EMBO/sd-character-level-ner huggingface dataset:
Figure 6A Schematic representation of GABAAR α3-subunit with residues of the cytoplasmic loop that were synthesized and used as a biotinylated peptide for downstream applications. Gephyrin core-binding motif is shown in red. B Representative pulldown-experiment and quantification of 6His-tagged gephyrin and G375D using immobilized α3-peptide. 6His-tagged gephyrin variants were expressed in E. coli cells, purified to homogeneity and incubated with α3-peptides immobilized to NeutrAvidin beads. n = 3, normalized to 6His-gephyrin. 38.9±1.5% G375D were pulled down. C Representative SPR sensograms of 6His-gephyrin and 6His-G375D binding to immobilized GABAAR α3-peptide. Response units are plotted against time. Injected gephyrin concentrations were 0.5, 2, 10, 25, 40 µM. D SPR binding isotherms of a representative experiment as in C. Calculated KD values of 3.2 and 10 µM for gephyrin and G375D, respectively, are indicated by vertical lines. The experiment was repeated three times with two independently purified protein batches and two sensor chips. E Representative ITC binding isotherms of the cytoplasmic loop of the GlyR ß-subunit (GlyR-ßL, residue 378-426) and 6His-tagged gephyrin or G375D. The GlyR-ßL-gephyrin binding data (black) were fitted in a two-side model, whereas G375D binding could only be fitted to a single binding site. Dissociation constants KD and stoichiometry (N = number of binding sites) are given for each binding component. n = 5 from two independently purified protein batches. Results are expressed as mean ± SEM.
Figure 2Microscopy of Tau:polyA, Tau:PEG, and Tau:PEG:polyA condensates (5 µM Tau, 10% PEG, 4.8 µg/ml polyA) formed at increasing NaCl concentrations and in X/2-buffer mimicking the axoplasm ion composition. Scale bar=10 μm. trDLS of Tau condensation in X/2-buffer. data point bubble sizes correspond to DLS amplitudes. Condensate growth kinetics in X/2 buffer assessed through power law, R=a*tb, fitting to trDLS data starting at the time of LLPS inducer addition. Representative trDLS of Tau condensation in low salt buffer, with subsequent stepwise increase of NaCl. data point bubble sizes correspond to DLS amplitudes.
a–c) Clearance of A53T α-synuclein in stable PC12 cells as in b—treated for 24 h with either DMSO (control), or with 47 μM SMER10 and its analogs (SMER10a–c) (a), 43 μM SMER18 and its analogs (SMER18a–l) (b), or 47 μM SMER28 and its analogs (SMER28a–k) (c)—was analyzed by immunoblotting with anti-HA antibody (above) and densitometry analysis relative to actin (below).figf5(a-c) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1b-treated for 24 h with either DMSO (control), or with 47 μM SMER10 and its analogs (SMER10a-c) (a), 43 μM SMER18 and its analogs (SMER18a-l) (b), or 47 μM SMER28 and its analogs (SMER28a-k) (c)-was analyzed by immunoblotting with anti-HA antibody (above) and densitometry analysis relative to actin (below). All the analogs were used in the cell culture medium at 1:400 dilution of 5 mg ml−1 stock solution (in DMSO). Error bars denote s.e.m. P = 0.0058 (SMER10a), P = 0.6736 (SMER10b), P = 0.9507 (SMER10c), P = 0.0481 (SMER10) (a); P = 0.0006 (SMER18a), P = 0.0249 (SMER18b), P = 0.0167 (SMER18c), P = 0.0117 (SMER18d), P = 0.0269 (SMER18e), P = 0.0165 (SMER18f), P = 0.0148 (SMER18g), P = 0.0011 (SMER18h), P = 0.7369 (SMER18i), P = 0.0012 (SMER18j), P = 0.1531 (SMER18k), P = 0.0006 (SMER18l), P = 0.0001 (SMER18) (b); P = 0.0014 (SMER28a), P = 0.0002 (SMER28b), P = 0.0001 (SMER28d), P = 0.0048 (SMER28h), P = 0.0002 (SMER28i), P = 0.0162 (SMER28j), P 0.0001 (SMER28c, SMER28e-g, SMER28k, SMER28) (c). (d-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates in COS-7 cells as in Figure 1c-treated for 48 h with either DMSO (control), or with 47 μM SMER10 and its analog (SMER10a) (d), 43 μM SMER18 and its analogs (SMER18a, SMER18c-h, SMER18j, SMER18l) (e), or 47 μM SMER28 and its analogs (SMER28a-k) (f)-were expressed as odds ratios. All the analogs were used in the cell culture medium at 1:400 dilution of 5 mg ml−1 stock solution (in DMSO). Error bars: 95% confidence interval. P = 0.003 (SMER10a), P = 0.004 (SMER10) (d); P 0.0001 (SMER18a, SMER18c, SMER18d, SMER18f, SMER18h), P = 0.009 (SMER18e), P = 0.001 (SMER18g), P = 0.382 (SMER18j), P = 0.067 (SMER18l), P = 0.031 (SMER18) (e); P 0.0001 (SMER28a, SMER28c, SMER28e-g, SMER28i-k, SMER28), P = 0.574 (SMER28b), P = 0.041 (SMER28d), P = 0.002 (SMER28h) (f). ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.
Development of orthoxenograft mouse models of MPNSTs.Figure 1Five orthotopic xenograft mice models were established from four different MPNSTs and one cell line. Two of the MPNSTs come from the same NF1 patient; the other two were sporadic cases. Primary tumor (2-3 mm3) or tumor cell line (3 × 106 cells) were grafted or injected in the leg of athymic nude mice, close to the sciatic nerve. Tumors were perpetuated along several passages and subsequently expanded. Several assays were performed on tumors in early passages (histopathological analysis, gene expression profiling, genomic profiling, and drug efficacy studies). *NF1-MPNST-001 at passage 4 (P4) and all primary tumors and orthotopic tumors (OT) at passage 1 (P1) were analyzed by exome sequencing and immunohistochemistry. All primary tumors and orthotopic tumors at passages 1 and 4 were analyzed by expression array (except NF1-MPNST-001 PT, SP-MPNST-001 OT P4, and NF1-S462 OT P4). Copy number analysis (CNA) was performed in all samples.
The apical α-βH Spectrin cytoskeleton may be mechanosensory in the wing imaginal disc.Figure 4The apical α-βHSpectrincytoskeleton may be mechanosensory in the wing imaginal discA Schematic diagram of central compression and circumferencial stretching in the third instarwing pouch.B-D Third instarwing imaginal disc stained for beta-galactosidase expressed from the expanded.lacZ reporter gene (B), Crb (C) and Kst-YFP (D). Quantification of line intensity shown below. Note inverse correlation of Crb and Kst-YFP with ex.lacZ. Scale bars, 50 μm.E, F Zoom of Kst-YFP cells under compression (E) and under stretch (F). Scale bars, 10 μm.G Schematic diagram of the effect of force on the apical spectrin cytoskeleton leading to declustering of Crumbs complexes and reduced Hippo signalling.
Figure 1A Expression of PTPN11 (gene encoding SHP2) in skin lesions in psoriatic patients compared with skin from healthy donors based on microarray data (No. GSE14905). B Expression of ptpn11 in human PBMCs of psoriatic patients (n=14) and normal controls (n=16). Data information: Data are represented mean ± SEM. P values are determined by two-tailed Mann-Whitney U test *P<0.05, **P<0.01. C Representative SHP2 staining in skin sections of psoriatic patients (n=13) and normal controls (n=5). Scale bars: 200 μm. D Western blot analysis of PBMCs lysates derived from psoriatic patients and normal controls. E The catalytic activity of SHP2 was measured in human PBMCs lysates derived from psoriatic patients (n=25) and normal controls (n=25). Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01. F Representative p-ERK staining of skin sections of psoriatic patients and normal controls. Scale bars: 200 μm. G Quantitative PCR analysis of Ptpn11 mRNA levels in the IMQ-treated or non-treated dorsal back of C57BL/6 mice at day 5 (n=6/group). Data were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01. H Representative histological sections of IMQ-treated or non-treated dorsal back of C57BL/6 mice at day 5. Scale bar: 100 μm. I Quantitative PCR analysis of mRNA levels in human PBMCs derived from psoriatic patients (n=20) and normal healthy controls (n=20). Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.
Effects of swapping actin for different variants on cell viability and cytoskeletal organization.Figure 3A Actin expression levels shown by western blotting for strains expressing S. cerevisiae's actin or other actins, with tubulin (Tub1p) as a loading control. B Quantification of actin expression levels showing varying levels of expression that do not correlate with evolutionary relationship. Data are presented as mean +/- SD (n = 8 for ScNI, n = 4 for all other strains; 2 biological replicates with n/2 technical replicates each). *P<0.05 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). C Doubling times of yeast strain cultures grown at 25ºC in YPD medium. Data are presented as mean +/- SD (n = 3 for all conditions; technical replicates). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). D Growth constant as a function of percentage identity of the actin variant, showing clear correlation (n = 3 for all conditions; technical replicates). Data are presented as mean +/- SD. r corresponds to the Pearson correlation coefficient with a two-tailed p value and a confidence interval of 95% E Phalloidin staining of F-actin organization. Images are maximum intensity projections of 3D stacks and contrasts were adapted due to the fact that phalloidin labeling had a very different efficiency depending on the actin ortholog expressed. Micrographs of Sc and Sc_NI cells are reproduced from Fig 2E. Scale bar: 3 µm. F In vivo actin network deviation indexes. Data are presented as mean +/- SD (n = 30 for all conditions). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). G Polarity indexes. Data are presented as mean +/- SD (n = 30 for all conditions). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). H Growth constant as a function of the in vivo actin network deviation index. Data are presented as mean +/- SD (for growth constants, n = 3 for all conditions; technical replicates; for indexes, n = 30 for all conditions). r is a Pearson correlation coefficient considered non-significant if its two-tailed p-value is > 0.05. I Effect of CK-666 (75 µM) on the organization of the actin cytoskeleton. Cells were stained with phalloidin after 30 min incubation with CK-666. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm J Quantification of actin patch resistance to CK-666 treatment. Bar graphs represent the percentage of cells with a given number of visible actin patches after CK-666 treatment. (n = 41 for ScNI, 55 for N2, 31 for Ca, 41 for Op, 55 for Hs).
Figure 4Immunoblot for Myc on lung lysates from wiltype (WT) and Vegfr2Y1212F /Y1212F FVB mice tail-vein injected with PBS or VEGFA followed by circulation for time points indicated. Each lane represents one individual mouse. Quantification of Myc/Actin expressed as Fold change to PBS. Min, minutes of stimulation. Error bars: SD; 2-way ANOVA p=0.0058; unpaired T test, *p < 0.05, ** p <0.01. n=3 experiments. Myc protein expression. C. Myc and Actin immunoblotting on lysates of lung isolated ECs from untreated wildtype (WT) and mutant (VEGFR2 Y1212F) C57Bl/6 and FVB mice. D. Quantification of Myc/Actin expressed as Arbitrary unit. Each lane (C) and each dot (D) represent one individual mouse. 2-way ANOVA p <0.0001; unpaired T test, ** p <0.01. n=5-7. Baseline transcriptional level of Myc from lung isolated ECs from untreated wildtype (WT) and mutant (VEGFR2 Y1212F) C57Bl/6 and FVB mice. Each dot represents one individual mouse. 2-way ANOVA p=0.0041; Sidak's multiple comparison test, * p <0.05. n=4-5. Proliferation of C57Bl/6 and FVB iECs. CD31 (white), ERG (red), EdU (green) and double-positive cells (yellow) in C57Bl/6 (F) and FVB (G) iECs. EdU signal was masked with ERG signal. Proliferation of C57Bl/6 and FVB iECs. EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs. Each dot represents 2 fields/well, one well/mouse. Scale bar, 100 µm. Error bars: SD; 2-way ANOVA p =0.0843 (H), p=0.0474 (I); unpaired T test, *p < 0.05; **p < 0.01. C57Bl/6 n=3-6 mice, FVB n=3-5mice. Rescue of angiogenic sprouting by Myc overexpression in E11.5 C57Bl/6 Vegfr2Y1212F /Y1212F explant tissue. Representative images of E11.5 explants from C57Bl/6 embryos taken from wildtype (WT) and Vegfr2Y1212F /Y1212F littermates with (MycOE/Cre +) and without (MycOE/Cre-) endothelial-specific overexpression of Myc. Endothelial cell marker (CD31) immunostaining highlights the vasculature in white. Embryonic tissue blacked out to highlight sprouting area. Rescue of angiogenic sprouting by Myc overexpression in E11.5 C57Bl/6 Vegfr2Y1212F /Y1212F explant tissue. Quantification as fold-increase of vessel sprouting area compared to WT MycOE/Cre -. Scale bar, 250 µm. Error bars: SD; unpaired T test, ** p <0.01, **** p <0.0001. n=4-11 explants, one explant/embryo. Rescue of Ec proliferation. Myc overexpression in E11.5 C57Bl/6 Vegfr2Y1212F /Y1212F explant tissue. ERG (red), EdU (green) and double-positive cells (yellow; arrows). Rescue of Ec proliferation. Quantification of EdU/ERG double positive ECs normalized to total ECs. Scale bar, 50 µm. Error bars: SD; unpaired T test, *** p <0.001, **** p <0.0001. n=3-7 explants, one explant/embryo.
MOSPD2 is an ER-resident protein able to tether synthetic vesicles by binding the FFAT motif A: HeLa/GFP-MOSPD2 cells were labeled with anti-Calnexin antibodies (top; magenta) or with anti-VAP-A antibodies (bottom; magenta).Figure 4 A: HeLa/GFP-MOSPD2 cells were labeled with anti-Calnexin antibodies (top; magenta) or with anti-VAP-A antibodies (bottom; magenta) Data information: In (A and C), the subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay. Scale bars: 10 µ  B: Pearson correlation coefficients between GFP-MOSPD2 and Calnexin (left) or GFP-MOSPD2 RD/LD and Calnexin (right) staining are shown. Each dot represents a single cell (20 cells from three independent experiments). Means and error bars (SD) are shown. Mann-Whitney test  C: HeLa cells co-expressing GFP-MOSPD2 RD/LD (green) and mCherry-MOSPD2 (magenta). Data information: In (A and C), the subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay. Scale bars: 10 µm  D: Description of the liposome aggregation assay experimental strategy. LA liposomes are decorated with an FFAT peptide owing to covalent links with MPB-PE lipids, and mixed with LB liposomes covered by 6 His-tagged MOSPD2 MSP domain attached to DOGS-NTA-Ni2+  E: Aggregation assays in real time. LA liposomes (50µM total lipids) decorated with conventional FFAT (380nM) were mixed with LB liposomes (50µM total lipids) covered with the wild-type (Top; 760 nM) or the RD/LD mutant (bottom; 760 nM) MSP domain of MOSPD2. Aggregation was followed by dynamic light scattering (DLS). Left panels: mean radius (black dots) and polydispersity (shaded area) over time. Right panels: size distribution before (gray bars) and after (black bars) the reaction. Representative illustration of at least 3 independent experiments
Figure 3A, In MET-pos-NS, the METhigh subpopulation retains GSC properties, and generates a heterogeneous progeny including also METneg pseudodifferentiated cells. B, LDA (sphere forming) measuring the GSC frequency after IR (5 Gy) in METhigh and METneg subpopulations sorted from BT308NS. *: 2 test, P=0.001. C, Flow cytometry analysis of MET in irradiated BT308NS, 24 h after the last irradiation (2 Gy × 3 days or a single dose of 5 Gy). Dotted lines: threshold to define the percentage of MET-expressing cells. Ctrl: non-irradiated cells. The MFI was as follows: ctrl, 18; 2 Gy × 3, 22.77; 5 Gy, 19.49. D, Flow cytometry analysis of MET in BT308NS weekly treated with IR (2 Gy) for 6 weeks. Dotted lines: threshold to define the percentage of MET-expressing cells. Ctrl: non-irradiated cells. E, Representative immunofluorescence staining of MET (red) on tumors generated by subcutaneous injection of BT308NS, irradiated (2 Gy × 3) and explanted 72 h after the last irradiation. Nuclei are counterstained with DAPI (blue). Ctrl: non-irradiatedtumors. Scale bar, 10 µm (63× magnification). F left, Quantification of the percentage of MET-expressing cells in tumor sections represented in (E) (n = 12 HPF/group). HPF: high-power field. *: t-test, P=0.001. F right, Quantification of MET mean fluorescence intensity in tumor sections represented in (E) (n = 12 HPF/group, fold vs. non-irradiated cells, ctrl). *: t-test, P=0.00016. G, representative METimmunohistochemistry staining of matched primary and recurrent tumors. Scale bar, 50 µm (40× magnification). H, Histogram representing the number of patient displaying different percentages of MET expressing cells in their primary or recurrent tumors: negative, 0-5%; low, 6-29%; moderate, 30-69%; high, 70-100% (see also Appendix Table 4). I, Dot plot representing the MET cumulative score (MET positive cell score + MET staining intensity score) in 19 primary and recurrent tumors (see Appendix Table 4). Wilcoxon, p<0.02. Data are represented as mean ± SEM or ± CI in B.
Inhibition of mitochondrial translation and decrease in mtDNA copy number sensitize resistant cells to IMT1 treatment.Figure 7A Depleted CRISPR guides (negative hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value. B Viability assessment of monolayer cultures of IMT1-resistant RKO cells treated for one-week with either DMSO, IMT1 (1 μM), CAP (1 μg/ml) alone or in combination with IMT1. Data are expressed as mean values ± SD of n=4 independent experiments each including four technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. IMT1 vs IMT1+ CAP: *p=0,0301. C Representative images and related plot of IMT1-resistant RKO spheroid areas in presence of the aforementioned compounds for two weeks (IMT1: 1 μM, CAP: 1 μg/ml); Scale bar: 1mm. Data show the mean values ± SD of n=4 experiments. Statistical significance was calculated with paired t-test: IMT1 vs IMT1+ CAP: *p=0,0242. D Log2 fold changes in viable cell count in a panel of five IMT1-resistant cancer cell lines in presence of IMT1 + CAP (1 μg/ml). Data represent mean values of n=5 (resistant RKO cells) and n=3 (PANC1, Capan2, HCT-29 and Calu6) ±SD independent experiments. Paired t-test of IMT1 vs IMT1+CAP: resistant RKO cells **p=0,0014; PANC1, CAPAN2, HCT-29 and Calu-6: non-significant. E Western blot analyses of TFAM protein steady state levels in IMT1-resistant RKO cells in presence siRNAs against controls (control #1, #2) and TFAM (TFAM #1, TFAM #2) and treated with DMSO or IMT1. Images are representative of n=3 independent experiments. ACTINB is showed as loading control. F mtDNA levels in IMT1-treated resistant RKO cells in presence of siRNA against controls (control #1, #2) or TFAM (TFAM #1, TFAM #2). Data are expressed as relative to DMSO-treated resistant RKO cells treated with controls siRNAs (dotted line = 1) and show the average of n=2 independent experiments. G Viable cells counts of IMT1-resistant RKO cells treated for one-week with either DMSO or IMT1 in presence of controls (control #1, #2) or TFAM (TFAM #1, TFAM #2) siRNAs. Data are expressed as mean values ± SD of n=5 independent experiments each including six technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. Controls + IMT1 vs TFAM #1 + IMT1: p= 0,2148; Controls + IMT1 vs TFAM #2 + IMT1: *p=0,0415.
Alternative oxidase (AOX) expression and function in WT and ∆4-CYB cells.Figure 7(A) SDS-PAGE, Western blot and immunodetection of AOXHA expression in whole-cell lysates from WT and ∆4-CYB cells transduced with the AOXHA/pWPXLd-ires-HygroR lentiviral vector. The transduction and selection controls were the same cell lines transfected with an empty pWPXLd-ires-HygroR vector (EV). (B) Growth curves of the AOXHA expressing cell lines and their corresponding EV controls. Cell growth was monitored every six hours after substituting the medium in two replicate 24-well plates, one plate with medium without uridine (Uridine-), and the second plate with medium supplemented with 50 µg/ml uridine (Uridine+). The graphs show the average confluence ± SD at each time point (n=6 wells per cell line). (C) Immunodetection of cI structural subunits and NDUFAF2 in the same samples (D) 1D BNGE, Western blot and immunodetection analyses of digitonin-solubilized mitochondria from WT and ∆4-CYB cybrids expressing AOXHA and their corresponding EV controls. (E) Complex I in gel activity assays (IGA) after BNGE The gels were incubated in the IGA reaction mixture for 5 hours. The asterisk indicates the presence of a high molecular weight cI-containing band of unknown nature (F) Spectrophotometric kinetic measurements of cI activity in WT and ∆4-CYB AOXHA and EV samples, normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n=5 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test **p=0.0077 (WT AOXHA vs. ∆4-CYB AOXHA); **p=0.0044 (∆4-CYB EV vs. ∆4-CYB AOXHA); ***p=0.0003; ****p<0.0001. (G) High resolution respirometry analyses performed in intact cells in an Oroboros instrument. ROUTINE: cellular basal oxygen consumption rate (in pmol O2/sec) per million cells in DMEM medium. LEAK is the non-phosphorylating respiration in the presence of the ATP synthase inhibitor oligomycin. ETS: Maximal respiration rate in the presence of the uncoupler CCCP. AOX: oxygen consumption in the presence of antimycin A, inhibiting cIII2 activity but not that of AOX. ROTENONE: Oxygen consumption in the presence of the cI inhibitor rotenone. In all cases this was equal to the background. Results are expressed as mean ± SD (n=2 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001. (H) Respirometry analyses , in WT EV controls and AOXHA- expressing cells untreated or treated with 2.5 µM antimycin A for 7 days (+AA). Results are expressed as mean ± SD (n=4 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001. (I) 1D BNGE followed by cI-IGA (right) or Western blot and immunodetection of cIII2 subunit CYC1 (left) in digitonin-solubilized samples from WT EV controls and AOXHA- expressing cells untreated (-) or treated (+) with 2.5 µM antimycin A for 7 days.