EMBO/sd-character-level-ner huggingface dataset:
Figure 3A, B RNF123 inhibits the activation of the IFN-β (A) promoters induced by the overexpression of RIG-I, RIG-I CARD, MDA5 CARD, and VISA, but has no effect on the TBK1- and IRF3-5D-induced activation of the IFN-β promoter (A) . HEK293T cells (2×105) were transfected with the indicated plasmids (100 ng), IFN-β promoter luciferase plasmid (100 ng), and pRL-TK Renilla luciferase plasmid (100 ng), together with an empty vector and RNF123 construct (100 ng). Luciferase assays were performed 24 h after transfection. A, B RNF123 inhibits the activation of the NF-κB (B) promoters induced by the overexpression of RIG-I, RIG-I CARD, MDA5 CARD, and VISA, but has no effect on the TAB2- and IKKβ-induced activation of the NF-κB promoter (B). HEK293T cells (2×105) were transfected with the indicated plasmids (100 ng), NF-κB promoter luciferase plasmid (100 ng), and pRL-TK Renilla luciferase plasmid (100 ng), together with an empty vector and RNF123 construct (100 ng). Luciferase assays were performed 24 h after transfection. C Knockdown of RNF123 potentiates the activation of the IFN-β promoter induced by RIG-I, RIG-I CARD, MDA5, and MDA5 CARD, but not that by VISA, TBK1, and cGAS/STING. 293T-GFPsh, shRNF123 #1, #3, and #4 stable cell pools (2×105) were transfected with the indicated plasmids. Luciferase assays were performed 24 h after transfection. D RNF123 interacted with RIG-I and MDA5, but not VISA, TBK1, and cGAS. HEK293T cells (1×106) were transfected with Flag-RNF123 (F-RNF123) together with HA-RIG-I, HA-MDA5, HA-VISA, HA-TBK1, or HA-cGAS plasmids (8 μg each). Cell lysates were immunoprecipitated (IP) with anti-HA (αHA). The immunoprecipitates were analyzed by western blotting (immunoblotting [IB]) with anti-Flag (αF) or anti-HA antibody (αHA). Whole-cell lysates (WCL) were analyzed by western blotting with anti-HA and anti-Flag antibodies to determine the expression levels of transfected plasmids. E Endogenous interaction of RNF123 with RIG-I upon SeV infection. HEK293T cells (3×106) were treated with SeV for 12 h or left untreated. Cell lysates were immunoprecipitated with rabbit anti-RNF123 antibody or control IgG. The immunoprecipitates were analyzed by western blotting with rabbit anti-RIG-I antibody. The expression levels of the endogenous proteins were analyzed by western blotting with anti-RIG-I and anti-RNF123 antibodies. F Endogenous interaction of RNF123 with MDA5 upon EMCV infection. HeLa cells (3×106) were treated with EMCV for 12 h or left untreated. Cell lysates were immunoprecipitated with rabbit anti-RNF123 antibody or control IgG. The immunoprecipitates were analyzed by western blotting with rabbit anti-MDA5 antibody. The expression levels of the endogenous proteins were analyzed by western blotting with anti-MDA5 and anti-RNF123 antibodies. Each histogram shows the mean ± SD of three independent experiments done in triplicate. ***P <0.001; **P <0.01; *P <0.1; ns, P >0.05.
PIKfyve is universally depleted in prion diseases.Figure 1A: Conspicuous downregulation of PIKfyve, but not FIG4 or VAC14, in brain lysates of terminally sick mice intracerebrally inoculated with prions (RML). Control: mice inoculated with non-infectious brain homogenate (NBH). Right: Quantification of immunoblot. Each dot represents one mouse. ***: p<0.001; unpaired t-test. Error bars represent s.e.m B: PIKfyve is selectively downregulated in mice infected with ME7 prions. Right: Quantification. Each dot represents one individual (n=4/group). ***: p<0.001; unpaired t-test. Error bars represent s.e.m. C: Time course of PIKfyve downregulation in brains of prion-infected mice (3 mice/time point). PIKfyve (but not FIG4, calnexin, NeuN and synaptophysin) was suppressed between 60 and 120 dpi. T: terminal disease stage. D: The PIKfyve/FIG4 ratio gradually decreased in prion-infected mice (unpaired t-test). **: p<0.01; ***: p<0.001. Each dot represents one mouse. E: Prion-infected Gt1 cells (75 dpi) stained with Alexa-Fluor647-succinimidyl ester which stains vacuoles negatively (arrows). F: Loss of PIKfyve, but not FIG4 and VAC14, in prion-infected Gt1 cells. Right: Quantification of immunoblots. Each dot represents an independent experiment. **: p<0.01; unpaired t-test. Error bars represent s.e.m. G: tga20 COCS were treated with pooled IgG or POM1 (72 hours). POM1 suppressed PIKfyve but not FIG4 and VAC14. Right: Quantification of immunoblots. Each dot represents an independent experiment. **: p<0.01; ***: p<0.001; unpaired t-test Error bars represent s.e.m. H: Immunoblots of human CJD brains (red: sampled region). Gt1Sc: prion-infected Gt1 cells. Loading controls: actin and calnexin (Cnx) I: Quantification of immunoblots revealed moderate and substantial PIKfyve loss in Type-1 and Type-2 CJD, respectively (unpaired t-test). **: p<0.01; ***: p<0.001. Error bars represent s.e.m. J: Brain homogenates from panel C were immunoprecipitated with anti-VAC14 antibodies. FIG4 and PIKfyve did not co-precipitate in prion-infected brains at terminal disease.
Figure 5 A Representative microCT 3D reconstruction and cut-plane images of the fracture site 3 weeks after bone fracture. Scale bar, 500 μm  B-E MicroCT analyses of trabecular bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). Results are shown as mean ± SEM; n=8; *: p<0.05 and **: p<0.01 by t test  F, G Representative images of H&E (F) and Safranin O (G) staining of the fracture callus 3 weeks after bone fracture. Scale bar, 200 μm  H, I Quantification of the mineralized bone callus (H) and cartilage callus (I) in the fracture site. Results are shown as mean ± SEM; n=8; **: p<0.01 and ***: p<0.001 by t test  J Representative microCT images of femoral cortical bone defects in Prx1-Cre;Usp34f/f and Usp34f/f control mice. The red dotted lines indicate the position of the original defect margin. Scale bar, 500 μm  K H&E staining of femoral cortical bone defects. The black dotted lines indicate the position of the original defect margin. Abbreviation: CB, cortical bone. Scale bar, 200 μm  L-N Bone mineral density (BMD), bone volume (BV/TV) and osteoblast surface (Ob.S/BS) of the regenerated bone in femoral cortical gaps. Results are shown as mean ± SEM; n=8; *: p<0.05, **: p<0.01 and ***: p<0.001 by t test
PDGFR-β activation leads to mesenchymal proliferation in vivo and in vitro (A) Activation of PDGFR-β in renal mesenchymal cells, denoted here as Foxd1Cre::Pdgfrb+/J mice, was achieved by crossbreeding the Foxd1-Cre mouse line (FoxD1GC) with a mouse line with a heterozygous knock-in mouse line with constitutively active Pdgfrb mutant (J) allele (Pdgfrb(S)J) instead of the wt Pdgfrb allele.Figure 2Activation of PDGFR-β in renal mesenchymal cells, denoted here as Foxd1Cre::Pdgfrb+/J mice, was achieved by crossbreeding the Foxd1-Cre mouse line (FoxD1GC) with a mouse line with a heterozygous knock-in mouse line with constitutively active Pdgfrb mutant (J) allele (Pdgfrb(S) J) instead of the wt Pdgfrb allele. Representative Ki67 immunofluorescence staining (green) in wt and Foxd1Cre::Pdgfrb+/J mice, showing increased proliferation in the transgenic mice. Glomeruli are outlined with circles, arrowheads point to Ki67 positive interstitial cells. Nuclei are stained with DAPI (blue). Scale bar = 50 µm. Quantification of proliferating Ki67-positive cells specifically in glomeruli (C), interstitium (D) and tubules (E) in Foxd1Cre::Pdgfrb+/J mice (black bars) and wt mice (white bars) of 6, 14, 25 and 35 weeks of age. Foxd1Cre::Pdgfrb+/J mice exhibited increased proliferation of glomerular and interstitial cells, whereas tubular epithelial cell proliferation was not altered. Data in C, D and E are shown as means ± SD of n = 3 animals per group. FoxD1-reporter mice (Foxd1::tdTomato) confirmed the expansion of mesenchymal cells in 14 weeks old Foxd1Cre::Pdgfrb+/J mice both in glomeruli and interstitium. Circles outline glomeruli. Scale bar = 50 µm. Quantification of FoxD1-Tomato positive cells in the cortical interstitium in 14 weeks old Foxd1Cre::Pdgfrb+/J and wt mice confirmed the significantly increased expansion of mesenchymal cells by 42% in Foxd1Cre::Pdgfrb+/J mice. Cells were counted in six viewfields at 40x magnification. Bar graphs show means ± SD of n = 3 mice per group. Isolated primary fibroblasts and mesangial cells from Foxd1Cre::Pdgfrb+/J mice have higher proliferation rates assessed by bromodeoxyuridine (BrdU) incorporation assay compared to cells from wt mice. Bar graphs show means ± SD, n = 4 per group, *: p≤0.05 compared to wt of same time point.
a) Hybridization of DNA from 90 HapMap YRI samples to the Affymetrix SNP 6.figf1(a) Hybridization of DNA from 90 HapMap YRI samples to the Affymetrix SNP 6.0 array reveals a correlated pattern of variation in intensity across six copy-number probes spanning the region upstream of IRGM, suggesting the existence of a common copy-number polymorphism. (Darker shades of red represent reduced hybridization intensity.) Quantitative PCR indicated that the copy-number-variable region was an insertion/deletion (Supplementary Fig. 1). (b) Mapping of the deletion breakpoints by microarray and PCR. Red indicates evidence for deletion; black indicates evidence to the contrary; blue arrows indicate locations of flanking PCR primers able to generate a PCR product in individuals with at least one deletion allele. (c) Sequence of the reference and deletion alleles. The CD-associated SNP rs13361189 is indicated in boldface red type. Physical coordinates are on the reference human genome (build 35/36); allele frequencies are for extended HapMap analysis panels, including 540 samples. (d) CD association of typed and imputed polymorphisms in the IRGM region in the NIDDK IBDGC genome scan. Blue trace indicates recombination map. The deletion polymorphism and rs13361189 are indicated by red arrows. rs13361189 (which is in perfect linkage disequilibrium with the deletion, r2 = 1.0) was also the most strongly associated SNP in the combined WTCCC genome scan and replication study.
Figure 4(A) Western blot analysis after treatment with Ku70 or control siRNA, followed by incubation with DMSO, etoposide (ETO) or bleomycin (BLEO) for 16 h (i). p53 levels after Ku70 depletion normalized to siRNA control for each condition (ii, n=3). All the experiments were performed with HCT116 cells. GAPDH: loading control in A, B, C. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM. (B) Western blot analysis as in A, except that tricostatin A (TSA) was used after Ku/control knockdown (n=4). All the experiments were performed with HCT116 cells. GAPDH: loading control in A, B, C. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM. (C) Western blot in cells treated either with DMSO, TSA, or ETO for 16 or 24 h. All the experiments were performed with HCT116 cells. GAPDH: loading control in A, B, C. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM. (D) RNA affinity chromatography using the 5'p53RNA and total extracts from cells treated with TSA, ETO or BLEO for 24 h, followed by western blot analysis of Ku70 quantified and normalized to the input (ii, n=3). All the experiments were performed with HCT116 cells. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM. (E) RNA affinity chromatography using the 5'p53RNA and total extracts from cells transfected or not with PCAF encoding plasmid for 48 h, followed by western blot analysis. All the experiments were performed with HCT116 cells. (F) RNA affinity chromatography using the 5'p53RNA or a dsDNA and total extracts from cells transfected with HA-tagged Ku70 wild-type (WT) or mutated (Mut6E) Ku70 [25] for 48 h, followed by western blot analysis. All the experiments were performed with HCT116 cells. (G) RNA affinity chromatography as in F, but with two additional Ku mutants (K282/338Q and K282/338R), followed by western blot of Ku70 quantified and normalized to the input and to the loading control, PTB (ii, n=3) All the experiments were performed with HCT116 cells.
Sporozoite antigen exposure elicits superior peripheral blood CD8+ T cell responses.Figure 2(A) Schematic overview of experimental design. C57BL/6 mice received 2x106 OT-I cells alone or were additionally immunised with 10,000 γ-radiation attenuated parasites intravenously. (B) Flow cytometry plots show the gating strategy for identifying Kb-SIINFEKL+ CD11ahi CD8+ T cells. (C) Temporal analysis of antigen-specific CD8+ T cell responses. Peripheral blood was obtained on days 4, 7, 14, 21, 42 and 88 post-immunisation with WT (triangles; n=3-9), CSPSIINFEKL (orange squares; n=4-8) or UIS4SIINFEKL (blue circles; n=4-10) sporozoites, or no parasites (diamonds; n=2-5) and stained for Kb-SIINFEKL+ CD11ahi CD8+ T cells. Line graph shows mean values (±SEM) from representative experiments. (*, p<0.05; **, p<0.01; ***, p<0.001; Welch's t-test comparing CSPSIINFEKL and UIS4SIINFEKL). See Appendix Table S1 for the number of mice used per timepoint and per group. See Appendix Table S4 for exact p-values. (D-H) C57BL/6 mice (n=4 per group), which received 2x106 CFSE-labelled OT-I splenocytes, were immunised with 10,000 γ-radiation attenuated WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites intravenously. 5 days later, mice were sacrificed, spleens harvested and splenocytes assessed for (D) CFSE dilution of CD8+ T cells, (E) CFSE dilution of antigen experienced Kb-SIINFEKL+ CD11ahi CD8+ T cells and stained ex vivo (F-H) for effector CD8+ T cell surface markers. Shown are flow cytometry plots of Kb-SIINFEKL co-staining with markers of effector phenotypes: (F) CD11ahi, (G) CD62Llo and (H) CD49dhi.
mRNA expression pattern in the early mouse embryo (A) E7.Figure 1(A) E7.0 whole embryo and transverse section (TSs) (dashed lines) through (a1) extra-embryonic tissue showing expression in a ring of extra-embryonic ectoderm (ee) and extraembryonic mesoderm (em) and absence of transcripts in the allantois (al); (a2) revealing expression in epiblast (ep) and in mesendoderm (me) emerging from the primitive streak (ps), dashed box (a2') defines higher magnification region shown in a2. (B) E8.5 whole embryo dorsal view and TSs (dashed lines) showing expression in: (b1,b2,b3) the open anterior neural plate (future fore, mid and hindbrain) including (b3) neural crest (nc) emerging from hindbrain; (b4) in the dorsal neural tube (prospective neural crest) and dorsal somites (s); (b5) in closing preneural tube neural (pnt) and epithelising somites; (b6) in caudal lateral epiblast (cle) adjacent to the node (n) at the anterior tip of the primitive streak, but not in presomitic mesoderm (psm). White arrows indicate open anterior and posterior neural plate . (C) E9.5 whole embryo side view and TSs (white dashed lines) showing expression in: (c1) forebrain (fb) and hindbrain , including optic vesicles (opv), (c1') seen at high magnification (and indicated by box grey dashed line) and otic vesicles (ov) (c1'') seen at high magnification (box grey dashed line); (c2) high expression in dorsal hindbrain (hb) and (c3) spinal cord (sc) and dermamyotome (dm) of the differentiating somite; (c5) throughout open posterior neural plate. (D) High magnification of E9.5 whole embryo showing expression in first branchial arch (ba) and forming cranial ganglia, which are just becoming apparent, (V), (VII), (VIII) (IX) and (X). OV, otic vesicle. (E) Expression in mesenchyme cells underlying the apical ectodermal ridge (aer) in the progress zone (pz) and in the zone of polarising activity (zpa) of the forelimb (fl). Data information: Images representative of n≥ 4 embryos for each stage. Scale bars 200 µm, except sections 100 µm.
ROCK2 inhibition is necessary and sufficient to control collective invasion A) Caco-2 cysts expressing an empty vector (pLKO) or shROCK1 (#02) or shROCK2 (#36) after 2 days of invasion were fixed and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI).Figure 5A) Caco-2 cysts expressing an empty vector (pLKO) or shROCK1 (#02) or shROCK2 (#36) after 2 days of invasion were fixed and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii and iii are shown at high magnifications. Arrowheads point to non-protruding cells. Arrow points to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. B) The number of protrusive Caco-2 cyst was quantified in each of the mentioned conditions and represented in a bar graph as percentage of total cysts (Means ± SEM of at least 3 independent experiments, paired t-test, ***p<0.001, *p<0.05 n.s: non significant). C) Caco-2 cysts stably expressing GFP alone or GFP-ROCK2DN#2, were submitted to a 2 days period invasion assay, then fixed and stained for F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii and iii are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. White and black stars show nuclei that engage in protrusions. Scale bars: 20μm. D) Bargraph representing the percentage of protrusive cysts transduced with GFP (CTRL, with or without Y27632) or with ROCK2DN#2 (Means ± SEM of at least 3 independent experiments, paired t-test, **p<0.01 n.s: non significant). E) T84 cells grown in 3D organotypic culture form polarized cysts with a central lumen that display protruding leader cells at the basal pole (arrows) reminiscent of collective invasion. T84 cysts were transduced with lentiviruses encoding inducible GFP or Venus-ROCK2-DA and treated with doxycycline 1μg/ml. Scale bars: 20μm. F) Bar graph representing the percentage of protrusive T84 cysts in control (WT), treated with ROCK inhibitor (Y27632) or transduced with lentiviruses expressing GFP or GFP-ROCK2-DA after induction by Doxycycline (Means ± SEM of at least 3 independent experiments) (paired t-test, ***p<0.001, *p<0.05). G) Overall Survival Prognostic value of ROCK2 expression in colon cancer patients of the GSE33582 data set (n=477 patients with survival annotation) using Kaplan-Meier survival analysis. The patient cohort was divided into tumours with or without a down-regulation of ROCK2 relatively to non-tumoural tissues (expression cut-off value corresponds to the 1st decile of ROCK2 expression in non-tumoural sample, i.e. 7.6). Log-rank test p-values for survival distribution difference are indicated at 3 and 5 years.
Figure 7Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were treated with IFN-β (10 ng/ml) for indicated times. Whole cell lysates were analyzed by WB with indicated antibodies. Primary astrocytes from OTUB1fl/fl (n = 3) and GFAP-Cre OTUB1fl/fl (n = 3) mice were treated with IFN-β (10 ng/ml) for 16 hours. Quantitative real-time PCR was performed to detect OTUB1 (B), CXCL10 (C), CXCL11 (D), CCL2 (E), and NOS2 (F) mRNA levels. Data show mean + SEM.
Figure 6(A, B) Comparison of arbitrary translation efficiency (TE) values of (A) late trophozoite stage (TP 40) and (B) schizont stage (TP 46) proteins across all experimental time points. The arbitrary TE is defined here as the normalized protein abundance per mRNA abundance to mimic the translational output per mRNA copy in a biological system. The top 5% up-regulated proteins (n = 100) at (A) late trophozoite stage (TP 40) and (B) schizont stage (TP 46) were used for this analysis. Differences among all groups were first tested by one-way ANOVA analysis, followed by Dunnett's test as multiple comparison post-test to find significant differences between pairs of (A) TP 40 or (B) TP 46 and other time points. P<0.01 is denoted as **. P<0.0001 is denoted as ****. Scatterplots of mRNA and protein fold-changes (log-transformed) at time points (C) 40 h and (D) 46 h in the erythrocytic cycle. Quadrants are designated with roman numerals. Data points falling into quadrant I are designated as high translationally efficient (HTE) proteins while those falling into quadrant IV are designated low translationally efficient (LTE) proteins PLS regression analysis of codon usage of HTE (n=100) versus LTE (n=100) proteins at ( (TP 4 ( Visualized by scores. Eclipse in represents Hoteling T2 limit at p-value of 0.01 (F-test) PLS regression analysis of codon usage of HTE (n=100) versus LTE (n=100) proteins a ( (TP 4 F) Visualized by scores. Eclipse in represents Hoteling T2 limit at p-value of 0.01 (F-test) PLS regression analysis of codon usage of HTE (n=100) versus LTE (n=100) proteins a G) TP 4 ( Visualized by X, Y correlation loadings. Outer and inner ellipses indicate 100% and 50% explained variance respectively. Percentages of observed variances explained by the PC-1 and PC-2 are indicated in parentheses along their respective axes PLS regression analysis of codon usage of HTE (n=100) versus LTE (n=100) proteins a H) TP 46 H) Visualized by X, Y correlation loadings. Outer and inner ellipses indicate 100% and 50% explained variance respectively. Percentages of observed variances explained by the PC-1 and PC-2 are indicated in parentheses along their respective axes
Figure 4(A) Nucleotide sequence alignment of putative stem-loop structures in the C. elegans sams-3, sams-4 and sams-5 genes together with six hairpin structures in 3'UTR of the human MAT2A gene. Conserved residues are shaded in black. Loop, transition and stem regions of the MAT2A hairpins (Doxtader et al, 2018) are boxed in magenta, green and grey, respectively. An arrowhead indicates adenine bases in the MAT2A hairpins that are specifically modified into m6A by METTL16 in vitro (Shima et al, 2017). (B) Schematic representation of the predicted hairpin structures in sams-3/sams-4 (left) and sams-5 (right) pre-mRNAs. The boundaries between intron 2 and exon 3 are indicated. Arrowheads indicate the m6A modification sites. (C) Alternative splicing of sams-3, sams-4 and sams-5 in smg-2 (yb979) and smg-2 (yb979); mett-10 (ok2204) mutants. Synchronized L1 larvae of each strain were incubated in S-complete medium alone (lanes 1 and 3) or with OP50 (lanes 2 and 4) for 3 hours at 20°C and the splicing patterns were analyzed and presented as in Figure 2B.
Figure 3(A) flg22-induced callose deposition is reduced in ddl-6 and parp1,2. Leaves of four-week-old plants were inoculated with 0.5 µM flg22 for 20 hr or hrcC at OD600=0.2 for 24 hr and stained with aniline blue. Callose deposits were visualized under UV light and quantified by Image J. The data are shown as mean ± SE (n=12). Bar=100 µm. (B) The hrcC-mediated induction of some late responsive genes is compromised in the ddl-6 and parp1,2 mutants. Fully expanded leaves of four-week-old plants were hand-inoculated with hrcC(OD600=0.5), and collected at 0, 3 and 24 hpi for qRT-PCR. The data are shown as mean ± SE from three independent repeats. (C) Pst DC3000-induced PR1and PR5transcripts are blocked in ddl-6 and parp1,2. Fully expanded leaves of four-week-old plants were inoculated with Pst DC3000 (OD600=0.01), and collected at 0, 8 and 24 hpi for qRT-PCR. The data are shown as mean ± SE from three independent repeats. (D) flg22-induced MAPK activation in Col-0 and ddl-6. Ten-day-old seedlings were treated with 100 nM flg22 and collected at the indicted time points. MAPK activation was analyzed by an immunoblot (IB) with -pERK antibody (top panel), and the protein loading is shown by Ponceau S staining for RuBisCo (RBC) (bottom panel). (E) flg22-induced ROS in Col-0 and ddl-6. Leaf discs from four-week-old plants were assayed for ROS production upon 100 nM flg22 treatment over 30 min. The data are show as mean± SE (n=24).