EMBO/sd-character-level-ner huggingface dataset:
Rab25 expression alters cells sensitivity to nutrient stress.Figure 1A. Rab25 expression decreases nutrient withdrawal (24 h) induced cell death in ovarian A2780 and IOSE29 cells measured using the Cell death ELISA plus assay (left); cells were cultured in complete media (containing 5% FBS), serum free media (SF), complete media plus 2DG, serum and glucose free media (SF-glu), or in amino acid, glucose and SF Earls buffer salt solution (EBSS). (a) p < 0.01 versus control and (b) p < 0.05 versus pcDNA. (Right) Percentage of apoptotic cells (sub-G0 population) by flow cytometry in A2780 cells following glucose and serum withdrawal (Glu/SF). (a) p < 0.001 versus A2780pcDNA control and (b) p < 0.05 A2780Rab25 versus A2780pcDNA control. B. Decreasing Rab25 expression increases sensitivity to nutrient stress induced cell death. Expression of Rab25 was down-regulated by siRNA or shRNA specific to Rab25. C. Rab25 regulates autophagy activity in ovarian cancer cells under serum and glucose deprivation conditions. Western blot for the 16kD LC3-II fragment, indicative of autophagy activity, in A2780 cells after 4 and 6 h of serum and glucose withdrawal. D. Electron microscopy of autophagy in ovarian A2780 cells after 4 and 24 h of serum and glucose withdrawal (upper panel, low magnification). High magnification images of boxed areas with arrowheads depicting autophagic vacuoles (lower panel, inset; N: nucleus). Average number of autophagosomes per cell was calculated by counting the number of autophagosomes in 16 individual cells from two-independent experiments. E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.E. RPPA detection of time-dependent activation of AMPK after nutrient withdrawal. E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.F. WB analysis of AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in HEY ovarian cancer cells (upper panel). WB of phospho-ACC levels in A2780, IOSE80ht and SKOV3 ovarian cells after 1 h of nutrient withdrawal (lower panel). E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.G. RPPA detection of phosphorylation of ACC after withdrawal of serum and glucose. E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.H. Effect of Rab25 down-regulation on AMPK (left panel) and ACC (right panel) phosphorylation. Total cellular protein, isolated from A2780pcDNA and Rab25 expressing cells 72 h post-transfection with either non-target (NT) RNAi or Rab25 specific RNAi, was separated by polyacrylamide gel electrophoresis (PAGE) followed by WB analysis. (a) p < 0.001 versus NT RNAi control and (b) p < 0.001, pcDNA versus Rab25.
gga-mir-12211 regulates the formation of PGCs by targeting LncBMP4 and BMP4.Figure 4A~B. Screening of miRNAs that target both BMP4-3'UTR and LncBMP4. (A) Two miRNAs (gga-mir-12211和gga-mir-12258) simultaneously target BMP4-3'UTR and LncBMP4. (B) Schematic diagram of base sequence and structure of gga-mir-12211 and gga-mir-12258. C. Morphological observation of the number of embryoid bodies in the PGC in vitro induction model after the overexpression and interference of gga-mir-12211. BMP4 induction was regarded as control. Scale bar: 60 µm (n = 3 independent experiments). D. The expression of reproductive marker genes (Cvh and C-kit), LncBMP4, and BMP4 were detected by qRT-PCR in the genital ridge after gga-mir-12211 overexpression and interference in vitro (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA). E. The efficiency of PGC differentiation was detected by IF, 4 d after induction, using C-KIT and CVH antibodies. Scale bar: 60 µm (n = 3 independent experiments). F. Detection of PGCs formation efficiency after the overexpression or interference of gga-mir-12211, in vitro, as assessed by flow cytometry using the CVH antibody (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA.).
Figure 6(A) Bur1 fused SV40-NLS does not rescue the rapamycin sensitivity of bur1-∆C compared to Bur1 without the NLS. bur1-∆C which expresses pRS416 (mock), pRS416 BUR1, pRS416 BUR1-mNG, pRS416 BUR1-mNG-NLS, pRS416 bur1-∆C, pRS416 bur1-∆C-mNG-NLS or pRS416 bur1-∆N, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0, 4, 8, or 16 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2 days. (B) Adding nuclear-export signal (NES) of PKI to Bur1 and/or deletion of the importin α-dependent NLS from Bur1 resulted in higher sensitivity to rapamycin compared to Bur1 without this NES. bur1-∆C which expresses pRS426 (mock), pRS426 BUR1, pRS426 bur1-∆N, pRS426 BUR1-mNG, pRS426 BUR1-mNG-NES, or pRS426 bur1-∆N-mNG-NES, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0, 4, 8, or 16 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2 days. (C) Model for the roles of Bur1, TORC1 and Sch9 for progression through G1 phase of the cell-cycle. Bur1 and TORC1 act in parallel to phosphorylate Sch9. We propose that similar to TORC1, Bur1 phosphorylates Sch9 on the vacuole membrane, or alternatively that Sch9 which is phosphorylated by TORC1 on the vacuole, translocates to the nucleus where it is further phosphorylated by Bur1. Then Sch9 acts in parallel with Cdc28/Cdk1-Cln3, in a process that results in the export of Whi5 from the nucleus and cell-cycle progression through early G1 phase.
Wde-mediated Egg recruitment to the chromatin leads to H3K9me3 deposition.Figure 5Rescue assays. Myc-Egg WT and Myc-SV40-NLS-Egg WT were expressed in OSCs upon endogenous Egg and Wde depletion (siEgg and siWde). Western blotting was performed using anti-Egg (left) and anti-Myc (right) antibodies. RT-qPCR quantified the expression level of Stalker2 transposon. The act5c gene was used for normalization. siEGFP: siRNA for EGFP was used as a negative control. Error bars represent SD from three independent experiments. *p < 0.05 (unpaired Student's t-test). Western blotting of Myc-SV40-NLS-Egg WT in whole (Whole), cytoplasm/nucleoplasmic (Cyto/Np) and chromatin (Chromatin) fractions before (-) and after Flag-Wde co-expression. Anti-Myc and anti-Flag antibodies were used. Histone H3 was detected as a marker of chromatin fraction. Immunofluorescence analysis of H3K9me3 (green), Myc-Egg WT (red), and Myc-SV40-NLS-Egg WT (red) in OSCs before and after Flag-Wde (yellow) co-expression. Anti-H3K9me3, anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm. Box plots indicating the H3K9me3 signal intensities per cell related to Figure 5B (p values based on unpaired Welch's t-test). Box plots show median (line) and 25th-75th percentile (box) ± 1.5 interquartile range (n > 25 cells for each box). Circles represent outliers.
Volumes of the binding pockets visualised by the van der Waals spheres of the ligand and coordinating residues of MhsT in complex with L-Phe, L-4-F-Phe, L-Tyr, L-Val, L-Leu and L-Ile.Figure 2Volumes of the binding pockets visualised by the van der Waals spheres of the ligand and coordinating residues of MhsT in complex with L-Phe, L-4-F-Phe, L-Tyr, L-Val, L-Leu and L-Ile.
Platelet specific deletion of MsrB2 leads to increased oxidative stress and intraplatelet apoptosis.Figure 3Levels of reactive oxygen species (ROS) quantified in the platelets using the fluorescent dye DCFH-DA. (ROS; *p=0.0139, MsrB2 fl/fl n=5, MsrB2 -/- n=6) C. Representative image and quantification of mitochondrial apoptosis, measured in freshly isolated platelets from the MsrB2 knockouts and age matched floxed mice. Results are expressed as % apoptotic cells. (TMRE; *p=0.0118, MsrB2 fl/fl n=5, MsrB2 -/- n=5) Representative images showing the intraplatelet expression of LC3 and active caspase-3 in CD41 stained platelets from the KO mice using fluorescent labelled antibodies against the respective proteins of interest. Magnification: 100X. Rightmost panel represents zoom in on an individual platelet showing the colocalization and expression of LC3 and active caspase-3. Graph indicates platelets positive for active caspase-3 signal. The y‐axis indicates percentage of caspase-3 positive cells from total cells calculated from n = 10 independent images per sample. (active caspase-3 signal ; **p=0.0003 Intracellular caspase-3 activity measured in freshly isolated platelets using the chromophore Ac-DEVD-pNA. Experiments performed in a minimum of n=6 samples per group. Values expressed as Mean ± SEM. (caspase-3 activity ; *p=0.0157) and H. Western blot analysis of Parkin, LC3I/II, MsrB2 and active caspasee3 in platelet after cell penetration peptide injection in chow and HFD mice. Control peptide (CP), LIF peptide (Foroud et al). Quantification analysis of individual band intensity. (Parkin; *p=0.02964, LC3II; *p=0.03200, MsrB2; **p=0.0059, Active caspase3; *p=0.0368 vs. chow or HFD group, each group n=3). Actin served as the loading control. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.
Redistribution of myosin II during nutrient starvation.Figure 7(A) The fat body of spaghetti‐squash (sqhAX3); sqh-GFP larva under fed or starved conditions were dissected and subjected to immunofluorescence analysis. Sqh-GFP and phospho‐myosin regulatory light chain (MRLC) were enriched in cell-cell junction under nutrient‐rich (fed) condition. Under starvation conditions, Sqh-GFP and phospho‐MRLC were localized to both cell-cell junction and perinuclear region. Actin was stained with TRITC‐labelled phalloidin (red) and nucleus was stained with DAPI (blue). Bar, 20 μm. (B) Starvation induced redistribution of myosin II in MCF7/GFP-LC3 cells. Both phospho‐MRLC and MRLC were localized in peripheral region of cells in nutrient‐rich medium (DMEM), whereas they redistributed to the perinuclear region and co‐localized with GFP-LC3 under starvation conditions (Earle's balanced salt solution; EBSS). Bar, 10 μm. (C) MCF7/GFP-LC3 cells cultured in nutrient‐rich (DMEM) or starved (EBSS) conditions were homogenized and subjected to centrifugation, and the resulting post‐nuclear supernatant (PNS) was fractionated by high‐speed centrifugation into membrane pellet and cytosol. Proteins were resolved by SDS-PAGE and immunoblotted with anti‐TGN46 antibody as a control for membrane‐association proteins, anti‐caspase‐3 as a control for cytosolic proteins. The levels of phospho‐MRLC and MRLC in each fraction were quantified using ImageJ and plotted relative to their amounts in PNS (n=3). Each value represents mean±s.e. of three experiments. *P0.05, **P0.01.
USP17 opposes the action of RNF26 and deubiquitylates p62.Figure 4A HeLa cells were transfected with negative control siRNA and RNF26 siRNA as well as constructs coding a non-targeting (NT) shRNA, USP17 shRNA1 or USP17 shRNA2, as indicated. 48 hours post transfection cells were stained for LAMP-1 (green) and DAPI (blue). Lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line. Scale bar represents 25μm. B The distribution of at least 400 LAMP1 positive vesiclesfrom a number of cells (n) from a series of confocal images across three separate experiments was plotted as vesicle relative position (mean value is red bar). Error bars represent standard error, ns indicate not significant, ** indicates a p-values less than 0.01, and **** indicates a p-values less than 0.0001. One way ANOVA used to determine if statistically significant differences between groups. C HeLa cells were transfected with expression constructs for HA-tagged p62 and FLAG-tagged ubiquitin as well as empty vector, USP17 or USP17CS. After 48hrs cell lysates were prepared and HA tagged proteins were pulled down using anti-HA agarose. Pull downs, and lysates, were immunoblotted with anti-HA, anti-FLAG, and anti-USP17 antibodies, as indicated, to confirm the presence of ubiquitinated p62.
Figure 3A Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A (red) and p-ATM (green). Scale bar=15 µm.B Higher magnification images showing representative OHC and IHCnuclei from control and CDDP-exposed cells. Scale bar=5 µm.C Histograms displaying green fluorescent signal intensity of x-projections of OHC and IHCnuclei from control and CDDP-exposed cells.D Quantification analysis of p-ATM foci numbers in both OHCs and IHCs exposed to either culture medium alone (light blue and red lines for OHCs and IHCs, respectively) or 10 µM CDDP (blue and red lines for OHCs and IHCs, respectively, n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (**P<0.008, ***P=0.00005, CDDP-exposed OHCsvs. CDDP-exposed IHCs).E Higher magnification confocal and 3D images showing the co-localization of p-ATM foci (green) with 53BP1 foci (red) in an OHCnucleus from the basal region of the organ of Corti culture treated with 10 µM CDDP for 1 day. Scale bar=5 µm. F Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A (red) and p-Chk2 (green). Scale bar=15 µm.G Histograms of green fluorescent signal intensity from OHC and IHCnuclei of a CDDP-treated organ of Corti explant.H Quantification of Chk2-positive nuclei in both IHCs (red bars) and OHCs (blue bars) from control and CDDP-treated organ of Corti cultures (n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (*P=0.045, ***P=0.0002, CDDP-exposed IHCsvs. control IHCs; CDDP-exposed OHCsvs. control OHCs). I Representative western blots using antibodies against p-p53 (serine 15), p53 and β-actin in control and 10 µM CDDP-exposed whole cochlear extracts.J-K Histograms representing the levels of total p53 protein (J) and p53 phosphorylation (K) in control (gray bars) and CDDP-exposed cochleae (green bars, n=6 cochleae per condition and per time point). Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (J: *P ≤ 0.023; K: *P=0.032, **P=0.007, ***P ≤ 0.0008, cochleae at the different times after CDDP exposure vs. control 12h).
Figure 2Biotinylated ssDNA oligonucleotides of indicated lengths were separately immobilised onto SPR CM5 chips via biotin-streptavidin interaction. Wild-type (WT) RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics. The dotted and solid curves show the normalised mean number of RAD51 bound to the ssDNA oligonucleotides, as measured by SPR (see Fig. 1 for equation) and the ODE model fits (see Fig. 3A for model description), respectively. As in A, except biotinylated dsDNA molecules of indicated lengths were used to measure association and dissociation kinetics. As in B, the dotted and solid curves show the mean number of RAD51 bound to the dsDNA oligonucleotides, as measured by SPR and the ODE model fits
Autophagosomes and amphisomes do form in ESCRT-depleted cells.Figure 5.Autophagosomes and amphisomes do form in ESCRT-depleted cells. HeLa cells stably expressing GFP-LC3 were transfected with control (A), Tsg101 (B, D, and E), or Vps24 (C) siRNA and processed for immuno-EM analysis. Cryosections were incubated with antibodies against GFP (15-nm gold) and LBPA (10-nm gold). Amphisomes (Am), characterized by their content of electron-dense LC3-positive areas and intraluminal vesicles, were observed in both control (A) and ESCRT-depleted cells (B and C). (D) In the Tsg101-depleted cells we found large clusters of more typical autophagosomes (Au) and (E) clusters of double-membrane structures, consisting of autophagosomes and tubular structures which might represent phagophores, all labeling strongly for GFP-LC3. We never observed similar clusters in control cells and very rarely in Vps24-depleted cells. Bars, 200 nm. (F) Quantification of amphisomes in control cells and cells depleted for Tsg101 or Vps24. Approximately 20 cells were included per siRNA depletion per experiment (n = 3). Error bars = SEM.
Fig.Figure 4BMDMs were co-loaded with 2 µM Calbryte590/AM (50 min) and 200 nM Lysotracker Green (LTG, 5 min) and, at room temperature, presented with 3-µm opsonized beads labelled with Alexa Fluor 647 (blue). A three-channel image was recorded every 10 s. The LTG brightness has been enhanced post hoc to visualize the dimmer, peripheral lysosomes. Time Zero is defined as the frame when the first Ca2+ signal was observed. (A) Images represent Ca2+ responses or an overlay of LTG and bead fluorescence. Times of each image are relative to the first Ca2+ spike. (B) Enlargement of the first contact point between lysosomes and bead (red arrow) as depicted by the dashed box, 80 s post-Ca2+. (C) Whole-cell Ca2+ responses from the cell in A. (D) Collation of the post-Ca2+ contact time, displayed as a scatter plot of individual cells and the mean ± S.E.M. (n = 27 cells). RAW246.7 cells were transfected with either TPC1-TagRFP-T or TPC2-TagRFP-T (red) and presented with 3-µm opsonized beads labelled with Alexa Fluor 647 (blue). Acute time-series of bead engagement were collected. Contact between TPCs and beads are depicted in the time-stamped micrographs, where time is arbitrary after the start of the image stack (E,F) i.e. 110 s is still prior to engagement. The spatial relationship between TPCs and beads was quantified using a target graticule of 0.5-µm bands centred on the engulfed bead; the mean fluorescence within each band was normalized to the fluorescence of the maximum band and plotted against this radial distance (G,H); n = 35-36 cells.
Figure 6(A-B'') Wing discs expressing the channelrhodopsin ChR2 were subjected to 0 minutes or 25 minutes of activating light in culture, then fixed and stained for Smo protein. In (B''), the yellow arrowhead indicates dorsoventral compartment boundary, and the white arrowhead indicates anteroposterior compartment boundary. (C) Quantification of the ratio of mean Smo fluorescence intensity in 25µm2 regions in the dorsal anterior compartment compared to the ventral posterior compartment. N=5 for 0min discs, n=9 for 25min discs, data were compared using an unpaired t test (***p<0.001), error bars are standard deviations. Identical analyses were carried out comparing dorsal posterior fluorescence to ventral posterior fluorescence (p=0.132), dorsal posterior fluorescence to dorsal anterior fluorescence (p=0.12), and ventral posterior fluorescence to ventral anterior fluorescence (p=0.36). (D-E') Discs expressing the red-light activated channelrhodopsin ReaChR under control of ap-Gal4 were raised on a 12hr light/dark cycle, dissected and stained for Smo protein (D, D') or Ptc protein (E, E').
Figure 2A,B. Representative pictures of ear sections from 10 week-old mice stained for δTcR (green); counterstained with Hoechst (blue) (A) and quantification of δTcR+ cells in the ear epidermis (B). Dotted lines indicate the epidermal-dermal border. Scale bar: 20μm. N=6 wt/wt mice, N=7 HPV8/wt mice, N=6 wt/Act mice and (N=8) HPV8/Act mice. C. Quantification of βTcR+ cells in ear skin cryosections from 10 week-old wt/wt (N=5); HPV8/wt (N=5); wt/Act (N=8) or HPV8/Act (N=8) mice. D-F. Percentage of viable CD4+ βTcR+ (D), CD8+ βTcR+ (E) or CD4+CD25+ (F) cells among all epidermal cells analyzed by flow cytometry. Results from 5 experiments with ears pooled from at least 3 mice per genotype are shown. Representative flow cytometry scatter plots are shown in Appendix Fig S1A-C. G. Representative pictures of ear cryosections from 10 week-old mice stained with antibodies against Foxp3 (red) and counterstained with Hoechst (blue). Insert shows higher magnification of the Foxp3-positive cells in wt/Act skin. Dotted lines indicate the epidermal-dermal border. Scale bar: 50μm. H. Kinetics of tumor incidence in HPV8/Act/wt (N=29), HPV8/Act/CD4KO (N=10), HPV8/wt/wt (N=23) and HPV8/wt/CD4KO (N=22) mice per genotype. Statistical significance was determined using the Log-rank (Mantel-Cox) test; ns = non-significant.
Figure 3A. Miro1 induces EYFP-Parkin translocation in the presence of PINK1 shRNA. PC6 cells were transfected with EYFP-Parkin and Miro1 or treated for 3h with A&O (both 10 µM) in the presence of scrambled shRNA or PINK1 shRNA. Miro1 induced Parkin translocation also in the presence of PINK1 shRNA while A&O treatment did not. ****P < 0.0001 compared with the respective control group, n = 5-6 dishes, 20 fields per dish, one-way ANOVA B. Miro1 induces EYFP-Parkin translocation in PINK1 -/- MEFs. MEFs obtained from PINK1 +/+ and -/- mice were transfected with EYFP-Parkin and Miro1 or treated with A&O (both 10 µM for 3h). Miro1 induced Parkin translocation in both MEF lines but A&O only in PINK1 +/+ MEFs. ****P < 0.0001 compared with respective control group, n = 6 dishes, one-way ANOVA C. Mutated EYFP-Parkin is recruited to mitochondria in response to Miro1 overexpression. PC6 cells were transfected with wild type, T240R or C431N EYFP-Parkin and Miro1 or treated for 3 hours with A&O (both 10 µM). Miro1 induced translocation of all EYFP-Parkin mutants whereas A&O treatment was not able to induce equivalent translocation of Parkin mutants. **P < 0.01 and ****P < 0.0001 compared with respective control group, n = 3-6 dishes, 20 fields per dish, one-way ANOVA D. Parkin mutants T240R and C431N interact with Miro1 both in DMSO- or A&O-treated (both 15 µM for 3h) HEK cells. Note that the higher molecular weight bands of Miro1 were observed only in WT Parkin-expressing groups.
Figure 1(A) Volcano plot representation of PARL interaction partners. Mitochondria isolated from PARL-/- FITR293Tcells expressing PARL-FLAG were subjected to co-immunoprecipitation (IP). Co-purifying proteins were separated by SDS-PAGE and identified by quantitative MS (n=3)(Dataset EV1). (B) Heat map of Log2 transformed LFQ intensities for three independent experiments. All significant (FDR < 0.05) proteins showing a positive ratio between PARLIP and control in (A) are shown. Grey color shows missing quantitative information. Clustering was performed using the complete method with Euclidean distance. (C) Complexome analysis of MEFmitochondria (n=3). Heat maps of the relative abundances of proteins significantly enriched in the eluate of PARLimmunoprecipitates at a FDR level of 0.05 (A) and identified by mass spectrometry after BN-PAGE analysis are shown after hierarchical clustering. (D) BN-PAGE analysis of mitochondria lacking SLP2, YME1L or PARL. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-13% blue native gradient gel and analyzed using SLP2-, YME1L- and PARL-specific antibodies. (E) High resolution complexome analysis of SPY complex members SLP2, YME1L and PARL using MEFmitochondria (n=3). Heat maps and migration profiles are shown after separation using a 3-9% BN-PAGE. SLP2, YME1L and PARL migrate in a high molecular weight complex (2 MDa), whereas SLP2 additionally is present in complexes of 1.6 MDa).