EMBO/sd-character-level-ner huggingface dataset:
Figure 4Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (A) Vacuolar morphology of Col-0 (n=64) and fer-4 (n=60). Mann Whitney U test (***p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values. Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. (B) 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green). Boxplot depicts vacuolar occupancy of Col-0 control (n=12) and fer-4 (n=10) mutant cells. Mann Whitney U test (**p < 0.01). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values. Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (C) Col-0 (n=52) and fer-4 (n=44) seedlings were treated with solvent DMSO or 50 µM EGCG for 22 h on solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values. Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (D) Col-0 (n=28) and fer-4 (n=28) mutant seedling roots were grown on the surface or into the matrix of 2 % agar-containing solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.
GFP–Atg8F77/79A double mutant shows a defect in the autophagic process.Figure 2(A) Cells were grown to mid‐exponential phase and then incubated in the presence of 3.5% galactose to induce expression of GFP-Atg8. Aminopeptidase I (API) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti‐Atg8 (right bottom panel), anti‐GFP (left bottom panel) or anti‐API antibodies (upper panel). (B) Cells were pulse-labelled with [35S]methionine and chased on non-radioactive starvation medium. Aliquots were taken at the indicated times and acid-soluble small peptides generated by proteolysis were determined. (C) Cells were grown in control or in starvation medium and visualized by light microscopy. Arrows point to the pre‐autophagosomal structure (PAS), and arrowheads point to GFP within the vacuoles. (D) Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblot analysis using anti‐GFP antibodies. GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.
ANO8 is required for maximal STIM1-Orai1 interaction and increases current inactivation under strong Ca2+ buffering.Figure 3(A, B) Knockdown of ANO8 (siA8) reduced CRAC current in cells transfected with Orai1 (O1) and STIM1 (S1) and buffered with 3 mM EGTA. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test. (C) Knockdown of ANO8 reduces the native store-dependent Ca2+ influx measured in store depleted cells by Ca2+ add-back. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test. (D,E) Knockdown of ANO8 reduced the number of store-dependent STIM1 puncta at the TIRF plane in cells expressing STIM1 and Orai1. Panel (D) shows representative images and (E) is the summary of 7 experiments. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test. (F, G) Current was measured with pipette solution contacting the fast and strong Ca2+ buffer 10 mM BAPTA in HEK cells transfected with STIM1, Orai1, and with (red) or without ANO8 (black). Panel (G) shows the increase in current density at peak current. Note the prominent current inactivation in the presence of ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test. (H) Knockdown of SARAF (red) in wild-type cells had no effect on current inactivation in the presence of 10 mM BAPTA. (I) Knockdown of SARAF did not prevent the ANO8-dependent current inactivation in the presence of 10 mM BAPTA. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.
Figure 5A-E Confocal single plane images of third instar larval wing discs stained with (A) anti-LamC (green) and anti-Zfh1 (red), (B) edl[k12907]-lacZ (green) and anti-Zfh1 (red), (C) anti-H (green) and anti-Zfh1 (red), (D) Cg25C[k00405]-lacZ (green) and anti-Zfh1 (red), or (E) vgk[G00454]-GFP (green) and anti-Ct (red).
Cholesterol levels affect SNARE trafficking.Figure 7(A) MSD, MPS‐IIIA and WT MEFs were subjected to a triple labelling with anti‐VAMP7, anti‐Vti1b and anti‐LAMP1 antibodies. The merges between VAMP7 and Vti1b (double merges in yellow) and between VAMP7, Vti1b and LAMP1 (triple merges in white) are shown (see also enlarged images showing the extent of co‐localization in different regions of the cells). The VAMP7-Vti1b co‐localization was quantified in 15 different cells and displayed as % of Vti1b co‐localizing with VAMP7 (means±s.e.m.). (B) Co‐localization of Vti1b with epsinR was quantified by double‐labelling experiments in MSD and MPS‐IIIA (untreated and MβCD treated) and in control WT (untreated and cholesterol treated) MEFs. The chart displays merge values (means±s.e.m.) that represent the percentage of Vti1b co‐localizing with epsinR measured in 15 different cells. (C) Vti1b trafficking was monitored by FRAP analysis in WT (untreated and cholesterol treated) and MSD (untreated and MβCD treated) MEFs transfected with GFP-Vti1b (see Materials and methods section for details). FRAP data are displayed as percentage of recovery with respect to the fluorescence before bleach (100%) and are representative of 10 recordings from different cells.
Mouse brains were obtained from wild-type (11 months old, one male; 5 months.f4Mouse brains were obtained from wild-type (11 months old, one male; 5 months old, one female; 12 months old, two female) and Nrf2 (−/−) (10 months old, two male and two female) mice. (a)Mouse brain tissues were homogenized in the lysis buffer, and 10 μg of lysates was incubated with 2 μg of GST-tau protein at 37°C for 30 min in the presence or absence of 1 mM ATP. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using a 12E8 and PHF1 antibodies, respectively. GST-tau was detected with a monoclonal GST antibody (GST-tau; Supplementary Fig. 4). Bar graph of the relative optical density of phosphorylated tau normalized to actin. Data shown are mean±s.e. of duplicated independent experiments and were analysed using Student's t-test. Mouse brains were obtained from wild-type (11 months old, one male; 5 months old, one female; 12 months old, two female) and Nrf2 (−/−) (10 months old, two male and two female) mice. (b) Mouse brain tissues were homogenized in the phosphatase storage buffer, and phosphatase activity in the lysates was quantified using the serine/threonine phosphatase assay system (Promega) by measuring the dephosphorylation of a phosphopeptide, RRA(pT) VA in the presence or absence of okadaic acid (OA, 20 nM). PP2A activity was defined as the activity inhibited by the addition of OA to the phosphatase reaction mixture. n=4. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test.
| Passive protection in vitro and in vivo conferred by isolated EV.Figure 4A, Scheme for protocol to determine the in vitro antiviral function of EVs from NoVΔB2-infected BALB/c suckling mice. B, C, 10μl or 50μl EVs stock from naïve [EVs (mock)] or NoVΔB2-infected [EVs (ΔB2), B] or SINV-infected [EVs (SINV), C] mice were added to BHK cell culture medium along with NoV-WT, and then BHK cells were harvested at 24 hpi. Accumulation of NoV RNA1 in BHK cells were measured by RT-qPCR. Normalization was done by β-actin mRNA levels. Data from at least three independent experiments were combined. Data are shown as mean±SD. ** indicates p<0.01, ns indicates no significant difference by student's t-test. D, Diagram showing the 462-nt sequence in length from NoV∆B2 RNA1 was inserted into the 3' untranslated region (UTR) of firefly luciferase reporter mRNA, to be targeted by antisense vsiRNAs with EVs from NoV∆B2-immunized BALB/c suckling mice. A translation initiation codon-deleted GFP sequence of the same length was inserted into the same site as control. E, F, Relative luciferase activity of the two reporter constructs after transfection of 293T cells (E) and 293T-Ago2 KO cells (F) and treatment by different pools of EVs, EVs (∆B2) or EVs (Mock). Error bars indicate standard deviation of three independent replicates. *** indicates p<0.001, ns indicates no significant difference by student's t-test. G, Experimental design for the in vivo functional analysis of EVs. In brief, mice were first immunized with live NoVΔB2, EVs (mock) or EVs (ΔB2), and challenged with WT NoV at 12 hours post-immunization. The NoV-challenged mice were sacrificed at 1 and 3 days after challenge infection for determining the viral load or monitored daily for survival. H, I, Accumulation of NoV RNA1 was determined by RT-qPCR in NoVWT-infected BALB/c suckling mice at 1 (H) or 3 dpi (I). n=5~7 per group. Data are shown as mean±SD. ****indicates p<0.0001 by student's t test. J, Total proteins were extracted from the hindlimb muscle tissue of NoVWT-infected BALB/c suckling mice to measure the viral coat protein (CP) and B2 protein by Western blotting. Staining of β-actin was used as a loading control. K, Survival curve of WT NoV-challenged suckling mice immunized with live NoVΔB2, EVs (ΔB2) or EVs (mock). n=7 per group. *** indicates p<0.001. All data were analyzed by log rank test.
Effects of silent mutations on actin expression levels, cell viability and cytoskeletal organization.Figure 2A Actin expression levels shown by western blotting for strains expressing S. cerevisiae's actin protein from various coding sequences, with tubulin (Tub1p) as a loading control. B Quantification of actin expression levels, showing a decrease when more silent mutations are present. Data are presented as mean +/- SD (n = 4 for Sc, n = 8 for ScNI; n = 12 for Sc[Ca], Sc[Sp] and Sc[At]; 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 strains cultures, grown at 25 ºC in YPD medium. Data are presented as mean +/- SD (n = 6 for Sc, n = 3 for ScNI, Sc[Ca], Sc[Sp] and Sc[At]; technical replicates). *P<0.05 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). D Level of actin expression as a function of growth constant does not show any clear correlation. Rather, there is an apparent level of actin expression (0.25 < expression < 0.35) below which growth rates drastically reduce. Data are presented as mean +/- SD (for actin expression values, n = 4 for Sc, n = 8 for ScNI; n = 12 for Sc[Ca], Sc[Sp] and Sc[At]; 2 biological replicates with n/2 technical replicates each; for growth constants, n = 6 for Sc, n = 3 for ScNI, Sc[Ca], Sc[Sp] and Sc[At]; technical replicates). r is a Pearson correlation coefficient considered non-significant if its two-tailed p-value is > 0.05. E Phalloidin staining depicting F-actin organization. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm. F In vivo actin network deviation indexes, defined to evaluate the patch-cable balance compared to S. cerevisiae haploid cells (value is 0 in S. cerevisiae's cells, 1 when cells contain only actin patches and -1 when cells contain only cables). Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). G In vivo actin network deviation indexes as a function of actin expression levels does not show any clear correlation. Rather, we observe a threshold of actin expression levels (0.25 < expression < 0.35) below which actin cytoskeleton organization is affected. Data are presented as mean +/- SD (for actin expression values, n = 4 for Sc, n = 8 for ScNI; n = 12 for Sc[Ca], Sc[Sp] and Sc[At]; 2 biological replicates with n/2 technical replicates each; for indexes, n = 30 for all conditions). Pearson correlation coefficient r is considered non-significant if p > 0.05. H Polarity indexes, defined to assess whether cell polarity is normal or affected (value is 1 when all patches of medium to large budded cells are present in the bud, and -1 refers when all patches are in the mother cell. Data are presented as mean +/- SD (n = 30 for all conditions). **P<0.01, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).
Figure 6(A) Experimental scheme to analyze the effect of Smed-exoc3 depletion on regeneration of head and tail amputated planarians. Planarians (S. mediterranea) were injected with exoc3(RNAi) or with control gfp(RNAi). (B) Representative photographs of morphology of exoc3 knockdown planarian vs. control planarian on days-29, -31 and -37 of the protocol Number ratios in the photographs indicate the photographically represented phenotypes. Organ regeneration was analyzed by immunostaining of different organ compartments in regenerating exoc3(RNAi)-treated planarians vs. gfp(RNAi)-treated control on day 37 of the protocol (C) Eyes were analyzed by staining against VC1. Representative images are shown (3 experimental replicates with 4-5 biological replicates per experiment). Arrows point to photosensitive cells (ps); pigmentary cells (pc) and to the optic chiasm (oc). Number ratios in the photographs refer to planarians with regenerative defects for the indicated phenotype (scale bar: 0.5 mm). Organ regeneration was analyzed by immunostaining of different organ compartments in regenerating exoc3(RNAi)-treated planarians vs. gfp(RNAi)-treated control on day 37 of the protocol (D) Representative photographs of staining with planarian pan-neural marker 3C11 (alias Synapsin) marking cephalic ganglia and sensory neurons Number ratios in the photographs refers to planarians with regenerative exhibiting the photographically represented phenotype, characterized by underdeveloped bilobed cephalic ganglia and atrophy of sensory neurons in exoc3(RNAi)-treated planarians versus controls (scale bar: 0.5 mm, cg: cephalic ganglia; sn: sensory neurons).
| Pten-deficient B cells are capable of acquiring an anergic phenotype A | Intracellular expression of IgM (Ig-μHC ic) in bone marrow (left) and spleen cells (right) was determined by flow cytometry and compared between the populations of B cells (green, identified by B220 and CD19 expression) and non-B cells (gray).Figure 2A | Intracellular expression of IgM (Ig-μHC ic) in bone marrow (left) and spleen cells (right) was determined by flow cytometry and compared between the populations of B cells (green, identified by B220 and CD19 expression) and non-B cells (gray). Numbers in the histograms indicate the percentages of the positive populations. Figures are representative of 11 - 35 individual mice per genotype. B | Intracellular Ca2+ influx was measured in CD90.2/Thy1.2- splenocytes derived from mice of the indicated genotypes following stimulation with 10 μg/ml α-κLC antibody. Figures are representative of at least 3 individual mice per genotype. C | Serum IgM concentrations measured in mice of the indicated genotypes. Mean ±SD, symbols represent IgM concentrations from individual mice (n). Statistical significance was calculated by using the Kruskal-Wallis test (see also Table S1), n. s. = not significant; **** p ≤ 0.0001. D, E | Absolute numbers of B cells in bone marrow (D) and spleens (E) from mice of the respective genotypes, determined by B220/CD19 surface expression. Mean ±SD, symbols indicate the numbers of B cells in individual mice (n). Statistical significance was calculated by using the Kruskal-Wallis test (see also Tables S2-3), n. s. = not significant; * p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001.
a, b, Haematoxylin-and-eosin-stained sections of uninvolved areas from ileo–colic resections from patients with Crohn’s disease homozygous for the safe (a) or risk (b) allele of ATG16L1 (blue dashed line denotes crypt unit).figf4a, b, Haematoxylin-and-eosin-stained sections of uninvolved areas from ileo-colic resections from patients with Crohn's disease homozygous for the safe (a) or risk (b) allele of ATG16L1 (blue dashed line denotes crypt unit). c-f, Immunofluorescence images of Paneth cells from control patients (c, e) and patients with the risk allele (d, f) stained for lysozyme (red; c, d) and double-labelled additionally for leptin (green; e, f; yellow dashed line denotes the crypt unit). g, Aberrant lysozyme expression was quantified using the same criteria as that used for mouse sections in Fig. 3 (n = 6,829 cells for at risk patients and 8,182 for control, n = 5 patients for each genotype). Leptin-positive D3 cells were quantified in patient samples (n = 4 patients per genotype) homozygous for the risk allele (76 out of 322 D3 cells were positive from a total of 580 crypts examined) and homozygous for the safe allele (11 out of 93 D3 cells were positive from a total 749 crypts examined). Scale bars: a-f, 10 µm. *P 0.05, **P 0.01, ***P 0.001. P values were calculated using two-tailed Student's t-test. Error bars, s.e.m.
is required for bronchial epithelial tight junction formation and function.Figure 4a-e) 16HBE cells were stably infected with lentiviral vector pLKO alone or expressing EMP1 shRNAs (1 or 2). All data are representative of n=3 independent experiments. (a) Total RNA was isolated and analysed for EMP1 expression using Taqman/qPCR with a GAPDH control. Wild type cells treated with 500nM GSK1120212 or siSOS1 for 4 days were also analysed. Error bars denote mean ± SEM, dots indicate individual data points. ***, p <0.0002 (GSK = 0.0002, siSOS1 = 0.0001); ****, p <0.0001. a-e) 16HBE cells were stably infected with lentiviral vector pLKO alone or expressing EMP1 shRNAs (1 or 2). All data are representative of n=3 independent experiments. (b) Cells were fixed and stained for ZO-1, E-cadherin and DNA. Scale bar represents 20m. a-e) 16HBE cells were stably infected with lentiviral vector pLKO alone or expressing EMP1 shRNAs (1 or 2). All data are representative of n=3 independent experiments. (c) Quantification of tight junction phenotype. >500 cells were counted per sample/experiment, across n=3 independent experiments (dots indicate individual data points). Error bars denote mean ± SEM. *, p = 0.0148; **, p = 0.006. a-e) 16HBE cells were stably infected with lentiviral vector pLKO alone or expressing EMP1 shRNAs (1 or 2). All data are representative of n=3 independent experiments. (d) Transepithelial resistance (TER) was measured in 16HBE on day 4 post-seeding. Error bars denote mean ± SEM, dots indicate individual data points. **, p = 0.0036; ***, p = 0.0005. a-e) 16HBE cells were stably infected with lentiviral vector pLKO alone or expressing EMP1 shRNAs (1 or 2). All data are representative of n=3 independent experiments. (e) Cells were seeded on glass-bottomed dishes for 4 days. FM4-64 dye was applied to the media and confocal z-stacks acquired. (f) 16HBE cells were fixed and stained for endogenous EMP1 and DNA using two different commercial antibodies. Scale bar represents 20m. (g) 16HBE cells were seeded on glass coverslips for 10 days to form a mature, polarised monolayer. Cells were fixed and costained for EMP1 and ZO-1, then analysed by confocal microscopy. A representative z-stack is presented; arrows indicate ZO-1 positive tight junctions.
Figure 4A. ST2 wild type and ACT1-deficient cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. As a control, cells were first solubilized and SF-IL-17 was added only post lysis. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in two independent experiments are shown. B. ST2 wild type, ACT1 KO or TRAF6 KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC and samples were analyzed by immunoblotting. C. ST2 cells deficient in TRAF6 reconstituted with TRAF6(WT) or enzymatically inactive TRAF6(C70A) were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in two independent experiments are shown. D. TRAF6 deficient cells reconstituted with TRAF6(WT), TRAF6(C70A) or empty vector were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated from lysates by anti-Flag immunoprecipitation and analyzed by immunoblotting. Short and long exposure of the same membrane stained for Act1 are shown for lysate and IP samples, respectively. TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) and stimulated with IL-17 (500 ng/ml) for indicated time. (E) The activation of signaling pathways analyzed by immunoblotting. TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) and stimulated with IL-17 (500 ng/ml) for indicated time. The induction of mRNA for selected genes analyzed by real time PCR. Mean + SEM from four independent experiments is shown, statistical significance was determined using unpaired two‐tailed Student's t‐test. G. ST2 cells wild type or NEMO KO were stimulated with IL-17 (500 ng/ml) for indicated time points, solubilized and analyzed by immunoblotting. ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC. Samples were analyzed by immunoblotting (H). ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 or were left unstimulated and IL-17 was added post lysis. results from four independent experiments were quantified by densitometry and mean + SEM from four independent experiments is shown (I).