EMBO/sd-character-level-ner huggingface dataset:
Figure 5In the EPM task, the time spent in the open arms was significantly decreased in cKO mice compared to that in the control (CTL) group (nCTL=20, ncKO=12, unpaired Student t-test). B. In the LDB task, the time spent in the light zone by cKO mice was significantly decreased compared to that spent by the CTL mice (nCTL=20, ncKO=12, unpaired Student t-test). C. In the PA task, the entry latency of cKO mice was significantly decreased compared to that of the CTL mice (nCTL=20, ncKO=12, unpaired Student t-test)..D. Nestlet shredding behaviors of the cKO group were also decreased compared to those of the CTL group. The weight of the shredded nestlets of the cKO group was significantly lower than that of the CTL group (nCTL=20, ncKO=12, unpaired Student t-test). In the 3-chamber test, the cKO mice exhibited a decreased interest for the stranger mouse (S1) against objects (O) compared to the CTL group (nCTL=12, ncKO=14, unpaired Student t-test). F. When cKO mice were exposed to a new stranger mouse (S2) against the familiar S1 mouse, they also showed a decreased interest for the S2 mouse compared to that of the CTL group. The preference index showed the decreased sociability of the cKO group in both sessions (nCTL=12, ncKO=14, unpaired Student t-test). Data information: All data are presented as mean values ± s.e.m.
Homedomain TFs are differential expressed in embryonic muscles.Figure 7(A) t-distributed stochastic neighbor embedding (t-SNE) plot of single-cell transcriptomes of GFP expressing somatic muscle cells sorted from Mhc-TAU-GFP expressing stage 16 Drosophila embryos. Colors correspond to clusters identified using hierarchical clustering that were annotated using marker gene expression of muscle subtypes. (B) Identification of highly variable genes using the method by (Brennecke et al, 2013). Scatter plot depicts for each gene the mean expression and squared coefficient of variation across cells using the Poxm+ cluster as input. The solid line indicates the fit, dashed lines the 95% confidence interval. Genes with a significantly elevated variance are shown as triangles, other genes as circles. Different gene classes are color coded. (C) Statistical model of Brennecke et al. (2013) was used to identify highly variable genes. Color code displays enrichment of Ig and homeodomain TF encoding genes among he variable genes according to a hypergeometric test. (D) Venn diagram displaying an overlap of Ig domain genes bound by Ubx in muscle and neuronal tissues, identified in a tissue-specific ChIP-Seq (Domsch et al, 2019). p-value was calculated using a hypergeometric test using as a reference all protein coding genes n=13920.
Figure 1A. Cryo-sections of human organoids were analyzed for the presence of enterocytes (E-cad), Goblet cells (Muc-2) and tight junctions (ZO-1) by indirect immunofluorescence. Nuclei are stained with DAPI. Scale bar 25 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test. B. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. 16hpi organoids were frozen, cryo-sectioned, and HAstV1 infected cells were visualized by indirect immunofluorescence (HAstV1 (red), nuclei (DAPI, blue). Scale bar 25 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test. C. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. Organoids at 16 hpi were fixed and the presence of HAstV1 infected cells (green) were visualized by indirect immunofluorescence. Apical and basolateral membranes were immunostained for actin (magenta) and Laminin (red) respectively. Nuclei are stained with DAPI (blue). Scale bar is 20 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test. D. Quantification of C with the percentage of infected cells determined. E. Human intestinal organoids were infected with HAstV1. At indicated time post-infection the increase in viral copy number was determined by q-RT-PCR. F. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. At 24 hpi the presence of IFNλ in the media was tested by ELISA. Dotted line indicates detection limit of the assay. G. Same as E but for the induced levels of either type I IFN (IFNβ1) or type III IFN (IFNλ). H. Human intestinal organoids were pre-treated for 24h with 2000IU/mL of IFNβ1 or 300ng/mL of IFNλ1-3. Interferons were maintained during the course of infection and the amount of HAstV1 copy numbers was assayed 24hpi by q-RT-PCR.
The perimeter of L2 cell dendritic trees after silencing Tor, Atg5 and Atg7 genes.Fig 5 Tor, Atg5 and Atg7 genes. After silencing the Tor (A) or Atg5 (B) under control of the 21D-promoter, the daily rhythm of the L2 cell dendritic tree size was abolished (mean +/- SD) [Tor: ANOVA, p>0.05; Atg5: Kruskal-Wallis Test: H (3, N = 401) = 7,722824 p =, 0521], while in case of Atg7 (C), the pattern of the daily rhythm of the L2 dendritic trees was changed. The dendritic trees were largest at the beginning of the day, and their size decreased during the day and at night [Kruskal-Wallis Test: H (3, N = 412) = 40.63845 p = 0.0000; multiple comparison test, ***-p≤0.01]. The differences between time points in control flies are indicated with "a" and "b" letters. The dendritic trees are largest at the beginning of the night, in ZT13-a [Kruskal-Wallis test: H (3, N = 423) = 23,31382 p =, 0000; multiple comparison test, the difference between ZT13 and ZT4-p≤0.01, differences among ZT13 and ZT1 and ZT13 and ZT16 p≤0.001].
Figure 4A. Cell-associated viral DNA levels (left), Tat mRNA levels (center), and RT activity levels (right) in cell culture supernatants in infected J34c cells vs. infected JØ cells at 24 and 48 hours post-infection. * p0.05 (paired two tailed t test). B. Representative flow cytometry plot of intracellular HIV-1 Gag protein staining (KC57) versus side scatter (SSC) in JØ and J34c cells, mock infected or infected with HIV-1NL4-3, after 48 hours of culture and corresponding fold change (J34c/JØ) plot in four biological replicate assays. Differences to control were found to be significant with a paired two tailed t test (p0.05). C. Fold change levels for miR-34c-5p, cell-associated viral DNA level, Tat mRNA, and RT activity levels in J34c cells treated with miR-34c-5p antagomiR versus control antagomiR, 48 hours post-infection. Differences to control were found to be significant with a paired two tailed t test (p0.05).
Ig genes affect synaptic wiring specificity in accordance with the motoneuronal homeodomain cluster map.Figure 6(A) Representative confocal images of stage 17 embryonic heads highlighting the expression of Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and in animals with reduced neuronal DIPkappa (elav>DIPkappaRNAi) as well as in animals, which mis-express DIPgamma or dpr1 in neuronal cells (elav>DIPgamma, elav>dpr1). Single channels focusing on the projections (FasII staining) of MN2a and/or MN3 to the MHE, MHD or LR muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right represents a schematic drawing of the confocal images shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and mis-expression conditions. (B) Chart highlighting the expression of the homeodomain cluster genes Dfd, Scr and mirr as well as the Ig genes DIPgamma and DIPkappa in MN2a and MN3, showing that both Ig genes are only expressed in MN2a and not in MN3. (C) Quantification of different phenotypes manifesting in MN2a and MN3 after neuronal RNA interference or mis-expression of Ig genes (DIPgamma, DIPkappa, dpr11, dpr1) in comparison to control animals: MN2a axons projecting to the MHE muscle, representing the wild-type (wt) innervation pattern of MN2a (magenta); MN3 axons projecting to the MHD muscle, representing the wild-type (wt) innervation pattern of MN3 (cyan); MN2a or MN3 axons projecting to muscles other than the MHE or MHD termed wiring defects (dark grey); abnormal synaptic morphologies at MN2a or MN3 derived axon terminals termed terminal defects (light grey). Each experiment was performed in triplicates. Note, each genetic experiment was performed in parallel to an adequate control experiment using the same driver line crossed to a line that controls expression of either UAS-RFP or UAS-GFPRNAi. p-values between two genetic conditions were calculated by a two-sided Fisher test. * p < 0.05, **<0.01, *** p < 0.005.
Functional VCP is required for autophagosome maturation.Figure 3.(A) Epifluorescent images for GFP and mRFP in U20S or VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells transfected with mRFP-GFP-LC3 (tfLC3) and treated with rapamycin for 2 h to induce autolysosome formation. (B) siRNA control (Scr) or VCP-KD- or Baf-treated control U20S cells transfected with tfLC3 and treated with rapamycin for 2 h to induce autolysosome formation. (A and B) Open arrows denote autophagosomes (both GFP and mRFP fluorescence), whereas closed arrows highlight autolysosomes (mRFP only fluorescence). (C) The graph represents Pearson's coefficient of GFP and mRFP colocalization from 10 independent fields of cells in two different experiments. Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.001. (D) Lysates from U20S or tetracycline-inducible VCP-WT, -RH, or -AE cells treated with vehicle or Baf for 4 h and immunoblotted for LC3 and α-tubulin. Note that Baf treatment does not increase the LC3II levels in IBMPFD mutant (RH and AE)-expressing cells. Bars, 15 µm.
Figure 6(A) The unweighted (left) and weighted (right) UniFrac distances based on OTU abundance (NC: gluten-free, M: gluten; n = 6). (B) The taxonomic composition distribution at phylum level (n = 6). Data information: P values were calculated by unpaired two-tailed t-test for comparison of two groups (C) The taxonomic composition distribution at genus level (n = 6). Data information: P values were calculated by unpaired two-tailed t-test for comparison of two groups (D) Duodenal colonizing P. aeruginosa and A. muciniphila as determined by RT-qPCR using bacterial-specific species gene primers (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups (E) The production of LasB (a protease of P. aeruginosa) was examined in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups (F) Dot plot of Pearson correlation coefficients among CRAMP, P. aeruginosa and A. muciniphila at species level. The size of each point represented the correlation coefficient and the color represented positive (red) or negative (purple) relationship (n = 6). (G) Animal protocol. (H) Duodenal colonizing PA14 and PAO1 as determined by RT-qPCR using P. aeruginosa-specific species gene primer (n = 9). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons (I) Western blot and densitometry analyses of pro-form CRAMP and mature peptide in duodenum (n = 8). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons
Figure 5(A) Relative cell viability of cells transfected with siCon (WT), siEWS (EWS KD), PARP1 (PARP-1 KD), and double siRNA (DKD) after MMS treatment. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. Analysis (***) indicates differences between DKD with EWS KD. (B) Additional Parp1 knockout in Ews knockout cells (DKO) were subjected to viability test after MMS treatment. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. *** indicated differences between DKO with EWS-KO. C) After transfection of siCon, siEWS, PARP1, and double siRNA, the effect of co-depletion of PARP1 and EWS on the γH2AX and phosphorylated CHK1 upon MMS treatment was analyzed by western blot. D) After treatment of MMS, the effect of additional Parp1 knockout in Ews knockout cells (Double KO) to the γH2AX and phosphorylated CHK1 were analyzed using western blot. E) Whole cell expression of PAR in mBA (WT: Ews-WT, KO: Ews-KO, DKO: double KO). F) Relative NAD+/NADH ratios were measured in WT, Ews-/- and DKO cells. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. G) PARGi (5 μM, 24 hours) treated WT and Ews-/- cells were subjected to cellular viability assay. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA. H) Relative cellular viability was measured after NMN treatment, with or without MMS pretreatment, in WT and Ews-/- cells. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. I) Chromatin-bound PARP1 was quantified by western blot in WT and Ews-/- cells treated with Olaparib (5 μM, 24 hours) treatment. J) Relative viability was measured in WT and Ews-/- cells upon treatment of Olaparib for 24 hours. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. K) Table of Ews-/- pups survival number in control (normal water) or NMN supplements (NMN containing water) groups 3 days after birth. Statistics (χ²) were determined by chi-square test.
Compartment-specific regulation of neuronal proteins by PTX.Figure 4(A) Volcano plots representing proteins down- (red) or up-regulation (yellow) after 48 hours PTX in the somatic and process compartment. FDR < 0.5. Representative proteins are highlighted and labelled. Linear model was used to fit proteome data and subsequently contrasted using empirical Bayes statistics for PTX-treatment effects across compartments. (B) Venn-Diagram of differentially expressed proteins between somatic and process compartment. (C) Spearman rank correlation between log2-fold changes of differentially expressed genes changing at the transcript and protein-level. The statistical significance of Spearman correlations was calculated in the standard way, using Spearman's rho statistic. Regression lines indicate fitted linear models, with the lightgrey shaded areas depicting the 95% confidence interval. (D) Representative plots of genes changing after PTX at the transcript and protein level. (Upper row: Scatterplots of transcript level; n=2; crossbar represents mean. Lower row: Boxplot of protein levels; n=4). Boxplots: central line: median; box: 25th to 75th percentile; whiskers: until last data point within 1.5x interquartile range (IQR).
Fig.Figure 5(A and B) Immunostaining assay of ASCL1 (green) in control, V1-PAKI and SOX1KO cells on day 25 of dorsal neural differentiation (A), and quantiﬁcation of ASCL1+ cells (B), p=0.0021 (V1-PAKI), p=0.0024 (SOX1KO). Scale bar, 100 μm. (C and D) Immunostaining assay of ASCL1 (green) in control, V1-PAKI and SOX1KO cells on day 25 of ventral neural differentiation (C), and quantiﬁcation of ASCL1+ cells (D), p=0.0003 (V1-PAKI), p=0.0004 (SOX1KO). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). **p<0.01, ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test). (E and F) Immunostaining assay of ASCL1 (green) after SOX1 overexpression in V1-PAKI cells on day 25 of dorsal neural differentiation (E), and quantiﬁcation of ASCL1+ cells (F), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm. (G and H) Immunostaining assay of ASCL1 (green) after SOX1 overexpression in V1-PAKI cells on day 25 of ventral neural differentiation (G), and quantiﬁcation of ASCL1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm. (I and J) Immunostaining assay of MAP2 (green) after ASCL1 overexpression in V1-PAKI cells on day 35 of dorsal neural differentiation (I), and quantiﬁcation of MAP2+ cells (J), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+dox). Scale bar, 100 μm. (K and L) Immunostaining assay of GABA (green) after ASCL1 overexpression in V1-PAKI cells on day 35 of ventral neural differentiation (K), and quantiﬁcation of GABA+ cells (L), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox group (one-way ANOVA).
Properties of the LAMP‐2A‐containing lipid microdomains at the lysosomal membrane.Figure 2(A) Lysosomes from livers of fed (Fed) or 24 h starved rats (Stv) were extracted with 1% Triton X‐114 and processed as in Figure 1 or the aliquots in the translucent region were pooled together as the detergent‐resistant (DR) fraction, and anything below this fraction was pooled, as the solubilized fraction (Sol) (B). Lanes 5-6 are one‐tenth of the amount of lysosomes loaded in the gradient. (C) Distribution of LAMP‐2A (L‐2A) and flotillin‐1 (Flot‐1) in fed rats liver lysosomes extracted with 1% Triton X‐114 at 4 or 37°C. DR fractions were pooled as in (B), and the aliquots below this region were divided in three fractions (a soluble (Sol; aliquot 15) and two intermediate fractions (light: aliquots 11-12; heavy: aliquots 13-14). (D) Fed ratliverlysosomes were incubated with MBCD or filipin, as described under Material and methods, and then processed as in (C). The mean value of the densitometric quantification of the LAMP‐ from two experiments is shown at the bottom in (C) and (D).
Figure 2Ventral furrow formation was inhibited upon photo-stabilization of myosin-II levels at the cell base. Embryos co-expressing the optogenetic module and the myosin-II probe Sqh::mCh were mounted with the ventral tissue facing the objective. A region of photo-activation (two-photon λ=950 nm) was defined at the cell base in the anterior half of the embryo, and whole-cell myosin-II signal was recorded (confocal setup; λ=561 nm) in an alternating fashion. Scale bars, 20 µm. (A) Top view showing apical myosin-II 10 min after the initial activation. While cells in the non-activated region have internalized and disappeared from the apical focal planes (right), cells in the activated region failed to internalize despite showing myosin-II accumulation at the apical surface (left). Red and Blue dotted lines indicate the location of the cross-section panels displayed in (C) and (D). (B) Quantification of apical myosin-II levels demonstrate that the defects in tissue invagination were not due to impaired apical myosin-II accumulation as the average apical myosin-II level per cell did not significantly differ between the non-activated (NR) and activated region (AR). The intensity values were normalized using the median value of the combined cell population in the NR and AR. (N = 3 embryos; nNR: 94, nAR: 78). Significances (by two-sample t-Test): n.s. indicates no statistically significant differences. In each box plot, the central mark, the bottom, and the top edge of each box indicates the median, the 25th and 75th percentile, respectively. Whiskers extent to the most extreme data point and the '+' symbol indicates an outlier. (C Ventral furrow formation was inhibited upon photo-stabilization of myosin-II levels at the cell base. Embryos co-expressing the optogenetic module and the myosin-II probe Sqh::mCh were mounted with the ventral tissue facing the objective. A region of photo-activation (two-photon λ=950 nm) was defined at the cell base in the anterior half of the embryo, and whole-cell myosin-II signal was recorded (confocal setup; λ=561 nm) in an alternating fashion. Scale bars, 20 µm (C, D) z-projections (transverse cross section) of myosin-II time-lapse recordings of the non-activated (C) and activated region (D). While cells in the non-activated region internalized, resulting in a complete closure of the furrow (C), stabilization of basal myosin-II levels prevented invagination (D). Scale bars, 20 µm
Figure 2A-E. The PFC was stained with CB1 receptor (A1-D1, green) or 5HT6 receptor (E1-H1, green), Bassoon (A2 and E2, red), GAD65 (B2 and F2, red), PSD-95 (C2 and G2, red), or SERT (D2 and H2, red) antibodies. Merge images are also depicted (A3-H3). CB1 receptors were mainly co-localized with the presynaptic marker Bassoon (white arrows, A3), mostly within GABAergic boutons (white arrows, B3) and to a lesser extend with PSD-95 (white arrows, C3) and SERT (D3), while 5-HT6 receptors were mainly co-localized with PSD-95 (white arrows, G3). No co-localization of CB1 and 5-HT6 receptors was detected (white arrows indicate the 5-HT6 staining, H3). Scale bar: 10 μm. The scatter plot represents the co-localization analysis for CB1 and 5-HT6 receptor immunostainings. Graph on the top, Mander's split coefficient was used to identify the fraction of CB1 receptors that co-localizes with Bassoon (Mander's coefficient: 0.66 ± 0.02, n=4), GAD65 (Mander's coefficient: 0.56 ± 0.01, n=4), PSD95 (Mander's coefficient: 0.15 ± 0.01, n=4) or SERT (Mander's coefficient: 0.082 ± 0.004, n=4). Graph on the bottom. Mander's split coefficient was used to identify the fraction of 5-HT6 receptors that co-localizes with Bassoon (Mander's coefficient: 0.09 ± 0.01, n=4), GAD65 (Mander's coefficient: 0.059 ± 0.004, n=4), PSD95 (Mander's coefficient: 0.58 ± 0.01, n=4) or CB1 receptors (Mander's coefficient: 0.0020 ± 0.0004, n=4). Error bars correspond to the mean ± SEM.
a) MEFs stably expressing EGFP-Parkin were exposed to CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis (IB) with antibodies to the indicated proteins.f1(a) MEFs stably expressing EGFP-Parkin were exposed to CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis (IB) with antibodies to the indicated proteins. The asterisk indicates a nonspecific band. (b) SH-SY5Y cells were exposed to CCCP (10 μM) for the indicated times and then subjected to immunoblot analysis. (c) MEFs stably expressing EGFP-Parkin were treated with CCCP (30 μM) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c and with a confocal microscope. The fluorescence of EGFP-Parkin was monitored directly. (d) Higher-magnification views of the boxed areas in c. Scale bar (c,d), 10 μm.
Figure 7(a, b) Recruitment reactions performed as in Fig. 2a. Sld7 was omitted in (b). (c) Recruitment reaction conducted as in Fig. 3a, with washes performed at 0.3 M KCl. (d) Replication reaction performed as in Fig. 1g. Experiments in (a)-(d) used MCM containing wild type (wt) or S/T-D substitution mutants of Mcm4 and Mcm6 as indicated. In (d), MCM loading was equalised across samples by using 5-fold more mutant than wild type MCM protein, as is shown in Fig. EV6c. (e) Model for Sld3/7-dependent Cdc45 recruitment to the MCM double hexamer. Sld3/7 may promote intra- (black arrows) or inter- (red arrows) hexamer Cdc45 recruitment. See text for full details of model in (e).