EMBO/sd-character-level-ner huggingface dataset:
Figure 6(A) Scheme derived from the results obtained throughout this work from the wild-type, the XccbphP (null mutant) and the complemented pXccBphP (overexpressing) strains. Systems affected are indicated in different colors and gene candidates involved in these systems found by RNA-Seq analysis that are regulated by XccBphP are depicted in the same color code. (B) Possible model for XccBphPlight-regulated EPS and biofilm results integrated with the spectroscopic results. XccBphP down-regulates biofilm formation, xanthan production and consequently Xcc virulence in a Pr:Pfr dependent manner. XccBphP exposed to sunlight filtered by a canopy (left), direct sunlight (middle) or darkness (right).
Figure 6A Quantitative RT-PCR evaluation of KLF10 mRNA expression in kidney samples of wild-type and KLF10-knockout (KO) mice. **P < 0.0001, significant difference versus wild-type controls (Wilcoxon two-sample test; n = 8). B Western blot analysis of KLF10 expression in kidney samples of wild-type or KLF10-KO mice treated with or without STZ at 8 weeks after the onset of diabetes. C Changes in body weight and blood glucose levels in wild-type or KLF10-KO mice with or without STZ treatment. No significant differences in body weights or blood glucose levels between diabetic wild-type mice and diabetic KLF10-KO mice were detected (Wilcoxon two-sample test; n = 8). D Levels of urinary protein excretion, weights of kidney and levels of HbA1c in wild-type or KLF10-KO mice with or without STZ treatment. The mean relative kidney weight (%) shown in the study is determined as the percent of kidneys out of total body weight, and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin (% HbA1c; DCCT unit). *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 8). E Immunofluorescence images of kidney sections stained with KLF10 and nephrin in wild-type or KLF10-KO mice untreated or treated with STZ. Scale bars, 20 μm. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3). F Immunofluorescence images of kidney sections stained with KLF10 and KDM6A in wild-type or KLF10-KO mice untreated or treated with STZ. Scale bars, 20 μm. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3). G Immunofluorescence staining of F-actin and KLF10 in primary podocytes isolated from the wild-type or KLF10-KO mice untreated or treated with STZ. Presented experiments were performed at least three times independently. Scale bars, 20 μm. H Immunofluorescence staining of F-actin and KDM6A in primary podocytes isolated from the wild-type or KLF10-KO mice untreated or treated with STZ. Presented experiments were performed at least three times independently. Scale bars, 20 μm. I Western blot analysis of KLF10, KDM6A, nephrin and WT-1 expression in primary podocytes isolated from the wild-type or KLF10-KO mice that were untreated or treated with STZ. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3). J Western blot analysis of KLF10, KDM6A, nephrin and WT-1 expressed in podocytes treated with TGF-β1 or the combination of TGF-β1 and KLF10 knockdown. *P < 0.05 versus normal controls, #P < 0.05 versus control siRNA with TGF-β1 treatment (Parametric ANOVA and a Bonferroni post hoc test; n = 3). K A model for the reciprocal regulation between KDM6A and KLF10 in podocytes under diabetic conditions. Under hyperglycemic conditions, increased KDM6A or KLF10 may regulate each other, and create a positive feedback loop that aggravates podocyte dysfunction.
Figure 1(A) BECN1 expression of microglia from Becn1+/- and wild type mouse pups in full medium without Bafilomycin A1 (Baf) was quantified by Western blotting. Microglia from Becn1+/- mice show a signifcant reduction of BECN1 (48%) compared to microglia from wild type mice; mean ± SEM, wild type n = 3, Becn1+/- n = 7; two-tailed t-test **: p < 0.01. Blot shows representative amounts of BECN1 after various treatments. (B) Microglia from newborn Becn1+/- and wild type mouse pups were kept for 2 h either in full medium or HBSS with or without Bafilomycin A1 (Baf). LC3 and ACTIN levels were determined by Western blot. LC3-II was significantly reduced in microglia from Becn1+/- mice after Baf treatment in full medium compared to wild type microglia; mean ± SEM, wild type n = 3, Becn1+/- n = 4; two-tailed t-test *: p < 0.05. (C) Microglia from newborn Becn1+/- and wild type mouse pups were kept for 2 h either in full medium or HBSS with Bafilomycin A1 (Baf) and stained for endogenous LC3. Exemplary images and the quantification of LC3-positive vesicles/µm2 cell area show reduced presence of LC3-positive vesicles in microglia from Becn1+/- and induction of autophagy by HBSS; mean ± SEM, wild type n = 4, Becn1+/- n = 4, 3-5 fields per view/n and condition and 73-271 cells/n and condition; two-tailed t-test *: p < 0.05; scale bar: 20 µm
Figure 4A. Splice forms of WBP2, numbered according to the Ensembl numbering scheme. The GRAM domain is marked in blue, the WW domains in green, and the locations of the variants in black (the p.Ala160Thr and p.Met163Leu variants are too close to show separately in this view). B. Agarose gel trace of cDNA obtained from mouse wt P28 inner ear (IE), P28 brain, adult and P4 organ of Corti (OC). Results show expression of two Wbp2 isoforms in the brain at P28 and very faint band for the short isoform together with a strong band for the long isoform in the inner ear at P28. If we look at just the organ of Corti (adult and P4), we observe a strong band for the long isoform in the P4 OC and an almost undetectable band for the short isoform, which was not even picked up by sequencing (see C). Wbp2 l: long isoform (550 bp); Wbp2 s: short isoform (480 bp). C. Cartoon illustrating the results from the sequencing of mouse wt cDNA performed at P4 and P28. While in the brain we detect both Wbp2 isoforms and in the organ of Corti only the long isoform, in the whole inner ear sample we detect the presence of the long isoform with a small band for the short one. D. Western blot showing the predominant presence of the Wbp2 long isoform in the cochlea at P28, with a short trace of the short isoform showing up only when a higher concentration of protein lysate (20μg) is loaded. Both isoform are absent in the Wbp2-deficient mouse. Gapdh was used as a loading control. Wbp2 l: long isoform; Wbp2 s: short isoform.
Figure 1Schematic of RNAi screens. SCP-1 cells expressing FUS-DDIT3 or EV were transduced with Module 1 of the DECIPHER Pooled Lentiviral Human Genome‑Wide shRNA Library. Half of the cells were harvested on day 3 (baseline sample) and day 12 (drop-out sample), respectively, and shRNA abundance was determined by next-generation sequencing (NGS). RIGER analysis to identify genes that are preferentially essential in FUS-DDIT3-expressing SCP-1 cells. EV-transduced SCP-1 cells and 20 FUS-DDIT3-negative cancer cell lines screened with the same shRNA library were used as reference set. Genes were ranked according to relative shRNA depletion, and YAP1 was identified as top FUS‑DDIT3‑specific essential gene. NES, normalized enrichment score. LFC change in YAP1 shRNA representation in 20 cancer cell lines and SCP-1 cells transduced with FUS-DDIT3 or EV. Error bars represent the mean ± SD of LFC scores for six independent shRNAs. Competition assays with SCP-1 cells transduced with RFP-labeled NTC or YAP1 shRNAs. Flow cytometric quantification of RFP-positive cells on day 9 relative to day 3 showed that YAP1 knockdown was preferentially toxic to FUS-DDIT3‑expressing cultures.
S.Figure 3S. sclerotiorum A2 strain induces autophagic structures in plants.S. sclerotiorum wild type and A2 strains were inoculated onto tomato leaves using colonized agar plugs. 24 hours post inoculation; leaves were stained with 100 µM final concentration of MDC (Sigma) in PBS for 30 min. Fluorescence was visualized using an Olympus IX81 inverted fluorescence confocal microscope (Olympus systems, Germany), with an excitation wavelength of 335 nm and an emission wavelength of 508 nm. Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software, version 2.2.1.227. Scale bar = 10 µm.
Transduced healthy (HDo) and Fabry donor (FDo)-derived T-Rapa "micropharmacies" (TRaMs) secrete functional lysosomal enzymes.Figure 1A. Schematic of TRaM production. CD4+ T cells were enriched by positive selection from leukaphereses of multiple HDos and 3 FDos - details are provided in Materials and Methods. B. Enzyme compartments that are measured in C-I and Fig EV4G; intracellular, total secreted and colloidal particles (enclosed in red and yellow circles). C. HDo-TRaMs were engineered with vectors relevant to treatment of Fabry, Gaucher, Farber and Pompe diseases. Intracellular lysosomal enzyme specific activities were measured in transduced T-Rapa and controls. Transductions varied across vectors/donors, as indicated. D, E. Enzyme activity was assessed in transduced T-Rapa supernatants compared to controls in activated/dividing (D) and quiescent/resting (E) states. F. Cells from all FDo were stably transduced, as indicated. Intracellular α-galactosidase A (α-gal A) specific activities were determined. G, H. Secreted α-gal A activity was measured from FDo-derived TRaMs in activated (G), and quiescent/resting (H) states. I I. α-Gal A specific activity in Fabry patient-derived skin fibroblasts after 6 hours of exposure to conditioned media from TRaMs in the presence or absence of 1mM M6P.
Figure 2(A) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV14 hippocampal neurons expressing PEX-mRFP-FKBP, KIF1C-MDC-FRB, KIF21A-MDC-FRB or KIF5B-MDC-FRB. The morphology of transfected cell is visualized using a BFP fill. Axons are marked with a blue line. Arrows mark peroxisome targeting to dendrites (gray) and axon (blue) 30 min after rapalog addition. Scale bar = 20 µm. (B) Plots representing intensity time traces of dendrites and axons before and after rapalog addition for KIF1C-MDC-FRB (n = 6 neurons), KIF21A-MDC-FRB (n = 4) and KIF5B-MDC-FRB (n = 8). Traces were normalized to the average intensity before rapalog addition, and to the background. C) Quantitative representation of percentage of DIV14 neurons transfected as in (A) with constructs showed in Figure 1A, in which peroxisomes after addition of rapalog redistributed to either axons ("a", blue bars) or to axons and at least 2 dendrites ("a+d", red bars). Kinesins were considered "dendrite targeting" if dendrites were targeted in ≥ 50% of cells and are highlighted in red. Kinesin constructs are transfected as indicated; the number of analyzed cells is summarized in Appendix Table S1. Error bars indicate SEM (D) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV2 hippocampal neurons transfected as in (A). Scale bars: 20 µm (E) Percentage of DIV2 neurons transfected with KIF1C-MDC-FRB (n=9 neurons), KIF21A-MDC-FRB (n=14) and KIF5B-MDC-FRB (n=14) in which peroxisomes redistributed into at least 2 neurites after addition of rapalog and (F) Percentage of neurites of DIV2 neurons transfected as in (E) targeted with peroxisomes after addition of rapalog. Error bars indicate SEM; Scale bars: 20 µm.
Figure 3HEp-2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY or the N- or C-terminally deleted toxin variants fused to β-lactamase (TEM) at 37°C for 4 h. Cleavage of the reporter dye CCF4-AM was used to visualize toxin delivery. After cell entry CCF4-AM is rapidly converted into the negatively charged form CCF4, which is retained in the cytosol and emits a green fluorescence signal (520 nm). In the presence of trans­located β-lactamase fusion proteins, CCF4-AM is cleaved, and disruption of FRET results in blue fluorescence (447 nm). Scale bar: 20 µm. Left upper and right panel: HEp-2 cells re­mained untreated or were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY or the N- or C-terminally deleted toxin variants for 4 h. Cells were lysed and the de­amidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS PAGE gels; left lower panel: HEp-2 cells were lysed and the cell extracts were incubated with full-length CNFY or the N-ter­minally deleted toxin variants for 4 h. The de­amidation of RhoA in the cell extracts was analyzed by the mobility shift of the modified Rho GTPase on SDS PAGE after detection with anti-RhoA antibodies. HEp-2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY or the N- or C-terminally deleted toxin variants for 24 h. The cell nuclei were strained with DAPI (blue) and the actin cytoskeleton was stained using FITC-phalloidin (green). The formation of large, multinuclear cells was ob­served by fluorescence micro­­scopy and the formation of thick actin stress fibers and membrane actin folding were only observed with CNFY-treated cells. The white scale bar is 40 µm. Cells incubated with extracts of YP147 (ΔcnfY) harboring the empty expression vector were used as negative controls.
amiRSUL moves intercellularly, and over long-distances via sieve elements(A) Schematized pSUC2::amiRSUL reporter.Figure 1Schematized pSUC2::amiRSUL reporter. T35S: CaMV 35S terminator. amiRSUL-silencing phenotype in pSUC2::amiRSUL transgenic- versus WT- Arabidopsis. GUS-staining pattern in pSUC2::GUS transcriptional reporter plants. RT-qPCR analysis of SUL mRNA levels in WT or pSUC2::amiRSUL (amiR) rootstocks grafted onto WT or amiR scions. Error-bars: SD. Welch's t-test p-value are indicated. n=5. amiRSUL northern analysis in WT or amiR scions (S) or rootstocks (R) in the indicated grafting combinations. U6 RNA hybridization provides an internal RNA loading control. Top: aphids' feeding-stylets selectively reach the phloem sieve-elements. Bottom: amiRSUL northern analysis, in biological duplicates, from total RNA extracted 14 days-post-infestation from aphids fed on WT or amiR plants. U6: as in (E). Same as (E) with amiRhen1-14 scions and non-transgenic WT or hen1-14 rootstocks. Average relative U6-normalized band-intensity quantifications from two biological replicates are indicated.
Figure 2W0, W6 and W12 refer to the week of study. Serum FGF-21 and GDF-15 levels before, during and after treatment (two-sided Wilcoxon signed-rank test, P-value = 0.031 in all significant cases) (see Fig. EV2 for details where P1 - P6 refer to the individual patients). Volcano plot showing differences in metabolite levels between 0 and 6&12 weeks of treatment. Metabolites in histidine, alanine, aspartate and glutamine metabolism, and the TCA cycle pathways are annotated at 10% False Discovery Rate (see Dataset EV1 for the whole list of differentially regulated metabolites. Although some other metabolites showed a more pronounced difference, these did not fit into a recognised pathway). Metabolite labels are colour-coded according to the identified KEGG pathways (dark red = "Alanine, aspartate and glutamate metabolism", dark green = "Histidine metabolism" and dark blue = "Citrate cycle (TCA cycle)", with transition colour-code if the metabolite belongs to more than one KEGG pathways). Scatterplot depicting P-values from the metabolites Pathway Enrichment Analysis (y-axis) and impact values from Pathway Topology Analysis (x-axis) (see Dataset EV2 for details). KEGG pathways at 10% adjusted Holm-Bonferroni P-value are highlighted. Volcano plot of acyl-carnitine metabolites between 0 and 6&12 weeks of treatment with highlighted differential metabolites at 10% FDR. Respiratory chain complex and citrate synthase activity in skeletal muscle before and after 12 weeks of treatment. No significant differences were detected before and after 12 weeks of treatment by using the two-sided Wilcoxon signed-rank test with an empirical level of significance.
Figure 6(A) Efficiency of Rab8a depletion confirmed by Western blot in RPE1 cells. (B) RPE1 cells were transfected by siRNAs as indicated, followed by incubation in serum-starvation media for 24 h, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (C) Quantification of the percentage of ciliated cells Data represent mean ± SD (n = 3 experiments), 250 GFP positive cells were scored per condition per experiment, *P < 0.05, Student's t-test. (D) Cells were transfected by siRNa as indicated followed by incubation in serum-starvation media for 24 h, and immunostained for EHD1 (red) and γ-tubulin (green). Scale bar, 10 μm. (E) Quantification of the percentage of cells with EHD1 in cilium or in the distal end of basal body as shown in (D). Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test. (F) Cells were transfected by siRNAs as indicated, followed by incubation in serum-starvation media for 24 h, and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm. (G) Quantification of the percentage of IFT20 fluorescent intensity at the centriole shown in (F). Data represent mean ± SD (n=3 experiments), 150 were scored per condition per experiment, *P < 0.05, Student's t-test. (H, I, J) Cells were transfected by siRNAs as indicated, followed by transfection with GFP-Rab8 WT, GFP-Rab8 Q67L, or GFP-Rab8 T22N, and further incubated in serum-starvation media for 12 h. Cell lysate was subjected to immunoprecipitation with anti-GFP antibody, followed by Western blot with antibody against GFP.
M.Figure 5Spearman's correlation between single-cell RecA-mCherrywt fluorescence range and fluorescence VMR over the lifetime of the cell (n = 360). The data shown are from 3 independent experiments. Dashed lines separate different levels of dispersion, and colors denote subpopulations that are underdispersed (gray), dispersed (plum), or highly dispersed (magenta). RecA-mCherrywt fluorescence of subpopulations segregated by fluorescence VMR (58 < n < 159). The data shown are from 3 independent experiments. Black lines indicate means ± SD. Asterisks denote significant difference by Kruskal-Wallis and Dunn's multiple comparison test: ****p < 0.0001. Single-cell time traces of RecA-mCherrywt fluorescence expressed as percent of generation time of subpopulations segregated by fluorescence VMR. The average fluorescence (solid black lines) and half of the generation time (dashed lines) are indicated. Population statistics (n = 360): 40% (0<VMR≤1); 44% (1<VMR≤10); 16% (VMR>10). The data shown are from 3 independent experiments. Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time. Bars shaded in plum indicate moderately pulsing cells (n = 185) and magenta bars indicate highly pulsing cells (n = 57). Significance by Mann-Whitney test. The data shown are from 3 independent experiments. Peaks mainly occur between 80% and 100% of the generation time. Single-cell size (E) and growth rate (F) averaged over the lifetime of the cell (58 < n < 159). The data shown are from 3 independent experiments. Black lines indicate means ± SD. Asterisks denote significance by Kruskal-Wallis and Dunn's multiple comparison test: ns, not significant; **p = 0.002; ****p < 0.0001. Cells experiencing more DNA damage (VMR>10) are slightly larger and grow at slower rates.
Figure 2Physical appearance of wild type (WT), Wwox null (KO) or KO injected with AAV-hWWOX (2x1010) at P19. Graph showing body weight of WT, KO and KO+AAV-hWWOX at different days after birth. n=3 mice per genotype. Kaplan-Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9-hWWOX [total n=16, alive n=6, spontaneously dead n=6, 4 mice (shown in yellow) were taken out for analysis) compared to the non-injected (n=8)] (P <0.0001, Log-rank Mantel-Cox test). D-F. Immunofluorescence images showing the expression of WWOX (magenta) in cortex (D), Hippocampus (E) and Cerebellum (F) at P19 from WT and Wwox null mice that were injected with AAV-hWWOX (2x1010) or Wwox null alone. Neurons are labelled with anti-NeuN antibody (green). The square white box shows the magnified area.   Graph represents the percentage of neurons showing the transgene (NeuN and WWOX positive) expression at different parts (cortex, hippocampus and cerebellum) of the KO brain at P19 after AAV injection. NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV-hWWOX injected mice (n=3) brains.
Figure 6By means of its affinity for cholesterol, uncleaved C99 at the ER induces the uptake and retrograde transport of cholesterol from the PM to the ER, resulting in the formation of a lipid raft domain, or MAM. These C99-dependent lipid rafts passively segregate and organize lipid-binding proteins, thereby facilitating their interaction and the regulation of specific signaling pathways. Failure to cleave C99 completely would result in a futile cycle of continuous uptake of extracellular cholesterol and its mobilization from the PM to the ER, resulting in the upregulation of MAM formation and activation, which in turn would cause the upregulation of SMases, ACAT activity, and LD deposition. Closing the cycle, this accumulation of cholesterol in membranes also induces APP internalization and its interaction and cleavage by BACE1, and the downregulation of α-secretase activity.