EMBO/sd-character-level-ner huggingface dataset:
FGMYBs promote female gametophyte development in both M.Figure 7A, B A bidirectional transcription module at the MpFGMYB/SUF locus acts as a toggle switch between male (A) and female (B) sexual differentiation in M. polymorpha. MpFGMYB expression is activated by one or more unknown cues associated with reproductive growth. In males, MpFGMYB expression is suppressed by constitutively expressed antisense SUF, allowing an unknown factor (M) to activate both autosomal and Y-chromosomal genes to promote male differentiation (A). A dominant "Feminizer" on the X chromosome directly or indirectly suppresses SUF expression, allowing expression of MpFGMYB and downstream autosomal and X-chromosomal genes to promote female differentiation (B). Genes on the X and Y chromosomes are dispensable for the sexual morphologies of the gametophytes, but required for the differentiation of functional gametes. C In A. thaliana, three FGMYB genes promote female differentiation in the embryo sac, a highly reduced female gametophyte of flowering plants. FGMYB expression is regulated at the transcriptional level after the formation of sporophytic female floral organs
MK-801-induced c-Fos expression is reduced in DG granular and CA3 pyramidal cells of Grin2aS/S mice.Fig. 5C-Fos expression 120 min after i.p. injection of MK-801. a C-Fos DAB immunosignals are depicted for one representative brain section of the different genotypes as indicated. The original images, including the cortical regions and results of PBS injected animals, are presented in Supplementary Fig. 6. The number of mice analyzed is given in brackets. b For quantitative comparisons the total numbers of c-Fos-positive cells in the cellular layers of the hippocampus were counted. C-Fos-positive cells in extra-hippocampal regions are given per mm2. Slices from six mice per genotype were used for the statistical analyses (two-tailed t-tests) and error bars represent mean ± SEM. CA1 and CA3 cornu ammonis regions 1 and 3, DG dentate gyrus, PC piriform cortex, RSC retrosplenial cortex, Neo neocortex, MTN midline thalamic nuclei, BLA basolateral amygdala. All scale bars: 0.2 mm.
Bob1-deficienct mice show a strong reduction in the frequency of GL7+ Tfh cells.Figure 3(A) Flow cytometry analysis of GL7 expression on Tfh cells. Mice were immunized with SRBC and seven days later analyzed for the presence of GL7-expressing CXCR5hiICOS+, CXCR5hiPD1+ and CXCR5hiBTLAhi Tfh cells in the spleen (SPL), mesenteric lymph nodes (MLN) and Peyer´s patches (PP) (n=4, mean ± SD). *, P < 0.05; unpaired Mann Whitney test. (B) Representative immunofluorescence analyses to detect GL7+ T and B cells in secondary lymphoid organs of Pou2af1-/- and Pou2af1+/- mice. Tissue sections obtained from mice seven days after immunization with SRBC were stained with anti-GL7 (red), anti-CD4 (blue) and anti-IgD (green) to reveal the localization of GL7+ B and T cells in relationship to the T cell zones and B cell follicles. Data is representative for at least 4 mice per group. (Scale bar = 100 µm).
Figure 3Wild-type or mutant versions of EGFP-tagged E2F7/8 were co-transfected with either empty vector or Flag-tagged cyclin F in HEK293 cells. Nocodazole was added to cells 8h before harvest. 48 hours after transfection, cells were collected and lysed for immunoblotting. Knockdown of cyclin F stabilized E2F7 and E2F8. HeLa and RPE cells were transfected with either scramble siRNA or pool cyclin F siRNA. Cells were harvested at 48 hours post transfection. Protein levels of E2F7/8 were analyzed by immunoblotting. Asterisk indicates the specific band of E2F7 detection. Cyclin F targets atypical E2Fs during G2/M. HeLa cells were transfected with either scrambled siRNA (scr) or cyclin F siRNA (sicyclin F) for 24 hours. Then cells were synchronized by double thymidine block and released into fresh medium after the second block. Cells were harvested at the indicated timepoints after the release. Protein expression was measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). Asterisk indicates the specific detection of endogenous E2F7. Cyclin F contributes to the ubiquitylation of E2F7 and E2F8 in vivo. HEK293 cells were transfected with HA-E2F7/8, with or without Flag-cyclin F, and with HA-tagged ubiquitin. 5 hours before harvest, cells were treated with MG132. 48 hours after transfection, HEK cells were harvested and lysed for immunoprecipitation pull-down assay with anti-HA resin followed by immunoblotting.
Figure 1Domain boundaries and sequence motifs mapped to the sequence of CNFY. Cartoon representation of CNFY, colored according to domain boundaries determined with PiSQRD (Aleksiev et al, 2009). Dark blue: domain D1, cyan: domain D2, dark green: domain D3, yellow: ADP-ribosyltransferase-like domain D4, pink: deamidase domain D5. Other colors indicate the position of sequence motifs that have been identified in E. coli CNF1, namely light blue: p37LRP/67LR receptor-binding motif, red: hydrophobic stretches predicted to form membrane-inserting α-helices, orange: cleavage site, magenta: main Lu/BCAM receptor-binding motif. The positions of N- and C-terminus are indicated by N and C, respectively. Surface representation of CNFY as seen from two different orientations with respect to B. Note that the cleavage site between D3 and D4 (orange) as well as the deamidase active site in D5 are partially blocked in the structure of full-length CNFY. The C-terminal domain D5 interacts mainly with D3 (610 Å­2), which partially hides the catalytic site of D5, but it interacts only weakly with D4 (380 Å2), which itself establishes an extensive interface with D1 (1390 Å2) by mainly hydrophilic interactions (17 hydrogen bonds and 6 salt bridges).
Figure 1, Localization of disease-causing missense mutations of Munc18-1b in its tertiary structure (PDB code 4JEU) Burkhardt et al, 2008(), with annotation of residues and binding sites of syntaxin-1 Habc domain and N-terminus. Highlighted in blue are the residues 406 and 544 that we have analyzed in this study in detail. Syntaxin-1 domain structure. The N-terminus (residues 1-28) binds to domain 1 of Munc18-1 while the Habc domain (residues 28-180) and the SNARE domain (residues 180-264) bind in Munc18-1's central cleft. The transmembrane domain (TMD; residues 264-288) does not participate in Munc18-1 binding. , Total protein levels of Munc18-1. HEK293T cells transfected with WT or mutant Munc18-1 variants and either GFP, syntaxin-11-264 or syntaxin-11-180 were lysed and lysates were analyzed by quantitative immunoblotting to indicated proteins, normalized to β-actin (Synt-1A = syntaxin-1A). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001 by Kruskal-Wallis test, followed by Dunn's multiple comparison test; n = 11 independent experiments; exact p values are shown in Appendix Table S1). Solubility of Munc18-1. HEK293T cells transfected as in (C) were solubilized in 0.1% Triton X-100 (TX) and equal volumes of soluble and insoluble fractions were analyzed by quantitative immunoblotting. TX-soluble Munc18-1 was measured as percent of total Munc18-1 by quantitative immunoblotting. Data are means ± SEM (*p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Kruskal-Wallis test followed by Dunn's multiple comparison test, or by one-way ANOVA followed by Bonferroni post-hoc test; n = 5-9 independent experiments; exact n and p values are shown in Appendix Table S1).
A time course experiment that reveals progressive appearance of senescence and loss of podocytes with age.Figure 3A: WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4-, 12-, 18- and 24-month-old mice. Quantification of WT1-positive cells (right upper panels), and glomerular and tubular p21-postive cells (right lower panels). Original magnification X400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules. B: WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4- and 12-month-old irradiated (RT+) and non-irradiated (RT-) mice. Quantification of WT1-positive cells (right upper panels), and glomerular and tubular p21-postive cells (right lower panels). Original magnification X400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules. Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test. C: Bioluminescence (BLI, upper panel) and quantification (lower panel) kidney BLI area in young or old mice (upper panel). n = 3 mice. Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test. D: Bioluminescence (BLI, upper panel) and quantification (lower panel) kidney BLI area in irradiated (RT+) and non-irradiated (RT-) mice over time. n = 3- 6 mice according to the different time points. Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test.
AURKA has extensive proximity interactions with centrosomes and centriolar satellites A-B Comparison of the AURKA proximity interactome with the proximity interactome of the centrosome (Gupta et al.Figure 3A-B Comparison of the AURKA proximity interactome with the proximity interactome of the centrosome (Gupta et al., 2015), the proximity interactome of centriolar satellites (Gheiratmand et al., 2019) and the physical interactome of centriolar satellites (Quarantotti et al., 2019). C. AURKA interacts with the centriolar satellite proteins PCM1, CEP131, CEP72, CSPP1, KIAA0586/Talpid3 and CP110. HEK293T cells were transiently transfected with FLAG-BirA* or FLAG-BirA*-AURKA. Following 18 h biotin incubation, cells were lysed, and biotinylated proteins were precipitated by streptavidin beads. The initial sample and immunoprecipitated biotinylated proteins were run on a gel and immunoblotted with fluorescent streptavidin and antibodies against FLAG, CEP72, CEP131, PCM1, CEP63, CSPP1, KIAA0586/Talpid3, CP110, BBS4 and SSX2IP. D. AURKA interacts with PCM1 in centriole-less cells. GFP-PCM1 or GFP-PCM1 was co-expressed with FLAG-AURKA in HEK293T cells treated with DMSO (control) or 500 nM centrinone B for 7 days. Complexes were precipitated with GFP-Trap beads. The initial sample and immunoprecipitated proteins were run on a gel and immunoblotted with antibodies against GFP and FLAG. E. Endogenous interaction of PCM1 and AURKA. Lysates from synchronous and serum-starved HEK293T were immunoprecipitated with AURKA antibody and rabbit IgG control. The initial sample and immunoprecipitated proteins were run on a gel and immunoblotted with antibodies against PCM1 and AURKA. F. AURKA co-localize with PCM1 at the peripheral satellite clusters upon chemical dimerization of satellites with Kif5b motor domain. U2OS cells were co-transfected with GFP-PCM1-FKBP and HA-Kif5b(1-269 a.a.)-FRB and treated with 500 nM rapamycin for 1 hour followed by fixation at 6 hours. Cells that were not treated with rapamycin were processed in parallel as a control. Fixed cells were stained with antibodies against GFP, AURKA and gamma-tubulin. DNA was stained with DAPI. Cell edges are outlined. Scale bar, 10 μm. G. Quantification of pericentrosomal levels of AURKA and gamma tubulin for F. n>25 cells per experiment. Data represent mean value from two experiments per condition ± SEM (****p < 0.0001; **p < 0.01; Unpaired Student's t-test.).
Striatal infusion of the high dose of cholesterol promotes lysosomal clearance and autophagy in the striatum of HD mice.Figure 6Representative confocal images showing p62 (A, red) in the infused and contralateral striatum on brain coronal sections from wt, R6/2 ACSF, and R6/2 chol-high mice (N=3/group). Hoechst (Ho, blue) was used to counterstain nuclei. Quantification of dots for p62 (B) in the contralateral and infused striatum of R6/2 ACSF and R6/2 chol-high mice. 20 images from 3 sections in the middle of the striatum for each animal were acquired and analyzed. The data in (B are shown as scatterplots with means±standard error, and each dot corresponds to the value obtained from each image. Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (***p<0.001; ****p<0.0001). Scale bars in (A 10 µm. Representative confocal images showing LAMP1 (C, green) in the infused and contralateral striatum on brain coronal sections from wt, R6/2 ACSF, and R6/2 chol-high mice (N=3/group). Hoechst (Ho, blue) was used to counterstain nuclei. Quantification of density for LAMP1 (D) in the contralateral and infused striatum of R6/2 ACSF and R6/2 chol-high mice. 20 images from 3 sections in the middle of the striatum for each animal were acquired and analyzed. The data in D) are shown as scatterplots with means±standard error, and each dot corresponds to the value obtained from each image. Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (***p<0.001; ****p<0.0001). Scale bars in C): 10 µm.
Figure 8A. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or NC mimics as indicated. Colonies were stained with crystal violet and counted after 14 days incubation. B. HeLa cells expressing either lncRNA-MIF shRNA or control shRNA were transfected with miR-586 inhibitors or NC inhibitors as indicated. Colonies were stained with crystal violet and counted after 14 days incubation. C. 2×106 HeLa cells expressing either lncRNA-MIF, lncRNA-MIF control RNA, lncRNA-MIF-shRNA or lncRNA-MIF control shRNA were individually injected subcutaneously into flanks of nude mice (n = 7 for each group). Representative photographs of xenografttumors in situ were taken 3 weeks after injection.D. Three weeks post graft, mice were euthanized, tumors were excised and weighed, and the mean tumor weight was plotted on the graph. Data shown are mean ± SD (n = 7; **P < 0.01, two-tailed t-test). E. A schematic illustration of the proposed model depicting a role of c-Myc-induced lncRNA-MIF in regulating glycolysis via suppression of c-Myc.
Figure 6A) HEK293A, VPS15 KO control and stably rescued VPS15 KOs (sgA-B3) were starved for 1 hr before WIPI2 (green), LC3 (red) and DNA (Hoechst, blue) were visualised. Quantification of WIPI2 puncta number per cell, mean +/-SEM, n=5. B) Stably rescued VPS15 KO (sgA-B3), HEK293A and VPS15 KO control cells were starved for 1 hr with (SB) or without (St) 100nM Bafilomycin A1, or cultured in full media (F). Quantification shows LC3II/Actin, mean +/-SEM, n=5. C) Autophagic flux examined over a four-hour starvation time-course. LC3II/Actin was quantified, mean +/-SEM, n=6.
Proteomic analysis of context-dependent DHHC5-associated protein complexes.Figure 4FLAG-DHHC5 immunoprecipitations were performed using lysates from Hela cells co-expressing either wild-type (WT) or palmitoylation deficient mutant (m) Golga7b. The interaction of DHHC5 with its substrate Flot2 was measured by immunoblotting and is significantly reduced in the presence of palmitoylation deficient mutant Golga7b, quantification normalised to input, *=p<0.05, ratio paired t-test, n=3. Venn diagram comparing the overlap of numbers of proteins immunoprecipitated when FLAG-DHHC5 is expressed alone or co-expressed with either WT or mutant Golga7b. IF images are taken from figures EV2 and EV3 to illustrate DHHC5 localisation. Volcano plot of DHHC5 interactors from AP-MS analysis from HeLa cells co-expressing FLAG-DHHC5 and WT Golga7b highlighting interacting proteins involved in clathrin-mediated endocytosis, cell:cell adhesion and known substrates of DHHC5. Validation of novel DHHC5 interactors by co-IP: immunoblots of Dsg2from Hela cells co-expressing DHHC5 and either Wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, *=p<0.05, ratio paired t-test, n=3. Co-IP immunoblots of clathrin heavy chain from Hela cells co-expressing DHHC5 and either wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, *=p<0.05, ratio paired t-test, n=3. Co-IP immunoblots of ZO-1 protein from Hela cells co-expressing DHHC5 and either wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, **=p<0.01 ratio paired t-test, n=3. Bar graph comparing the number of the cell:cell adhesion proteins identified in each protein interaction dataset. All scale bars 10μm. All error bars represent s.e.m.
MITF binds and represses the GATA6 locusA.Figure 7A. Gene track of 3HA-MITF signal occupancy showing a significant MITF binding peak (P1) in the GATA6 gene body in 501mel (GSE61967). Additional tracks indicate potential regulatory regions highlighted by ATAC-seq and H3K27ac, BRG1, SOX10 and H2A.Z deposition (GSE94488 and GSE61967). H3K27ac deposition is also shown in MM099 at the GATA6 locus. The scale bar indicates the size of the genomic region in kilobases (Kb). A magnification of the P1 region for MITF occupancy is shown in which the "intGATA6r" region is indicated in red and the two E-boxes in black. B. ChIP experiment monitoring the fold enrichment (compare to control IgG) of MITF protein and H3K27ac mark at the "intGATA6r" region. Proteamine 1 (PRM1) and Tyrosinase (TYR) regulatory regions were used as negative and positive controlled, respectively Data information: data are presented as mean values + SD for three biological triplicates. The p-value (Student's t-test) is indicated, *<0.05, **<0.01; ***<0.001. C. qRT-PCR analysis showing average TBP-normalized fold expression of MITF and GATA6 in 501mel treated with either siCTL or siMITF for 48h. Data information: data are presented as mean values + SD for three biological triplicates. The p-value (Student's t-test) is indicated, *<0.05, **<0.01; ***<0.001. D. MM099MITF-SOX10-PAX3 expressing inducible MITF-SOX10-PAX3 genes was treated or not with Doxycycline (1μg/ml) for 24h and protein lysates were immuno-blotted for the indicated protein. The numbers below the gel lanes represent relative protein level, which was determined from the band intensity using ImageJ software, and normalized relative to VINCULIN. E. qRT-PCR analysis showing average TBP-normalized fold expression of GATA6, ABCG2, AMIGO2 or SERPINE1 in MM099MITF-SOX10-PAX3 treated or not with Doxycycline (1μg/ml) for 24h. Data information: data are presented as mean values + SD for three biological triplicates. The p-value (Student's t-test) is indicated, *<0.05, **<0.01; ***<0.001. F. Left panel; Schematic representation of pCDNA-ieCMVenh-CMV-GFP (C1) or pCDNA-intGATA6r-CMV-GFP (C2) reporter vectors. The ieCMVenh sequence in C1 was replaced by the "intGATA6r" sequence to generate C2. Right panel; qRT-PCR analysis showing average TBP-normalized fold expression of GFP in MM099MITF-SOX10-PAX3 transfected with C1 or C2 vectors for 48h before treatment or not with Doxycycline (1μg/ml) for 24h. Data information: data are presented as mean values + SD for three technical replicates (n=6). The p-value (Student's t-test) is indicated, ***<0.005, ns=non significant (>0.5).
Figure 5A,B Tethering assay using the 8nt-F-Luc-HhR-poly(A) reporter in S2 cells. Firefly luciferase activities and mRNA levels were normalized to those of the Renilla luciferase and analyzed as described in Fig 2A and B. C,D Tethering assay using the TISU-R-Luc-6xMS2-poly(A) reporter in human HEK293T cells. Samples were analyzed as described in Fig 2C and D. E-G Tethering assay using the β-globin-6xMS2-poly(A) reporter in human HEK293T cells treated with silvestrol or DMSO. Panel E shows the inhibitory effect of silvestrol on the translation of a cotransfected R-Luc reporter. In panels F and G, samples were analyzed as described in Fig 2C and D.
VHHASC disrupts the "prionoid" activities and disassembles ASC specks.Figure 2(A) Time-lapse confocal imaging of the in vitro nucleation of soluble ASC-mTurquoise (red) by ASC-TagGFP (ASC-GFP) specks (green), that were left untreated (None), or pre-incubated with VHHASC or mutVHHASC (200 µg ml−1 for 15 min). Scale bars: 10 μm. (B) Median fluorescence intensity (MFI) of mTurquoise graphed over time showing its polymerization seeded by ASC-TagGFP specks. Each line shows the mTurquoise MFI around a seeding ASC-TagGFP speck. Data is one representative of three independent experiments. (C) WES capillary electrophoresis and immunoblotting of DSS cross-linked in-vitro-generated human ASC-mTurquoise (ASC) specks that were incubated for 1h (RT) with 2, 20 or 200 µg ml−1 of VHHASC, mutVHHASC or with 20 µg ml−1 of a polyclonal anti-ASC Ab (a-ASC pAb). Data is from one representative of four independent experiments.
Figure 6A Schematic of putative mechanism underlying Nox4-mediated regulation of calcium transfer via InsP3R channels to mitochondria. Akti: Akt inhibitor; XeC: Xestospongin C (InsP3R blocker). B Basal mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs in the presence or absence of Akti (1 µmol/L) normalized versus WT MEF control. n=6/group with >20 cells imaged per experiment. C Effect of Akti (1 µmol/L) or XeC (1 µmol/L) on histamine-induced changes in mitochondrial calcium levels in MEFs. The time course of changes in calcium levels is shown on the left and the mean data for peak mitochondrial calcium levels on the right. n=3/group with >40 cells imaged per experiment. D Quantification of cell death in serum-starved WT and Nox4KO MEFs in the absence or presence of Akti or XeC. n=6/group. E Cardiac troponin I (cTnI) levels as a marker of cardiomyocyte necrosis in the perfusate of isolated WT and Nox4KO hearts subjected to ischemia-reperfusion (I/R). XeC was added at a final concentration of 2 µmol/L prior to ischemia. n=6-7/group. F Immunoblotting for the phosphorylation levels of Akt and InsP3R in crude mitochondrial fractions from WT and Nox4KO hearts after I/R. InsP3R was first immunopreciptated and then the precipitate was immunoblotted for total InsP3R and for the phosphorylated Akt-substrate motif RXRXX(pS/T) (p-InsP3R). Mean data shown to the right. n=3/group. G Cardiac left ventricular contractile function in isolated Langendorff-perfused WT and Nox4KO hearts at baseline (Basal) and then after I/R. Hearts were treated with XeC (1 μmol/L) or vehicle control for 20 min prior to ischemia. RPP, heart rate x left ventricular pressure product; DEVP, left ventricular developed pressure. n=6-7/group.
GemC1 induces multiciliate cell differentiation of airway epithelial cells ex vivo.Figure 2A. and B. MTEC cultures were infected with a lentivirus expressing either GFP-GemC1 (GemC1) or GFP as a control and two days later transitioned to ALI culture conditions. Cells harvested at different time points during differentiation were co-stained with antibodies against GFP (green) to mark infected cells and endogenous McIdas(A) or Foxj1(B) (red). Arrows indicate infected cells that are McIdas or Foxj1 positive. Scale bars, 10 µm.C. and D. The percentage of infected cells expressing McIdas(C) or Foxj1(D) at each time point was quantified. Data are presented as the mean values of at least 10 independent fields obtained from at least two independent experiments for each condition. Error bars indicate ± SEM. *p<0.05, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test.