EMBO/sd-character-level-ner huggingface dataset:
Figure 7 (A) HeLa-GFP-Parkin cells expressing mCherry-NDP52 D439R/C443K mutant or WT NDP52 were incubated with valinomycin and nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of two independent experiments  (B) Selected frames from time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 D439R/C443K mutant under valinomycin and bafilomycin A1 treatment. The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 9. Cyan, GFP-Parkin; magenta, mCherry-NDP52 D439R/C443K. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structures. Scale bars, 1 µm  (C) Schematic model of damaged mitochondria recognition systems by autophagic membrane via NDP52-MTPAP interaction. On depolarized mitochondria, Parkin is activated and generates polyubiquitin chains on the OMM. The accumulation of polyubiquitin chains on the OMM induces the recruitment of mitophagy receptors and OMM rupture by proteasomes. NDP52 irrupts into mitochondria and interacts with MTPAP OMM rupture-dependently. The autophagic membrane recognizes damaged mitochondria by (i) poly-ubiquitin bound mitophagy rerceptors on the OMM (p62-like receptor mediated system), (ii) PHB2 on the IMM (Prohibitin-2 mediated system) and (iii) the NDP52-MTPAP complex (NDP52-MTPAP mediated system) via affinity with Atg8s (LC3 and GABARAP family)
Figure 5(A) Fork velocity as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). More than 200 tracks were considered except for RAD1 +RNH1 (n=125), ATR +RNH1 (n=182) and RAD18 +RNH1 (n=146). Median values are indicated. *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases. (B) Fork asymmetry as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). From 40 to 200 total measurements were considered for each candidate. Median values are indicated. *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.
Schematic model of results Lack of bisecting GlcNAc relocates BACE1 from early endosomes (Aβ generation site) to lysosomes, leading to a reduction in both Aβ generation and the level of BACE1 protein.Figure 8Lack of bisecting GlcNAc relocates BACE1 from early endosomes (Aβ generation site) to lysosomes, leading to a reduction in both Aβ generation and the level of BACE1 protein.
Depletion of NAV3 accelerates migration and invasion and alters 3D morphology of mammary cells.Figure 2MCF10A cells, pre-transfected with the indicated siRNAs, were assayed for migration or invasion in the absence or presence of EGF. Cells that reached the filter's bottom were stained and photographed. Scale bars, 150 μm (migration) and 100 μm (invasion).The results from (A) are depicted as means ± SD (four experiments). Phase microscopy analysis of shCTRL and shNAV3 MCF10A cells (scale bars, 50 μm), which were plated in Matrigel and grown for 8 or 12 days in the presence of EGF (20 ng/ml). Acini were photographed and quantified for their volumes using ImageJ. The right panel shows analysis of acinus size after 8 days in culture (data represent three different experiments, and P-value was calculated using t-test). shCTRL or shNAV3 acini (day 10) were immunostained for activated caspase-3 (red) and DAPI (scale bar, 20 μm). Clusters positive for activated caspase-3 were counted and quantified in three experiments (means ± SD).
Figure 5Figure 5. Nucleoid disorganization in patient fibroblasts leading to a defect in mtDNA repair under conditions of oxidative stress. A. Total extracts from control (C) fibroblasts and intact isolated mitochondria from control and patient fibroblasts (P1, P2) were analyzed by immunoblotting using antibodies against PCNA (nuclear protein), GAPDH (cytosolic protein) or SMAC (mitochondrial intermembrane space protein) (upper panel). Mitochondria from patient and control fibroblasts were incubated with NP-40 and separated into pellets (P) and supernatants (S). The fractions of each extraction were subjected to western blot analysis. VDAC and SMAC were used to identify behaviors of well defined mitochondrial proteins that are integral membrane and soluble proteins, respectively (middle panel). Ratio of mtDNA amplified from supernatant/mtDNA amplified from pellet, by qPCR, was quantified in control and patient fibroblasts (lower panel). B. Determination of the mtDNA copy number after H2O2 treatment. mtDNA/nDNA values in control, patient 1 and patient 2 fibroblasts. mtDNA : mitochondrial DNA, nDNA: nuclear DNA. C. mtDNA repair after H2O2 treatment. A long-range PCR was used to evaluate the oxidative damage, induced by H2O2 treatment, in mtDNA. The relative PCR amplification of a 15.6kb mtDNA fragment was normalized to relative PCR copy number that was evaluated by PCR amplification of a 172bp mtDNA fragment. mtDNA repair activity in control, patient 1 and patient 2 relative PCR. B-C. Cells were exposed to 150µM H2O2 for 30 min and either harvested immediately or allowed to recover in conditioned medium for the indicated times. Untreated control cultures were incubated in serum-free medium alone. Results represent the mean of relative PCR amplification ± SD of three independent experiments in which three PCRs per point were performed. Values were normalized to untreated cells and differences were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01), very significant (**:0.01>p>0.001). Patient 1 versus control: *: p=0.015 (recovery 1h), *: p=0.022 (recovery 2h), **: p=0.003 (recovery 4h). Patient 2 versus control: *: p=0.0230 (recovery 2h), *: p=0.041 (recovery 4h). NT: cells not treated with H2O2
Figure 5The high-resolution structure of MC complex in different orientations. TssJ is in orange, TssM is in green and blue according to their position as an inner or outer pillar respectively. Pseudoatomic model of the N-terminus of TssM.i (green), showing it is composed of 8 helices, coloured in different shades of red and numbered from the C-terminus (1) to the N-terminus (8). The pore radius formed by the inner pillar of TssM protomers is highlighted as dots and mapped using HOLE. The atomic model of TssM is coloured according to secondary structure (Helices in pink and strands in brown). The graph shows the pore radius calculated along the centre of the inner pillar of TssMs. Ribbon diagram of the region surrounding the periplasmic gate, with the amino acids involved in the formation of the gate in atom form. Gln779 and Asn780-781 from the inner pillars of TssM (green) are labelled in one of the protomers. Fluorescence of prey cells (green) indicating their survival against attacker cells after 4h. The image of a representative bacterial competition spot is shown on the upper part. The relative fluorescent level (in AU) and the number of recovered E. coli recipient cells (in log10 of colony forming units (cfu)) are indicated in the lower graphs. The assays were performed from at least three independent cultures, with technical triplicates and a representative technical triplicate is shown. Error bars in the left graph represent ± standard deviation (SD) from technical triplicate. The circles in the right graph indicate values from technical triplicate, and the average is indicated by the bar. Fluorescence microscopy recordings showing sfGFPTssM foci in the parental (WT) and TssM mutated strains (TssM Q779C/N780C, TssM ∆777-783). TssM foci containing cells are indicated by arrowheads. Microscopy analyses were performed independently three times, each in technical triplicate, and a representative experiment is shown. Scale bars, 1 µm. Fluorescence microscopy recordings showing TssBsfGFP sheath in the parental (WT) and TssM mutated strains (TssM Q779C/N780C, TssM ∆777-783). Fluorescent sheath containing cells are indicated by arrowheads. Microscopy analyses were performed independently three times, each in technical triplicate, and a representative experiment is shown. Scale bars, 1 µm.
Figure 2(A, B) CP20, OVCAR4, and OVSAHO cells were transfected with non-target micro RNA control (miR-CTL) or miR-195, 24 h post-transfection, cells were recounted and plated as single cells for anchorage-dependent (A) or anchorage-independent (B) clonal growth. After 10 (CP20) or 14 (OVCAR4 and OVSAHO) days, colonies were quantified using an Optronix GelCount colony counter. The left panel shows representative images of the colonies and the right panel depicts a graphical representation of data presented as percent clonal growth relative to the control (miR-CTL). (C) Migration of miR-CTL, miR-195, and miR-195+ MICU1 (pLYS1-MICU1-Flag) transfected cells, towards serum gradient was examined and the number of cells per field was counted. The left panel shows representative images and the right panel depicts a graphical representation of relative migration compared to the control (miR-CTL). (D) Invasion of miR-CTL, miR-195, and miR-195 + MICU1 cells through fibronectin-coated filters was examined using Boyden chamber and number of cells per field was counted. Left panel shows representative images and the right panel depicts a graphical representation of relative invasion compared to the control (miR-CTL).
Figure 7(A) The SNF1-Mig1 axis induces NADPH upon HU treatment. The relative ratios of cellular NADPH/NADP before or after HU treatment were measured as described in Methods and Materials. Error bars represent SD from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01< P-values < 0.05; **0.001 < P-values < 0.01). (B) The effects of α-KG on the HU sensitivity of snf1Δ and other checkpoint mutants. Yeast spot assays were performed as in Fig 2A. (C) α-KG restores the levels of Rfa1-GFP foci in snf1Δ. Rfa1-GFP foci were measured as in Fig 3. At least 200 cells were analyzed for each experiment. Error bars represent SD from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05). (D) α-KG restores Rad53 phosphorylation in snf1Δ. CBB indicates coomassie bright blue (CBB) staining of the membrane as the loading control. (E) The effects of α-KG on the HU sensitivity of Rfa1 zinc finger mutants (C486S, C491S, C505S, C508S). Yeast spot assays were performed as in Fig 2A. (F) The proposed working model. In brief, we uncover that sugar metabolism is rewired to protect genome stability by means of a redox mechanism. Mechanistically, an SNF1-Mig1-ZWF1-NADPH axis stimulates the ssDNA-binding of RPA, thereby promoting a fast activation mode of Mec1-Rad53 kinase cascade to fight against replication threats.
Fig.Figure 6(A) Scheme representing the study design. FIX deficient mice (FIX -/-, n=5/group) were administered with factor IX (FIX, 40 µg/mouse) in the pre-immunization phase and with AAV vectors (2x1011 vg/mouse) in the treatment phase. Control mice were injected with PBS. Red symbols represent timing of blood collection. (B) Table reporting the different mice groups of the study. (C) Measurement of anti-hFIX mouse IgG in plasma samples collected from mice injected with FIX or PBS (n=5/group) at different time points. Data represent mean±SEM. (D) Representative western blot analyses on plasma samples (n=4) collected from animals 8 weeks post-AAV injection. (E) Antithrombin activity in plasma samples collected from mice at different time points. Data are presented as mean±SD (n=3-4 per datapoint). Data were analyzed in a 2-way ANOVA with Tukey's correction for multiple comparisons. ***p<0.01 ****p<0.001 (2-way ANOVA with Tukey's multiple comparison test) (F) Graph reporting the volume of blood loss (µL) over 30 minutes following the tail clip. WT: wild-type littermates of F9-/- mice. The grey area represents the range of blood loss of F9-/- mice that received AAV-vectors expressing human FIX. Data are presented as mean±SD (n=3-4/group), and were analyzed in a 1-way ANOVA with Tukey's correction for multiple comparisons. **p<0.01 Boxes represent 25%-75% quartiles, with the line indicating the median. Whiskers span min to max. G Measurement of anti-KB-AT-23 mouse IgG in plasma samples collected from mice (n=5/group) 8 weeks post-AAV administration. Data represent mean±SD.
Figure 2(A) Fear conditioning test from Nedd4-2 WT (n = 8) or Nedd4-2 cKO (n = 8) mice (6-8 weeks old). Schematic representation of fear conditioning test (left). Mice were placed in the fear conditioning chamber for 180 s with a metal grid floor and received two electrical shocks at 120 and 150 s (0.5 mV, 2s). Twenty-four hrs later, mice were placed in the same chamber for 180 s in the absence of the shock. Freezing behavior during contextual fear conditioning test at 24 hrs after training is shown (right). Freezing rate from Nedd4-2 WT (n = 3) and Nedd4-2 cKO (n = 3) mice without shocks during the training (unshock) was also shown (right). (B) Barnes maze test from Nedd4-2 WT (n = 8) or Nedd4-2 cKO (n = 8) mice (6-8 weeks old). Schematic representation of Barnes maze (top left). Mice were trained for 4 days with three training trials on each day to locate the escape box (black circle). Learning curve during 4 training days was shown by the escape latency and the number of errors before escaping (top right). Schematic representation of Barnes maze on probe trials (bottom left). Quadrant occupancy on probe trial at day5 and at day12 (bottom right) were assessed by the time spent in the target area (green quadrant). (C) Novel object recognition test from Nedd4-2 WT (n = 8) or Nedd4-2 cKO (n = 8) mice (6-8 weeks old). Schematic representation of the test (left). Mice explored the chamber with two identical objects for 10 min during the training day. Twenty-four hrs later, mice returned to the chamber with one of the objects replaced by a novel object. Exploration time of familiar and novel objects on testing session (middle) and the preference index for the novel object (right) were measured. The dash line represents the 50% chance of exploring either of the objects. (D) Open field test from Nedd4-2 WT (n = 7) and Nedd4-2 cKO (n = 8) mice. Representative image showing the explorative behavior of Nedd4-2 WT and Nedd4-2 cKO mice during 5 min open field test period (left). Quantification of locomotor activity and anxiety-like behavior are shown by average velocity, total distance traveled, immobile time, time spend in center zone, number of entries in center zone, and defecation number (right).
Figure 3Repression of lnc-MARCKS using CRISPRi increased MARCKS expression and decreased IL-6 induction. (A) Illustration of CRISPRi system and the relative locations of the two sgRNAs. lnc-MARCKS (B) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs. , MARCKS (C) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs. IL-6 (D) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs. (E) The lnc-MARCKS gene locus. The full-length sequence of lnc-MARCKS was obtained by rapid amplification of cDNA ends (RACE). lnc-MARCKS is located on human chromosome 6 and encompasses 2 exons. Overexpression of lnc-MARCKS decreases MARCKS expression RT-qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector (E.V.) or lnc-MARCKS, treated with PMA overnight, and left unstimulated or stimulated with Pam3 for 8 h. Lnc-MARCKS and MARCKS expression (F) Overexpression of lnc-MARCKS enhances IL-6 expression. RT-qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector (E.V.) or lnc-MARCKS, treated with PMA overnight, and left unstimulated or stimulated with Pam3 for 8 h. IL-6 expression (G). lnc-MARCKS regulates MARCKS in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect lnc-MARCKS (H) lnc-MARCKS regulates MARCKS in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect MARCKS (I), lnc-MARCKS regulates IL-6 in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect IL-6 expressions (J).
CCCP-induced parkin-mitochondrial aggregate formation is an intermediate step for mitophagy.Figure 2.CCCP-induced parkin-mitochondrial aggregate formation is an intermediate step for mitophagy. (A) MEFs expressing WT GFP-parkin were treated with CCCP or CCCP and nocodazole (NOC, 10 µM) for 8, 16, and 24 h as indicated. Cells were immunostained with anti-Tom20 to visualize mitochondria. GFP-parkin-positive cells are marked by dotted lines. Arrows indicate parkin-positive mitochondria or mitochondrial aggregates. Bar, 25 µm. (B) The average percentages of cells with mitochondrial aggregates or without mitochondria from three independent experiments from A are presented with standard deviation as error bar. **, P < 0.01.
Figure 8A) An illustration of the quantitative framework constructed to relate expected log2(H/M) measurements to the known half-life of a particular protein. For the sake of simplicity, the natural logarithm (e) is used here but values were ultimately converted to base 2 (which is the base used in the half-life terminology). B) Dependence of expected log2(H/M) values at t=24h on the proteins half-life, given the factor (α) by which its catabolism is inhibited. Note that beyond α=10, the curves are nearly identical. C) Expected log2(H/M) values over the course of 24 hours for proteins with half-lives ranging from 1 to 10 days. Compare with the actual measurements shown in Fig. 7 A-C.
Figure 6(A) Representative western blots of whole cell lysates from MCF7 cells treated with palbociclib (1 μM) for 1, 4 or 7 days, or treated identically, and then washed out for the indicated times (see Figure 1C). (B) Analysis of adjusted relative density from 3 independent western blot experiments. Bars display mean values -/+ SD. Significance determined by unpaired Student's T test comparing treated target protein to asynchronous target control. (* < 0.01, ** < 0.001, *** < 0.0001). (C,D) Quantification of the nuclear morphologies (C) or γH2AX-positive DNA damage foci (D) from MCF7, MCF7 p53 KO or T47D cells that were treated with palbociclib (1μM) for 0, 1, 4, or 7 days and then analysed 48hr after drug washout. Either 100 cells (nuclear morphology) or 50 cells (γH2AX) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments. (E) Cumulative mitotic entry of cells following washout from 0, 1, 4 or 7 days treated of palbociclib (1μM). 50 cells were quantified per experiments and graphs display mean -/+ SEM from 3 experiments. (F) Quantification of colony forming assays of MCF7, MCF7 p53 KO and T47D cells treated with CDK4/6 inhibitor for 0, 1, 4 or 7 days and then grown at a low density without palbociclib for 14-21 days. Bar graphs displays mean data + SEM from 4-5 experiments.
A Schematic summary of the findings.Figure 7Under normal conditions, ppMLC promotes the recruitment of LUZP1 to TJ-associated CRs, where LUZP1 inhibits myosin phosphatase in a MT-facilitated manner to up-regulate ppMLC levels. Without LUZP1, myosin phosphatase actively dephosphorylates ppMLC within CRs, leading to apical constriction defects. MT depolymerization causes attenuated LUZP1-mediated inhibition of myosin phosphatase, allowing myosin phosphatase to dephosphorylate ppMLC.
Figure 4A 3D images of OHCnuclei taken from the basal regions of organ of Corti cultures treated with medium alone, 10 µM KU55933, 10 µM CDDP or 10 µM CDDP in combination with 10 µM KU55933 for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar=5 µm.B Quantification analysis of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions (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 ≤ 0.0004, CDDPvs. control, or CDDPvs. CDDP+KU55933). C Confocal images showing the basal region of the organ of Corti cultures treated with medium alone, 10 µM KU55933, 10 µM CDDP or 10 µM CDDP in combination with 10 µM KU55933 for 5 days and immunolabeled for Myosin 7A (red). Scale bar=24 µm.D Histograms representing the numbers of surviving IHCs (red bars) and OHCs (blue bars) for all treatment conditions after 5 days (n=5 cochleae per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (*P=0.03, **P =0.004, ***P ≤ 0.0008, CDDPvs. control, or CDDPvs. CDDP+KU55933). E 3D images of OHCnuclei from the basal regions of organ of Corti cultures treated with culture medium alone, 100 µM PFT-α, 10 µM CDDP or 10 µM CDDP in combination with 100 µM PFT-α for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar=5 µm.F Quantification of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions (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 ≤ 0.0007, CDDPvs. control).
Figure 2(A) FU-induced reductions in ER-mitochondria associations can be detected using SIM. NSC34 cells were transfected with either EGFP control vector, EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and immunostained for TOM20 and PDI to label mitochondria (Mito) and ER respectively; FUS were detected via their EGFP tags. Merge (ZOOM) show zoomed images of boxed regions and co-localisation shows co-localised pixels. Scale bar=2 m. Bar chart shows ER-mitochondria co-localisation (Manders coefficient) normalized to control in the different samples. Data were analysed by one-way ANOVA with Tukey's post-hoc test. 10-14 cells were analysed per condition from 3 independent experiments; error bars are s.e.m., **p<0.01 and ***p<0.001. (B) ER-mitochondria associations and the VAPB-PTPIP51 interaction are disrupted by wild-type and ALS/FTD mutant FUS. NSC34 cells were transfected with EGFP control vector (CTRL), EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and proximity ligation assays performed using VAPB and PTPIP51 antibodies. FUS were detected via their EGFP tags. Scale bar=10 m. Bar chart shows relative number of proximity ligation assay signals/cell. Data were analysed by one-way ANOVA and Tukey's post-hoc test; n=47-53 cells from 5 experiments. Error bars are s.e.m.; ***p<0.001. (C) overexpression of FUS reduces the binding of VAPB to PTPIP51 in transfected cells. Cells were transfected as indicated with either control empty vector (CTRL), HA-PTPIP51+CTRL, or HA-PTPIP51+ either EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K. PTPIP51 was immunoprecipitated using the HA tag and the amounts of endogenous bound VAPB detected by immunoblotting. Both inputs and immunoprecipitations (IP) are shown and no immunoprecipitating VAPB signals were obtained in the absence of HA-PTPIP51. Bar chart shows relative levels of VAPB bound to PTPIP51 in the immunoprecipitations following quantification of signals from immunoblots. VAPB signals were normalized to immunoprecipitated PTPIP51-HA signals.