Kruskal-Wallis test was applied to compare relative fluorescence values between Ctl and P1, and Ctl and P2. (E) Representative pictures of nuclei from two controls and patient's primary fibroblasts showing 53BP1 foci (green) and telomeres (red, detected by Telo-FISH). Yellow arrows indicate telomere dysfunction-induced foci (TIF). The scale bar corresponds to 5µm. (F) Quantification of (E) in primary fibroblasts from two healthy controls (passages > 4) and from patient 1 (passages < 4). Control 1: n = 224; Control 2: n = 263; Patient 1: n = 231. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied when indicated. (G) Representative pictures of SA-β-galactosidase staining in control (passage 15) and P1's primary fibroblasts (passage 6). Results are expressed as the percentage of SA-β-galactosidase-positive cells (averages, lower panel). Control: n = 436; P1: n = 436. A test to compare two population proportions was applied. (H) Representative pictures of chromosomes with normal or aberrant telomeres detected by Telo-FISH. The scale bar corresponds to 1µm. (I) Quantification of terminal deletions and sister telomere losses (J) from three independent experiments (counted chromatids: Ctl1: n=2224; Ctl2: n= 5696; P1: n=4764; P2: n=7908). Averages are shown and χ2-tests were applied to compare Ctl1/2 with either P1 or P2.
Figure 7Loss of cyclin F induced E2F7/8-dependent Homologous Recombination deficiency. HeLa cells that were stably transformed with pDR-GFP were transfected with siRNA as indicated.48h after the initial transfection, cells were harvested for Flow-cytometry. GFP positive cells were gated (Fig EV5A). Relative HR efficiency were adjusted to the scramble siRNA condition. Data represent averages ± SEM (n=3); **P<0.01 (Student's t-test). n.s.: not significant. Loss of cyclin F induced E2F7/8-dependent γ-H2AX accumulation. HeLa cells were transfected with indicated siRNA for 24 hours, and then treated with nocodazole for 16 hours before fixation for immunofluorescence staining of γ-H2AX. DAPI was used to stain the cell nucleus. Relative intensity of γ-H2AX was quantified by Image J software, and 150 cells were quantified for each condition. Red bars represent averages; **P<0.01 (Student's t-test). n.s.: not significant. Scale bar 20 μm. Loss of cyclin F increased DNA damage recovery time before cell division. RPE cells integrated with the 53BP1 construct were transfected with indicated siRNA for 24 hours, and then treated with HU for 16 hours. At the beginning of the imaging, only the single cells with at least one 53BP1 foci were traced, till the time frame that no 53BP1 foci was observed. The mitotic progression of the cells was defined as the duration from damage recovery to cell division. Histogram shows the damage recovery time (green) and the mitotic progression (black) of 50 cells for each condition. Chi-square analysis was performed to test the statistical significance (P<0.01). Knockdown of cyclin F caused a delay in DNA lesion recovery that is dependent on E2F7/8. The cumulative curves represent the add-up number of cells that overcome the DNA damage lesions, for a time frame of 24 hours. For each condition, 50 cells were quantified. Log-rank tests were performed to analyze the statistical significance.
DAPk antisense RNA protects from IFN-γ–induced autophagy of HeLa cells.Figure 9.(A) Quantitation of autophagy in HeLa cells. Cells were either grown in complete medium (1), treated with IFN-γ (1,000 U/ml) (2), or treated with IFN-γ in the presence of the broad caspase inhibitor BD-fmk (100 μM) (3) for 3 d. (B) Photographs of the MDC-stained HeLa cells corresponding to the experiment in A. Treatments: complete medium (1), IFN-γ (1,000 U/ml) (2), and IFN-γ (1,000 U/ml) + BD-fmk (3). (C) PARP cleavage in HeLa IFN-γ-treated cells. Proteins extracted from IFN-γ-treated (left) or TNF-α-treated (100 ng/ml) cells (right) were subjected to Western blot analysis with anti-PARP antibodies. The proteins were prepared from the same experiment shown in A. (D) Transmission electron micrographs of nontreated (top) and IFN-γ-treated (bottom) HeLa cells. Arrows point to autophagic vesicles. (E) DAPk antisense-transfected and control DHFR-transfected polyclonal populations of HeLa cells were incubated with hygromycin B (200 μg/ml) in the presence or absence of IFN-γ (1,000 U/ml) and BD-fmk (50 μM). Detection of autophagic vesicles was performed 2 d later using the MDC dye. Graphs represent mean ± SD of MDC-positive cells calculated from triplicates of 100 cells each.
Cell type specificity of enhancer transcription.Figure 5(A) Unsupervised clustering of all pairwise Spearman correlations of normalized TT-seq signal for mRNAs (left) and eRNAs (right) for the union of transcripts over all fourteen cancer cell lines Grey and colored bars on top indicate gender and cancer type, respectively. (B) Heatmaps showing mRNAs (left) and eRNAs (right) as being synthesized (red, normalized TT-seq signal FPK ≥ 15) or not (blue, normalized TT-seq signal FPK < 15) for all fourteen cancer cell lines (columns). Heatmaps show only transcripts (rows) with normalized FPK ≥ 30 in at least one of the cell lines (mRNAs, n=14,040; eRNAs n=48,105). Heatmaps are ordered by increasing cell type specificity, as indicated by the grey bars to the right showing the number of cell lines a transcript is observed in. (C) Distribution of entropy-based cell type specificity scores for mRNAs (grey) and eRNAs (green) as in (B). The scores range between 0 and 1, where 0 means unspecific (i.e. uniform synthesis level across all cell lines) and 1 means specific (i.e. specific synthesis level in one cell line). (D) Boxplots showing normalized TT-seq signal for nearest active promoters in the cell line in which enhancers are exclusively transcribed (from left to right: n=890; 500; 494; 214; 238; 239; 378; 62; 153; 879; 586; 173; 601; 232) compared to the same promoters in all other cell lines in which the respective enhancers are not transcribed. All pairwise comparisons are significantly different (P-values < 1e-9, two-sided Mann-Whitney U-test). Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown. (E) Barplot showing percentage of nearest active promoters for which transcription is observed exclusively in the same cell line as the respective exclusively in this cell line transcribed enhancers (exclusive) or is observed in all fourteen cell lines (ubiquitous). Percentages are expressed as mean over all 14 cell lines ± SD. (F) Heat scatterplot comparing log2 cell type specificity scores for enhancers (x-axis) and nearest active promoters (y-axis) over all cell lines. Spearman correlation = 0.39. Dashed green line denotes linear regression line with slope = 0.28.
Alanine scanning approach to identify amino acids in the active subdomain of TMEM59 that are essential for LC3 activation.Figure 5(A) Identification of critical amino acids for HA-LC3 conversion. 293 cells were transfected with the indicated CD16:7-263-281 mutants and HA-LC3A, subjected to anti‐CD16 aggregation and lysed for western blotting against the indicated molecules. Asterisks mark mutations with reduced activity. Shown is one representative experiment of six repetitions. (B) Identification of critical amino acids for GFP-LC3 activation and colocalization with the endocytosed chimera. JAR cells were transfected with the indicated CD16:7-263-281 mutants and GFP-LC3A, aggregated and stained for the endocytosed chimera (red). Preparations were scored by blindly counting the number of red vesicles (chimera) and green vesicles (GFP-LC3A) per cell, as well as the number of red vesicles colocalizing with green ones. The percentage of green vesicles colocalizing with red ones was close to 100% for all mutants, that is, virtually no GFP-LC3A vesicles were unrelated to endocytosed chimera (not shown). At least 50 cells were scored per experimental point. The experiment was repeated three times. The graph shows the number of GFP-LC3A vesicles per cell expressed as the percentage of the value obtained for the wild−type chimera (left axis), and the percentage of chimera vesicles labelled with GFP-LC3A (right axis). Data are expressed as means ±s.d. of the triplicates. Asterisks indicate significant differences with respect to wild−type values (paired Student's t−test; P<0.01). (C) Representative confocal microscopy pictures of the phenotype produced by the indicated mutations. Procedures were as in B. (D) Simultaneous mutation of the four essential amino acids to alanine blocks HA-LC3 conversion induced by CD16:7-263-281. 293 cells were transfected with the indicated chimeras (4M, quadruple mutant) and HA-LC3A, aggregated and lysed for western blotting. (E) Simultaneous mutation of the four essential amino acids blocks GFP-LC3 activation and colocalization with the endocytosed chimera. JAR cells were transfected with the indicated CD16:7-263-281 constructs and GFP-LC3A, aggregated and stained for the endocytosed chimera (red). Representative confocal microscopy images are shown. (F) Quantification of the phenotype in E. Data gathering and expression were as in B.Source data for this figure is available on the online supplementary information page.
Fig.Figure 2A. The indicated recombinant INVS proteins (lanes 1-4) were used for IVK. Akt phosphorylates WT (lane5), 1-970 (lane 6), and 1-898 (lane 7) INVS, but failed to phosphorylate 1-670 (lane 8) INVS. The indicated recombinant INVS proteins (lanes 1-10) were used for IVK. Akt phosphorylates WT (lane 6) and 1-898 (lane7) INVS, but failed to phosphorylate other subfragments of INVS (1-822, 1-746, and 1-670, in lane 8, 9, and 10, respectively), as detected using a phospho-Akt substrate antibody.. C. Using the indicated subfragments of INVS for IVK, we confirmed that Akt phosphorylates 675-1065 (lane 4), but not 675-822 (lane 5), or 675-746 (lane 6) INVS. D. Amino acid alignment of 824-898. Motif scan analysis using the Scansite3 database identified three amino acids, T864, S865, and T866, as putative Akt phosphorylation targets. E. Purified recombinant WT or 3A INVS were generated (top panel, lanes 1-4). In IVK, Akt phosphorylated the WT fragment (824-898, middle panel, lane 3), but failed to phosphorylate 3A INVS (middle panel, lane 4), as detected by using a phospho-Akt substrate antibody. F. Akt phosphorylated WT (lane 10), 1-898 (lane 12) and 675-1065 (lane 15) INVS, but failed to phosphorylate both full-length and 675-1065, ∆727-896, 1-857, or 1-822, 3A INVS (lanes 16, 11, 14, and 15, respectively), all of which lacked the T864/S865/T866 target amino acid.
Figure 4A Ten-fold serial dilutions of the indicated mutant cells and subsequent colony formation at the temperatures indicated. Note how mutations on both the BC motifs and PX in bem1 compromise growth at the restrictive temperature. B Western blots probed with anti-HA antibody to detect the indicated bem1 constructs tagged with 3XHA (top panel). Detection of Ade13 was used as loading control (bottom panel). C Representative images of the indicated bem1 mutants tagged with GFP at the BEM1 locus. The inverted fluorescence images are average intensity projections. D Frequency of cells with polarized Bem1-GFP. E Scatter dot plot showing the fluorescence levels of the indicated bem1 strains at the cell pole. The measurements were made on average intensity projections.