EMBO/sd-character-level-ner huggingface dataset:
Figure 4A) Representative cytometry plots of CD11b+ populations present in the cortex of CX3CR1GFP/+ or CX3CR1GFP/+;Sfrp1-/- mice 3 days after saline or LPS intraventricular infusion. Gating was set for isolating the following myeloid cell populations: CD11b+;CD45lo;GFP+ surveying microglia, CD11b+;CD45+;GFP+ activated microglia and CD11b+;CD45+;GFP- infiltrated monocytes. B) Quantification of the percentage of activated microglia and infiltrated monocyte present in Sfrp1-/- and controls brains 3 days after infusion of saline or LPS (n=5 animals per genotype and condition). Error bars represent Standard Error. Statistical significance: **P<0.01 by two-way ANOVA followed by Bonferroni's multiple comparisons test. C) Quantification of the total fluorescence from phagocytized pHrodo-labelled E. coli bioparticles and percentage of phagocyting cells in the CD11b+;GFP+ microglial population (n=3-5 animals per genotype and condition). Error bars represent Standard Error. Statistical significance: **P<0.01, ***P<0.001 by two-way ANOVA followed by Bonferroni' s multiple comparisons test. D) ELISA determination of SFRP1 levels present in the media of microglia (n=4 cultures per genotype), astrocytes (n=4 cultures per genotype) or mixed astrocytes and microglial (2:1, n=7 cultures per genotype) cultures derived from wt or Sfrp1-/- pups exposed for 24h to saline or LPS (1μg/ml). Error bars represent Standard Error. Statistical significance: *P<0.05; ***P<0.001; ****P<0.0001; One-way ANOVA followed by Bonferroni Test. Secretory profile of cytokines present in the medium of microglia, astrocytes or mixed astrocytes and microglial (2:1) culture as above (n=5-6 cultures type -microglia, astrocytes or mixed cultures- and genotype). The content of IL1β, IL10 was determined by Multiplex ELISA. Values are represented in absolute concentration. Error bars represent Standard Error. Statistical significance: ns P>0.05; *P<0.05; ***P<0.001; ****P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test. F) Secretory profile of cytokines present in the medium of microglia, astrocytes or mixed astrocytes and microglial (2:1) culture as above (n=5-6 cultures type -microglia, astrocytes or mixed cultures- and genotype). The content of IFNγ, TNFα, IL4, IL6 was determined by Multiplex ELISA. Values are represented in absolute concentration. Error bars represent Standard Error. Statistical significance: ns P>0.05; *P<0.05; ***P<0.001; ****P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test.
Figure 2A. ReDDM activation in Dronc heterozygous (+/-) intestine 7 days after temperature shift at 29 Co; esg expression (green) labels the intestinal progenitor cells, Histone-RFP (red) is a semi-permanent marker retained in differentiated cells and Pdm-1 (grey) labels differentiated ECs. The red arrows indicates the enlarged areas depicted in the insets in the entire figure. DAPI labels the DNA In the entire Figure (blue). All of the experiments in the entire figure have been made under experimental conditions that protect the epithelial integrity (intestine reared in Oxford Medium and flies transferred every two days into vials with fresh food). B. ReDDM activation in a Drosophila intestine in which all of the progenitor cells are fully mutant for Dronc (see full genotype in methods); note the increased cell size of esg-expressing cells (GFP+, green), the co-expression of Histone-RFP (red) and the EC marker Pdm-1 (gray), and the coexpression of Pdm-1 and GFP in enlarged cells (compare panels and detailed insets from A with B). The image is a representative example of a Drosophila adult gut 7 days after temperature shift at 29 Co. Genotype: w1118; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry C. Relative number of esg-expressing cells normalised to DAPI; notice that the relative percentage of esg-labelled cells is significantly higher in Dronc fully mutant conditions (-/-) at 3 days (***; P = 0.0007) and 7d (****; p <0.0001) post temperature shift at 29 Co (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t-test, +/- n=32, -/- n=25). D. Average cell size of esg-expressing cells (μm²); note the increased cell size of Dronc fully mutant progenitor cells (-/-) at 3d (**; P = 0.0014) and 7d (***, P = 0.0007) post temperature shift at 29 Co (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed parametric t-test, 3d +/- n = 19, 3d -/- n = 28, 7d +/- n = 29, 7d -/- n = 28). E. Relative number of esg-negative cells expressing Histone-RFP normalised to DAPI; notice that the number of Histone-RFP cells without esg expression is significantly higher in Dronc fully mutant (-/-) conditions at 7d (*; P = 0.0046) (Quantifications were made using N biological replicates ≥ 2; Mann-Whitney test, +/- n = 24, -/- n = 20.
Pharmacological inhibition of YAP impairs mutant p53-driven proliferation.Figure 6A. CCNA and CCNB2 expression transcripts were analyzed by RT-qPCR in cDNA derived from tumours of control (saline) or zoledronic acid (ZA)-treated mice (ref. 31). N=3 mice per group. P-values were calculated with two-tailed t-test. B. ChIP analysis of mutant p53 -bound chromatin from MDA-MB-231 cells treated or not with 1 µM cerivastatin (Cer) for 24h. The CCNB2 promoter occupancy was analyzed by RT-qPCR. Normalization was performed to the amount of input chromatin. The experiment was performed in biological triplicate. P-values were calculated with two-tailed t-test. C. MDA-MB-231 cells treated with the indicated concentrations of Cer for 24h were transfected with pCCAAT-B2LUC (100 ng) luciferase reporter vector. Error bars represent mean + s.d., from three biological replicates. P-values are indicated in the figures;p-values were calculated with two-tailed t-test. D. CCNA, CCNB2 and Cdk1 expression transcripts were analyzed by RT-qPCR in cDNA derived from MDA-MB-231 (left panel) and SKBr3 (right panel) cell lines treated or not with 1 µM Cer for 48h. Error bars represent mean + s.d., from three biological replicates. P-values were calculated with two-tailed t-test. P-value<0.01. E. Western blot analysis for CCNA, CCNB and Actin (loading control) expression in lysates from MDA-MB-231 cells treated or not with 1 µM Cer. F. CCNA, CCNB2 and Cdk1 expression transcripts were analyzed by RT-qPCR in cDNA derived from MDA-MB-231cell line treated or not with 30µM ZA for 48h. Error bars represent mean + s.d., from three biological replicates. P-values were calculated with two-tailed t-test. P-value<0.01. G. Viability assay of siGFP and sip53 -MDA-MB-231 cells after treatment with increasing amounts of cerivastatin (0, 0.01, 0.1, 1 and 10 µM) for 48 h. Data are normalized to untreated. Error bars represent mean + s.d., from three biological replicates.
3' UTR dependent mechanisms of regulation by RBPs and miRNAs: (A) Schematic of cis-acting RNA-binding proteins (RBPs) and microRNAs (miRNAs) and their effects on mRNA stability(B) Cumulative distribution of 3'-UTR lengths of down- and up-regulated genes in either compartment.Figure 6(A) Schematic of cis-acting RNA-binding proteins (RBPs) and microRNAs (miRNAs) and their effects on mRNA stability (B) Cumulative distribution of 3'-UTR lengths of down- and up-regulated genes in either compartment. Two-sample Kolmogorov-Smirnov test; ns > 0.05, *p-value < 0.05. (C) Enrichment analysis for RBP motifs in down- and up-regulated genes in either compartment. Fisher's exact test was used to perform enrichment analysis; FDR-corrected. (D) Schematic of miRNA-binding site (MBS) enrichment. PTX mediated synaptic downscaling might lead to a local increase in miRNA abundance or activity. These effects can be potentially captured by testing for an enrichment of MBS in 3'UTRs of downregulated genes in comparison to the sequencing background (expressed genes). (E) Log2-Ratios of the number of miRNA-binding sites found in significantly up- vs. downregulated genes in the somata (red) or processes (blue). Elevated ratio in processes indicates that miRNA-binding sites are more frequent in transcripts downregulated by PTX in processes. Non-parametric Mann-Whitney U-test was used to test difference (mean somata: 0.37 vs mean processes: 0.783); ***p-value < 0.0001; n=80 miRNAs, represented as colored datapoints. (F) Enrichment of MBS in genes that are exclusively down-regulated in somata or processes compared to the sequencing background. The significant miRNA families (p-value < 0.05 and log2-fold enrichment > 0.5) are labelled by a representative miRNA. See Methods for detailed statistical analysis.
a–k) Confocal analyses of the subcellular co-localization of endogenous UVRAG with ER markers, including endogenous calnexin (red, a), endogenous PDI (red, b), and overexpressed DSred-ER (red, c); with Golgi markers, including endogenous GM130 and p115 (red, d,e, cis-Golgi), overexpressed GFP–Man II (green, f, medial-Golgi), endogenous TGN46 (red, g,.figf2(a-k) Confocal analyses of the subcellular co-localization of endogenous UVRAG with ER markers, including endogenous calnexin (red, a), endogenous PDI (red, b), and overexpressed DSred-ER (red, c); with Golgi markers, including endogenous GM130 and p115 (red, d,e, cis-Golgi), overexpressed GFP-Man II (green, f, medial-Golgi), endogenous TGN46 (red, g, trans-Golgi), and overexpressed PtdIns(4) P probe, GFP-FAPP1-PH (green, h); with endogenous coatomer proteins, including Sec31 (red, i, COPII-related) and β′-COP (red, j, COPI-related); and with the endosome marker GFP-p40phox(PX) (green, k) in HeLa cells. The insets show a high magnification of the selected areas. Scale bars, 10 μm. (l) Confocal microscopic quantification of co-localization of UVRAG with the indicated markers (data are mean±s.d., n = 150 cells obtained by gathering data from three independent experiments); **P0.01; ***P0.001.
Figure 7Each circle represents a distinct protein pair (ortholog or isozyme) between species. Each plot shows identical point values but highlights a different functional class of proteins with relevance to oxidative stress. The y=x diagonal line is a reference to compare intrinsic vulnerability to carbonylation between orthologs. The y=x/3.78 diagonal line is a reference to compare combined intrinsic and extrinsic carbonylation properties between orthologs. Elliptical dotted line encircles the points falling within 3 standard deviations of the mean coordinates and 3 standard deviations of the distance from reference line y=x, encompassing ~91% of all data points. This reference region distinguishes outlier points that are distant from the main population. Outliers with associated experimental evidence related to hypersensitivity to oxidative stress are labeled with their protein names. See also Appendix Fig S3 and Fig EV5.
Model for Parkin-mediated inhibition of intrinsic apoptosis Model of the effect of Parkin-mediated BAK ubiquitination on intrinsic apoptosis.Figure 7Model of the effect of Parkin-mediated BAK ubiquitination on intrinsic apoptosis. Following mitochondrial damage, PINK1 is stabilised at the mitochondrial outer membrane (MOM) whereby it phosphorylates ubiquitin to recruit Parkin. Phosphorylation by PINK1 activates Parkin to ubiquitinate a variety of substrates including Mfn2 and VDAC. Parkin ubiquitinates BAK on a conserved lysine (K113) to inhibit BAK oligomerisation and its apoptotic function in releasing cytochrome c release from the intermembrane space (IMS). Additionally Parkin ubiquitinates VDAC2 thereby limiting BAX association with mitochondria. This limits apoptosis mediated by both BAK and BAX, thus enabling the effective engulfment of the mitochondrion by the autophagosome without errant activation of apoptosis.
Figure 3(A) WT and SKO cells were treated with OA for 1 h and processed for ET. Representative single tomographic slices of ER-LD contacts. Orange arrowheads indicate narrow ER-LD contacts, blue arrowheads indicate widened ER profiles seen in SKOcells. (B) 3D-models of LDs and ER-LD contacts based on tomograms in (A). Yellow: ER, brown: LD, red: ER-LD contacts. (C) Exemplary TEM image of a WT cell incubated with OA for 1h. A grid is randomly placed on top of the image and the width of perpendicularly oriented ER profiles passing through grid lines are measured. Yellow lines indicate measured sites. (D) ER profile analysis of (C). Bars: average profile width/cell, ± SEM, n= 11-9 cells (149-224 profiles), *p<0.05 (unpaired T-test).
Figure 1A. Strategy used to screen for mRNAs enriched in motile protrusions of HUVECs migrating through Transwell membranes. B. RNAseq data are plotted in log2 fold change (FC) levels of protrusions over cell bodies against adjusted -log10 false discovery rate (FDR) (n=2 replicates, average FC values are represented). The horizontal dashed line marks the FDR (q) = 0.05 threshold; vertical dashed lines mark the FC = 0.625 (left) or FC = 1.6 (right) thresholds. C. Heat map represents the k-means clustering of transcript log2 FC levels (protrusions over cell bodies) extracted from RNAseq datasets published elsewhere. The corresponding HUVEC FC levels are shown in parallel. D. smFISH co-detection of k 5 mRNAs and GAPDH in subconfluent motile HUVECs. arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µm E. Polarisation Index (PI) of k 5 mRNAs and GAPDH in co-detected in HUVECs (n≥28 cells; **P<0.01, ***P<0.001, ****P<0.0001; Wilcoxon test). F. k 5 mRNAs PIs plotted against respective GAPDH PIs. The slope of the coloured lines represents the average k 5 mRNA/GAPDH PI ratio; the dashed grey line represents a 1:1 ratio (n≥28 cells). G. Top: distribution pattern of mRNAs clustered in k 2, k 5, k 7 and GAPDH. Bottom: smFISH co-detection of exemplar k7 / k 2 mRNAs and GAPDH in subconfluent motile HUVECs. arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µm H. PIs of k 7 mRNAs and GAPDH co-detected in HUVECs (n≥25 cells; *P<0.05, **P<0.01, ***P<0.001; paired t test). I. PIs of k 2 mRNAs and GAPDH co-detected in HUVECs (n≥19 cells; *P<0.05; Wilcoxon test).
Tgif1 inactivation deepens KrasG12D-driven PDAC A Formalin-fixed paraffin-embedded (FFPE) sections from control or Tgif1KO mice were immunostained with anti-pSmad2 antibody and revealed by immunofluorescence (IF) and DAPI.Figure 1Formalin-fixed paraffin-embedded (FFPE) sections from control or Tgif1KO mice were immunostained with anti-pSmad2 antibody and revealed by immunofluorescence (IF) and DAPI. Representative pictures at 40x are shown (n=30). Scale bars, 100 μM. FFPE pancreatic sections from control or Tgif1KO mice were stained with hematoxylin and eosin (H&E) or immunostained with antibodies to CK19 or Amylase and revealed by IHC. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM. Kaplan-Meyer survival analysis of control, Tgif1KO, KC and KTC mice. A regular mosaic two-colors line was used to discriminate between control and Tgif1KO mice. Pictures of whole pancreas and spleen tissues from control, Tgif1KO, KC or KTC mice (left). Weight of pancreas was measured and presented as dot plot (n=30). FFPE pancreatic sections from control, Tgif1KO, KC or KTC were stained with H&E or subjected to IHC analysis using antibodies to CK19 or α-SMA. Yellow arrows indicate PanINs in KC mice. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.
Bcl-2 overexpression blocks BSC apoptosis and proliferation in K15ΔNLef1 mice.Figure 6A qRT-PCR for Bcl-2 mRNA expression in sorted (CD34+) BSCs of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice (n=2 biological replicates). B Immunofluorescence staining of epidermal tail whole mounts of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice, stained for keratin 15 (Krt15, green), activated Caspase 3 (aCas3, red) and DAPI (blue). Note increased number of aCas+ cells in the bulge of K15ΔNLef1 mice (arrows). B: bulge, HG: secondary hair germ, SG: sebaceous gland. Scale bar, 100µm. C Quantification of hair follicles (HF) with activated Caspase 3 (aCas3+), apoptotic cells in control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice (n=6 biological replicates; mean ± standard deviation (SD); *** p<0.001, two-way ANOVA test). D Immunofluorescence staining of epidermal tail whole mounts of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice, stained for keratin 15 (Krt15, green), BrdU (1 h pulse, red) and DAPI (blue). Note increased number of BrdU+ cells in the bulge of K15ΔNLef1 mice (arrows). B: bulge, HG: secondary hair germ, SG: sebaceous gland. Scale bar, 100µm E Quantification of hair follicles (HF) with BrdU+, proliferating cells in bulge of control (Ctr), Bcl-2EOE, K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice (n=6 biological replicates; mean ± standard deviation (SD); **** p<0.0001, multiple t-test comparisons). nd: not detected.
The IGF1R pathway influences retrograde axonal transport of signalling endosomes PMN were treated with 1 µM PPP (blue) or 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT.Figure 2PMN were treated with 1 µM PPP (blue) or 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Example images of DIV6 PMN treated with 1 µM PPP or 50 ng/ml IGF1. PMN were stained with 30 nM AlexaFluor555-HcT. The scale bar is 50 µm. PMN were treated with 1 µM PPP (blue) or 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Example of HcT-containing organelle tracking in the axon of PMN. Each colour denotes a single endosome tracked over time. The scale bar is 10 µm. PMN were treated with 1 µM PPP (blue) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Graph shows the average velocity of HcT-containing organelles, after PPP treatment compared to controls (DMSO: 138 endosomes, 24 axons, 6,433 movements; PPP: 130 endosomes, 23 axons, 5,222 movements) (** P= 0.0061, Student's t-test, N=4 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Percent of time HcT-containing organelles pausing per axon. There was no difference in pausing between PPP-treated neurons and controls (P=0.45, Student's T-test, N=4 independent experiments data shown are mean ± SEM). PMN were treated with 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Graph shows the average velocity of HcT-containing organelles after IGF1 treatment compared to control (water: 138 endosomes, 22 axons, 5,119 movements; IGF1: 106 endosomes, 20 axons, 5,202 movements; *** P=2.83x10-5, Student's T-test, N=3 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Percent of time HcT-containing organelles spent pausing per axon. IGF1 significantly increased the pausing time of HcT-containing organelles (* P=0.011, Student's T-test, N=3 independent experiments, data shown are mean ± SEM). PMN were treated with 1 µM PPP (blue) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Speed distribution profile of PPP-treated neurons and controls. PPP caused an increase in instantaneous velocities of HcT-containing organelles (N=3 independent experiments, data shown are mean ± SEM). PMN were treated with 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Speed distribution profile of IGF1-treated neurons and controls. IGF1 caused a decrease in instantaneous velocities of HcT-containing organelles compared to controls (N=3 independent experiments , data shown are mean ± SEM).
Treg-specific USP44 deficiency stunts tumor growth and enhances anti-tumor immunity.Figure 7MC38 colon carcinoma, B16F10 melanoma cell lines were s.c. injected into the shaved flanks of Usp44fl/flFoxp3Cre+ mice and their wild type (Usp44fl/flFoxp3Cre-) littermates (1x105 cells/mouse; n=5-9 mice/group). Tumor dimensions were measured every 2 days, and changes in the mean tumor volume over time were observed (lower panels). Upper panels depict representative photographs of excised tumors. EL4 thymoma cell lines were s.c. injected into the shaved flanks of Usp44fl/flFoxp3Cre+ mice and their wild type (Usp44fl/flFoxp3Cre-) littermates (1x105 cells/mouse; n=5-9 mice/group). Tumor dimensions were measured every 2 days, and changes in the mean tumor volume over time were observed (lower panels). Upper panels depict representative photographs of excised tumors. D. 21 days post-implantation, proportions of FOXP3+ Tregs in the CD4+ compartment of all MC38 tumor-bearing mice in each experiment were found by flow cytometry. Suspensions of cells obtained from spleen, tumor-draining lymph nodes, and tumor-infiltrating leukocytes (TILs) were stained for surface CD4 and intracellular FOXP3 prior to analysis by flow cytometry. E, F. Frequencies of CD4+ and CD8+cells producing the proinflammatory cytokine IFNγ were found among the tissues of tumor-bearing mice in the MC38 modelby intracellular cytokine staining Kralovics et al(, 2005) and flow cytometry after ex vivo re-stimulation of recovered leukocytes by PMA and ionomycin in the presence of brefeldin A. G, H. The capacity of CD8+ cells for effector function in MC38 tumors was further assessed by measuring (G) production of TNFα by ICS and (H) intracellular straining of the cytotoxic mediator granzyme B followed by flow cytometry.
Figure 2A Alignment of the amino acid sequences of IDOs restricted to the stretch of amino acids containing the YxxM motif (where x is any amino acid). Residues are highlighted based on increasing conservation level of symbols in the multiple alignment (light gray ≥ 44.4%; dark grey ≥ 66.6%; black ≥ 88.8%). IDOs, homologues of IDO1. Proto-IDOs, homologues of IDO2. Accession number for each specific gene is indicated. B Cartoon model of human IDO1 (gray) and the SH2 domain of PI3K p85 (orange) complex as resulting from docking study. Residues of the YENM motif are colored in yellow and shown in sticks. Residues of phosphotyrosine binding site in p85 are colored in cyan and shown in sticks. Heme and l-tryptophan are shown in sticks and colored in red and green, respectively. C Pull-down (PD) assay of lysates from P1.HTR cells with unphosphorylated (YENM) and tyrosine-phosphorylated IDO1 peptides (pYENM, pITIM1, and pITIM2), analyzed by sequential immunoblotting with anti−p85, −SHP-1, and -SHP-2 antibodies. D Immunoblotting with anti-IDO1 and -p85 antibodies of IDO1 immunoprecipitates obtained from P1.HTR cells transfected with IDO1.WT or IDO1.Y149F. Mock-transfected P1.HTR cells were used as a control. E Tyrosine phosphatase activity in anti-IDO1 immunoprecipitates from lysates of P1.HTR transfectants as in D. F Kyn release over 24 h by P1.HTR transfectants as in D.
Figure 9 Expression of CD69 on PBMCs from three healthy donors (A, B, C- n=3) was measured by flow cytometry following 24 h treatment with 3 µM of PH02 alone, or in combination with the indicated concentration of PEP005. Positive control samples were treated with PMA. Data information: Statistical significance, as determined by one-way ANOVA is indicated: *, P<0.05; **, P<0.005; ***, P<0.0005; ****, P<0.00005; ns, no significant difference. The exact P-values are indicated in the Appendix Table S1.