EMBO/sd-character-level-ner huggingface dataset:
Figure 3A Alternative-codon constructs for pure and interrupted polyGln assays. B, C Cell death (B; LDH, n=8) and viability (C; MTT, n=6) assays in T98 cells 42-hrs post-transfection. NT: not transfected; EV: empty vector; n=independent experiments; * p < 0.05; unpaired t test; mean ± SEM. D qRT-PCR of Alt. polyGln and Alt. Int. polyGln construct transcript levels, n=3 independent experiments; p=0.9516, ns: not significant, unpaired t-test, mean ± SEM. Protein blots of polyGln proteins expressed in transfected HEK293T cells with interrupted or pure polyGln repeats; EV: empty vector; n=3 transfections quantification of polyGln proteins expressed in transfected HEK293T cells with interrupted or pure polyGln repeats; EV: empty vector; n=3 transfections, ** p<0.01, *** p<0.001, unpaired t test, mean ± SD. H Immunofluorescence of polyGln expressed from Pure 104 and Int.102 constructs in HEK293T cells; scale bar: 20μm; EV: empty vector. Immunofluorescence of intranuclear inclusions for polyGln expressed from Alt. polyGln and Alt. Int. polyGln constructs in HEK293T cells; scale bar: 50μm; green arrow heads indicate cells positive for Alt. Int. polyGln intranuclear inclusions; dashed white box in merge panels indicates the region show in the orthogonal (Ortho) zoomed images which show cross section and localization of diffuse polyGln staining and nuclear polyGln(Arg) inclusions with alpha tubulin as a cytoplasmic marker; n=4 experiments with n>55 cells per experiment quantification of intranuclear inclusions for polyGln expressed from Alt. polyGln and Alt. Int. polyGln constructs in HEK293T cells , * p<0.05, **** p<0.0001, two-way ANOVA, data presented as mean percentage of polyGln positive cells ± SD. K PolyGln(Arg) proteins but not pure polyGln proteins colocalize with nucleophosmin in HEK293T cells; scale bars: 20µm; dashed white box in merge panels indicates the region shown as an orthogonal projection in the ortho zoom panels which show the co-localization of polyGln inclusions and nucleophosmin in three dimensions.
Figure 2A Immunofluorescent staining of cGAS in both wild-type (WT) and G3BP1-/- U937 cells. Leica TCS SP8 Confocal Microscopy was used to acquire images. The white arrows indicate the cGAS-G3BP1 condensates. Data information: Representative images are shown Scale bars, 5 μm. Hoechst (blue), nuclear staining. B Immunofluorescent staining of cGAS in both WT and G3BP1-/- U937 cells. 3D images were reconstituted with Leica LAS X software. C Quantitative analysis of total cGAS puncta number (left) and volume (right) per cell of (B). n = 51 cells. D Data information: Representative images are shown Scale bars, 5 μm. Hoechst (blue), nuclear staining. Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type. D Immunoblotting of fractionated lysates from WT and G3BP1-/- U937 cells. GAPDH and Lamin B1 blots were used as controls for cytoplasmic and nuclear fractions, respectively. E qPCR analysis of IFNB mRNA levels in HT-DNA-treated WT and the indicated rescued cells (U937) (top). Immunoblotting analysis of indicated proteins (bottom). n = 3 biological replicates. Data information: Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type. IB, immunoblotting. HT-DNA, Herring testes DNA. F ELISA analysis of the secreted IFN-β in HT-DNA-treated WT or G3BP1-/- U937 cells. n = 3 biological replicates. Data information: Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type. HT-DNA, Herring testes DNA. G cGAMP production in ISD-treated WT or G3BP1-/- U937 cells were analyzed by LC-MS/MRM. ND, not detected. n = 3 technical replicates. Data information: Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type. ISD, interferon stimulatory DNA.
Interferon signaling in mice bearing heterozygous Stat1Y701F mutation resembles that of human cells with heterozygous Stat1Y701C mutation.Figure 1A Western blot analysis STAT expression and phosphorylation. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 250 IU/mL of IFNβ or 5 ng/mL of IFNγ for 0.5, 6, 12 and 24 h. Whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 (Y701) and STAT2 (Y689) and total level of STAT1 and STAT2. The blots are representative of more than 3 independent experiments. B Effect of STAT1Y701Fheterozygosity on the expression of type I IFN-induced genes (ISG). BMDMs of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 250 IU/mL of IFNβ for 4 and 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.C Effect of STAT1Y701Fheterozygosity on the expression of IFNγ-induced genes. BMDMs of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 5 ng/mL of IFNγ for 4 and 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.
The ER Morphologically Changes during the UPR.Figure 2The ER Morphologically Changes during the UPR(A) Control cells and UPR-induced cells were used to analyze and follow the ER within a single cell using EM. Boxes indicate the areas magnified in (B). Cells shown here correspond to the full section of the images labeled "+140 nm" in (B).(B) Serial section of control and UPR-induced cells. Sections are separated by 70 nm on the z-axis. ER is represented in magenta and NE in blue.(C) Electro micrographs from control and UPR-induced cells showing that the distance between ER membranes increases during the UPR. For a better preservation of the ultrastructure, samples for this experiment were prepared using high-pressure freezing/freeze substitution techniques (see Material and Methods).
Figure 1A, B. MDCK-SIAT1 cells co-incubated with Luc-WSN/33 and MH-S CM fractions. Replication was quantified by luminescence. (A) Replication curves display average luminescence reads from 5 wells per condition (measured at 7.5 min intervals - displayed here at 30 min intervals) from 1 experiment representative of 7 independent experiments. (B) Mean calculated area under the luminescence curves (AUC) for individual wells normalized to the corresponding mean of the control condition from these 7 experiments. C. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with flow cytometry-quantified MH-S AM-EVs at specified EV:cell ratios. D. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with CM fractions of J2 retrovirus-immortalized primary mouse AMs. E. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with primary rat AM-derived EVs. F. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with human THP-1 macrophage-derived EVs.
Figure 5(A) Epifluorescence microscopy images of supernatants from LLO-treated (0.5 nM 15 min) shCtlr, shGp96 or shNMHCIIA HeLa cells collected into poly-lysine-coated cover slips, fixed and stained with FITC-WGA (green) and DAPI (blue). Insets show PM blebs of variable sizes and DAPI staining confirms the absence of cell nuclei. Scale bar - 10 μm. (B) Quantification of released PM blebs (total FITCWGA/field). Values are the means ± SEM (n≥3). ***p<0.001. (C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (C) 0.5 nM LLO for the indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001. (C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (D) 1.5 or 3 nM LLO for 15 min; or (E) 0.1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001. (C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (E) 0.1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.
The N-terminal and linker intrinsically disordered domains and the C-terminal dimerization domain are essential for robust N LLPS in vitro.Figure 5A) DIC micrographs of 50 μM N and domain deletion variants in 50 mM Tris 70 mM NaCl pH 7.4 without and with TEV protease (to cleave MBP from N) and without or with 0.5 mg/mL desalted total torula yeast RNA. Scale bars represent 80 μm. Phase separation over time as monitored by turbidity of 50 μM full-length N or deletion variants after addition of TEV protease in the absence (B) or presence (C) of 0.5 mg/ml desalted total torula yeast RNA. Error bars represent standard deviation of three replicates.
Figure 1Statistical analysis of stx mutant stx34 sleep profile compared with that of the Canton-S control. 24-hour-sleep curve (y-axis was average sleep time in every 30 minutes), n=16. Statistical analysis of stx mutant stxd77 sleep profile compared with control w1118. 24-hour-sleep curve, n=16. Statistical analysis of stx mutant stx34 sleep profile compared with that of the Canton-S control. (B) total sleep (From left to right mean±SEM: 275.8±17.08, n=47; 358.6±23.47, n=39; 517.0±16.49, n=47; 519.7±15.89, n=39). (C) sleep bout duration (From left to right mean±SEM: 13.04±1.287, n=47; 18.01±2.212, n=39; 66.70±9.569, n=47; 59.41±6.909, n=39). (D) number of sleep bouts (From left to righ mean±SEM: 23.61.5±0.882, n=47; 25.95±1.353, n=39; 12.64±0.713, n=47; 12.44±0.888, n=39). (E) sleep latency (From left to right mean±SEM: 49.00±2.041, N=4; 33.09±5.100, N=4). Statistical analysis of stx mutant stxd77 sleep profile compared with control w1118. (G) total sleep (From left to right mean±SEM: 359.0±12.91, n=64; 444.5±10.98, n=74; 478.9±10.76, n=64; 458.1±13.98, n=74). (H) sleep bout duration (From left to right mean±SEM: 21.01±1.312, n=64; 37.63±2.988, n=74; 58.81±5.202, n=64; 61.0±6.555, n=74), (I) number of sleep bouts (From left to right mean±SEM: 20.23±0.627, n=64; 16.38±0.693, n=74; 11.66±0.605, n=64; 11.93±0.556, n=74). (J) sleep latency (From left to right mean±SEM: 52.20±5.496, N=7; 22.76±2.621, N=9). Sleep deprivation test of stx mutants. 12-hour mechanical night sleep deprivation was performed in stx34 and Canton-S control. (For K, from left to right mean±SEM: 34.53±9.85, n=53; 334.2±14.34, n=53; 378.8±14.20, n=53; 108.8±20.73, n=47; 361.1±16.71, n=47; 450.8±14.84, n=47; for L, from left to right mean±SEM: 45.60±13.43, n=53; 104.9±17.70, n=47). Increased sleep amount in the Recovery sleep comparing to the Baseline which was calculated by the subtraction of the day sleep amount after the deprivation (Recovery) by the day sleep amount of previous day (Baseline). Sleep deprivation test of stx mutants. 12-hour mechanical night sleep deprivation was performed in stxd77 and w1118 control. Sleep amount calculation of deprived night sleep (Deprivation), the day sleep amount of previous day (Baseline) and the day sleep amount after the deprivation (Recovery) (For M, from left to right mean±SEM: 35.06±10.76, n=50; 370.8±16.07, n=50; 434.5±17.42, n=50; 59.36±11.59, n=53; 420.3±15.60, n=53; 523.9±15.12, n=53; For N, from left to right mean±SEM: 61.86±13.35, n=50; 104.0±11.86, n=53) Increased sleep amount in the Recovery sleep comparing to the Baseline which was calculated by the subtraction of the day sleep amount after the deprivation (Recovery) by the day sleep amount of previous day (Baseline).
Caco-2 cysts collective invasion relies on the Rac-GEF FARP2.Figure 7A) siRNA screen performed in Caco-2 cysts treated with Y27632: Caco-2 cells transfected with control siRNA or pairs of siRNA targeting RhoGEFs were seeded in the modified 3D Boyden Chamber and grown as cysts for 3 days. Then, Caco-2 cysts were treated with Y27632 and the serum gradient was induced. After 2 days, the number of cells present on the bottom chamber were quantified by automated segmentation after Hoechst staining. The bar graph represents the mean invasion rate. Each condition was duplicated. The red dashed line represent the median invasion rate, the red full line indicates the 3 fold decrease in invasion rate. Three hits were identified: #2:ARHGEF18+RNEF, #7: SGEF+ARHGEF16 and #24: FARP1+FARP2 (Mean ±SD) . B) Caco-2 cysts stably expressing empty vector (pGIPZ) or 2 distinct shFARP1 or shFARP2 were treated with Y27632, allowed to invade for 2 days, fixed and stained for F-actin (Phalloidin). Boxed regions i, ii, iii and iv are shown at high magnification. Arrowheads point to non-protruding cells, arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. C) Bar graphs representing the percentage of protrusive cysts formed from Caco-2 cells transduced with lentiviruses encoding pGIPZ or shRNA against FARP1 and FARP2 after treatment with Y27632 from at least 3 independent experiments (Means ± SEM, paired t-test, ns: non significant, **p<0.01). D) Representative acquisition of Caco-2 cells overexpressing GFP-FARP2 grown in 2D and stained with ZO-1, in non-treated (NT) and after 2h with Y27632 or H1152. The dashed lines crossing the cell-cell junctions (i, ii and iii) are representative of the linescans measurements. Scale bars: 20μm. E) Representative fluorescence intensity of ZO-1 (red) staining and GFP-FARP2 (green) along the dashed line i, ii and iii represented corresponding to non-treated, Y27632 or H1152 treatments) F) Bar graph representing the average ratio of GFP-FARP2 over ZO-1 fluorescence intensities measured with linescans crossing cell-cell junctions. The quantification was performed on 71 cells expressing GFP-FARP2-FL from 3 independent experiments. For each cell, between 10 and 15 lines (12 pixels wide) were drawn across random region of cell-cell junctions positive for ZO-1 staining (Mean ±SEM Unpaired t-test **p<0.01, *p<0.05).
The FXR1 regulates nuclear morphology during G1 cell cycle phase.Figure 3A HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels. Arrowheads point to nuclear blebs observed in FXR1-deficient cells. B, HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by Western blot (B) C HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by immunofluorescence microscopy (C). The percentage of cells with irregular nuclei was quantified, 1000 cells were analysed (mean ±SD, **P < 0.01, ***P < 0.001; N = 3). HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Time from prophase till anaphase (D), from prophase till metaphase (E), from metaphase till anaphase (F) and from anaphase till chromatin decondensation (G) was quantified. HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. The selected frames of the movies are depicted and time is shown in minutes (H). Arrowheads point to nuclear blebs appearing during nuclear expansion of FXR1-deficient cells. HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Percentage of daughter cells with irregular nuclei was quantified in (I) HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. time from anaphase till nuclear blebs was quantified in (J).
Characterization of the human primary GBM, then analysis of targeted gene delivery.Figure 6Fluorescence-activated cell sorting (FACS) analysis with antibodies against Sox-2, Nestin and CD133 stem cell markers. Immunofluorescent staining for αv, β3 or β5 integrins in primary HSJD-GBM-001 and G26 GBM cells. Targeted transduction of HSJD-GBM-001 and G26 cells, over a time course, by RGD4C/AAVP-Luc. Non-targeted/AAVP-Luc was used as negative control. Results represent RLU/1μg protein of triplicate wells. Data shown are representative of two experiments, n=3. Scale bar, 100 μm.
Figure 5A. Schematic representation of Lrig1 mutants are shown on the left. Expression levels of these mutants were analysed in transfected cell extracts by immunoblotting with anti-Flag antibodies. B. Representative images of rathippocampal neurons transfected at DIV8 with empty vector, full length (FL) Flag-tagged Lrig1, or Lrig1 mutant lacking the LRR domain (LRR) in combination with an enhanced green fluorescent protein (GFP) expression vector. After transfection at 9 DIV, neurons were cultured in the absence or in the presence of BDNF (30 ng/ml) for 48 h. Then, hippocampal cultures at 11 DIV were fixed and stained with anti-Flag antibodies to control Lrig1 expression. Scale bar, 15 m. C, D. Quantification of the effects of Flag-Lrig1 constructs on BDNF-induced primary (C) and secondary (D) dendrite formation of hippocampal neurons treated as indicated in A. The results are shown as mean SEM of 3 independent experiments. *p<0.05, **p<0.01 by one-way ANOVA followed by Tukey´s multiple comparison test. NS, not significant. E. Representative confocal images of dendritic shafts containing spines from hippocampal neurons transfected at 15 DIV with either control vector or Flag-Lrig1 construct together with GFP. After transfection, neurons were cultured in the absence or in the presence of BDNF (30 ng/ml) for 48 h (15+3 DIV). Scale bar, 5 m.F. Quantification of the effect of Lrig1 overexpression on neurotrophin-induced spine density. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by one-way ANOVA followed by Student-Neuman Keuls multiple comparison test.
Figure 2(A) 293T cells were transfected with a vector encoding V5-tagged full length SPATA2, along with empty vector (EV) a vector encoding FLAG-tagged HOIP wild-type (wt) or a vector encoding a FLAG-tagged PUB domain mutant of HOIP (N102A). After FLAG-IP, the blot was probed with antibodies recognizing HOIP, CYLD, V5 and actin. (B) 293T cells were transfected with empty vector or a vector encoding FLAG-tagged HOIP along with either V5-tagged SPATA2 (wt) or a V5-tagged PIM mutant (PIMmut) of SPATA2. After V5-IP, the blot was probed with antibodies recognizing FLAG, CYLD, V5 and tubulin. (C) 293T cells were transfected with a vector encoding FLAG-tagged HOIP and increasing amounts of V5-tagged SPATA2. After FLAG-IP, the blot was sequentially probed with antibodies recognizing OTULIN, HOIP, V5 and tubulin. (D) 293T cells were transfected with empty vector, FLAG-SPATA2 or FLAG-HOIP. After FLAG-IP, the blot was probed with antibodies recognizing OTULIN, CYLD, FLAG and actin. (E) 293T cells were transfected with empty vector, V5-tagged SPATA2 (wt) or a V5-tagged PUB domain mutant PUBmut of SPATA2. After V5-IP, the blot was probed with antibodies recognizing CYLD, FLAG, V5 and tubulin. (F) A549 cells were treated with FLAG-TNF (2µg/ml) for the indicated time. The purified TNF-RSC was sequentially probed with antibodies recognizing SPATA2, CYLD, RIPK1, TRADD, SHARPIN, TNF and tubulin as indicated.
GemC1 is a central regulator of multiciliate cell differentiaton.Figure 7Model illustrating GemC1 as an early regulator of multiciliogenesis through the control of transcription factors McIdas and FoxJ1.Abbreviations: MCC: multiciliated cells.
Figure 1A Bacterially purified recombinant EDC3 (0.2μg) was incubated in the presence and absence of biotinylated recombinant Pim1 (0.5 μg) protein with Streptavidin-coated magnetic beads. The beads were precipitated and subjected to Western blotting with EDC3 and Pim1 antibodies (Abs). B Pim1, 2, or 3 and GFP-EDC3 expression vectors were transfected into HEK-293T. GFP-Trap Nano-body beads (GFP-Trap) were used to immunoprecipitate (IP) GFP-EDC3, and the associated proteins were analyzed by Western blotting with the indicated Abs. C Western blot of endogenous immunoprecipitation (IP) with EDC3 in MDA-MB-231 cells using Protein G Mag Sepharose with either control IgG or EDC3 primary antibody. D Pim1 kinase dead (KD) and GFP-EDC3 plasmids were co-transfected into HEK-293T cells. The lysates were precipitated with GFP-Trap beads. The IP products were subjected to Western blotting with the indicated Abs.
Figure 3(A) Representative images for the time course of PKCα nuclear translocation, subsequent nuclear envelope rupture, and DNA release in human dPMNs exposed to 50 nM PMA for 5, 15, 30, 60, and 180 min, then stained concomitantly for DNA (DAPI), nuclear lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), and PKCα (primary anti-human PKCα, and PE-labeled secondary antibody), following by confocal fluorescent microscopy analysis. White-empty arrows indicate cytoplasmic distribution of PKCα at 5, 15, and 30 min, yellow arrows indicate site of discontinuity/rupture of nuclear envelope at 60 min, light blue arrows display the sites of nuclear envelope rupture and chromatin release at 180 min (A). Scale bars, 20 μm. The time course started 5 min after PMA stimulation in order to allow the adherence of neutrophils on the bottom of dish for immunocytostaining. (B) Summary analysis of the nuclear envelope continuity was analyzed based on staining of the nuclear envelope with primary anti-lamin B, and FITC-labeled secondary antibody. (C Representative immunoblots of total PKCα and p-PKCα in primary human pPMNs that were treated by PMA (C) for 0, 0.5, 1, 2, 3 h. D) Representative immunoblots of total PKCα and p-PKCα in primary human pPMNs that were treated by PAF (D) for 0, 0.5, 1, 2, 3 h.