EMBO/sd-character-level-ner huggingface dataset:
Miro regulates peroxisomal size, morphology and number.Figure 5A) Representative images of catalase staining (peroxisomes) in WT, Miro1KO, Miro2KO and DKO MEFs. B) Bar graph comparing average peroxisomal area of WT Miro1KO, Miro2KO and DKO MEFs ( n = 60 cells per condition over three independent experiments). Data information: Calculation of statistical significance in B) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm. C) Comparison of total peroxisomal number between WT, Miro1KO, Miro2KO and DKO MEFs. n = 60 cells per condition over three independent experiments. Data information: Calculation of statistical significance in C) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm. D) Representative images of Control (GFP-tagged 1-70 of Tom70), GFPMiro1 and GFPMiro2 in WT MEFs stained with catalase (peroxisomes). E) Representative images of Pex14 (peroxisomes) in DKO MEFs expressing either GFP (control) or GFP-tagged variant 4 of Miro1 (GFPv4). F) Bar graph comparing average peroxisomal area of Control, GFPMiro1 and GFPMiro2 overexpressing WT MEFs (n=60 cells per condition over three independent experiments). Data information: Calculation of statistical significance in F) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm. G) Comparison of average area of individual peroxisomes between DKO MEFs expressing either GFP (control) or GFP-tagged variant 4 of Miro1 (GFPv4) (n=30 cells per condition over three independent experiments). Data information: Statistical test used in G) was a two-tailed Student's t test. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.
a) MCF7 cells stably expressing Myc–TEV–Flag–Beclin 1 (MEF–Beclin 1) and parental controls were lysed and subjected to MEF-tag-based purification.figf1(a) MCF7 cells stably expressing Myc-TEV-Flag-Beclin 1 (MEF-Beclin 1) and parental controls were lysed and subjected to MEF-tag-based purification. Proteins bound to MEF-Beclin 1 were isolated and detected by SDS-PAGE and silver staining. Seven bands specific for MEF-Beclin 1 are numbered. (b) Six bands were identified by nanoflow LC-MS/MS and MASCOT software. (c) MCF7 cells were lysed and subjected to immunoprecipitation using the indicated antibodies. (d) Differential subcellular fractionation. Post-nuclear supernatant of A549 cell lysates were spun at 20,000g for 10 min and the supernatant was further spun at 100,000g for 60 min. Equivalent quantities of each supernatant and pellets from the same number of cells were subjected to western blotting with the indicated antibodies, including anti-Sec61β (ER), cathepsin D (lysosome), EEA1 (early endosome), transferrin receptor (plasma membrane and recycling endosome) and GAPDH (cytosol). (e) Gel filtration analysis. The supernatant fraction of A549 cell (or Atg14L knockdown) lysates were subjected to a Superose 6 column and each fraction was immunoblotted with the indicated antibody (see Supplementary Information, Fig. S2e). Relative amounts of each fraction determined by densitometry were plotted. Vo, void fraction. The elution pattern of each protein was reproduced in several experiments. Full scans of the gel and blots in a, c and d are available in Supplementary Information, Fig. S6.
Figure 5Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg/kg of the MMP2/9-inhibitor SB3-CT (MMPi). , Co-immunostaining for pSMAD3 (green) and IGFBP7 (magenta) on representative tumor-containing colon sections derived from Apcfl/fl-Cdx2CreERT2 mice treated with vehicle (A) MMPi (B). Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg/kg of the MMP2/9-inhibitor SB3-CT (MMPi). Quantification of pSMAD3+ cells (left panel) and pSMAD3 staining intensity per pSMAD3+ cell (right panel) on at least five arbitrarily selected tumor areas per section of MMPi (n=6) or vehicle (n=6) treated mice. effect of in vivo TGFBRi and MMPi treatment of mice for two weeks on colon tumor formation. D, Experimental setup. Apcfl/fl-Cdx2CreERT2 mice were treated with Tamoxifen and, one day post treatment, injected with either MMPi (n=10) or the TGFBRi SB431542 (n=10) at 40mg/kg for two weeks with five injections per week. Vehicle treated mice: n=11. effect of in vivo TGFBRi and MMPi treatment of mice for two weeks on colon tumor formation. Apcfl/fl-Cdx2CreERT2 mice were treated with Tamoxifen and, one day post treatment, injected with either MMPi (n=10) or the TGFBRi SB431542 (n=10) at 40mg/kg for two weeks with five injections per week. Vehicle treated mice: n=11. Average tumor size per mouse was assessed during colon resection one week after the end of treatments (E). Model of activation of latent TGFβ stored in extracellular matrix (ECM) by neutrophil-secreted MMP9, leading to suppression of effector T cells and promotion of Tregs.
Fig.Figure 2(A) Cartoon representation of the NHE9 homodimer shown from the extracellular-side in the endosomal lumen (left), and along the membrane plane (right). Ion translocation 6-TM core transport domains (blue), dimerization domains (orange) and linker helix TM7 (grey), are coloured as in Fig. 1E with the respective helices numbered. (B) Cartoon representation of dimeric NHE9 ΔCTD from the cytosolic side with the electrostatic surface representation through the ion-binding site of one monomer (coloured blue to red, for positive to negative charges respectively). The strictly conserved ion-binding residue Asp244 is labelled and shown in yellow as sticks. The inward open ion-binding cavity is encircled. (C) NHE9 ΔCTD as in (B), but coloured according to conservation scores from the alignment of 650 mammalian NHE1-9 representative sequences calculated with ConSurf server (Ashkenazy et al., 2016) (see Materials and Methods). (D) left: cartoon representation of the NHE9 ion-binding site in the 6-TM core transport domain, which is made up of two broken helices TM5a-b (green) and TM12a-b (purple) accessible to a sodium ion from the cytoplasm (yellow sphere). right: ion-binding site residues are shown as yellow sticks and labelled with the corresponding residues in PaNhaP (PDB id: 4cz8) shown as grey sticks.
The complex intracellular trafficking of Mpl.Figure 9Mature Mpl reaches the surface through the conventional ER‐Golgi pathway and bears fully processed N‐linked carbohydrate. Immature Mpl appears to be routed through an autophagic pathway for unconventional secretion, providing an alternate pathway for membrane expression of core‐glycosylated Mpl. Once delivered to the cell surface, both forms of Mpl can be recycled or stored in calcium‐dependent and independent compartments capable of fusion with the plasma membrane. In some cells, such as K562 cells, Mpl is packaged into exosomes and secreted.
Figure 3(A) Streptozotocin-induced diabetic C57BL/6 (H2b) mice with IL-33 treatment were transplanted with BALB/c (H2d) islets. Mice were sacrificed at day 7, 30 and 80 post-islet transplantation. (B-D) Proportion or numbers of CD4+Foxp3+ Tregs in the spleens, kidneys and islet grafts of normal, islet transplant mice receiving vehicle and islet transplant mice with IL-33 treatment (at day 7, 30 and 80 post-islet transplantation). Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; ***P<0.001. (E) Representative confocal microscopy images of immunostaining for CD4, Foxp3 and insulin in islet grafts. Scale bar = 50 µm. (F-H) Proportion or numbers of ILC2s in the spleens, kidneys and islet graft of mice with or without IL-33 treatment. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001. (I) Representative confocal microscopy images of immunostaining for CD127, ST2, CD3 and insulin in islet grafts. Scale bar = 50 µm. (J) The mRNA expression of IL-25, IL-33 and TSLP in islet grafts of mice with or without IL-33 treatment was examined by qPCR, and expressed relative to the control of each experiment. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.
Figure 1U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining with DAPI, anti-human centromere autoantibody (ACA), and antibodies for Bub1 and TOP2A. Example images are shown (A). The immunofluorescence intensity ratios of centromeric TOP2A/ACA (B), centromeric TOP2A/arm TOP2A (C), and arm TOP2A/DNA (D), were determined on around 600 (B and C) or 400 (D) chromosomes in 20 cells. ns, no statistical significance. Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm. U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining with DAPI, and antibodies for Bub1 and TOP2A. Lysates of nocodazole-arrested mitotic cells were immunoblotted with the indicated antibodies (E). ns, no statistical significance. U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining. Example images are shown (F). U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining. The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 500 chromosomes in 15 cells (G). Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm. U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. Example images of the immunofluorescence staining are shown (H). U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. The immunofluorescence intensity ratios of centromeric Bub1/ACA (I) were determined on around 300 chromosomes in 15 cells. Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm. U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. The immunofluorescence intensity ratios of centromeric TOP2A/ACA (J), and centromeric TOP2A/arm TOP2A (K) were determined on around 300 chromosomes in 15 cells. Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm. L U2OS cells stably expressing CB-GFP or CB-Bub1-K-GFP were treated as in (H). Example images of the immunofluorescence staining are shown.
Figure 5A MEF eEF2K WT, KO or KO reconstituted with human eEF2K (Flag-eEF2K) were analyzed for cell growth at indicated times. Fold change (means ± s.e.m of triplicate) of the cell number just before treatment are shown. Histogram shows percentage of growth inhibition after 72h of 10 μM NFR. P values are 1way ANOVA with Bonferroni posttest calculated from 3 independent experiments. *** P value ≤ 0.001. B-F Dose response curves for cell viability after 48h NFR measured using MTS assay. Curves and bar graph for EC50 are mean ± s.e.m. of 3 independent experiments performed in triplicate. For curves, p values are 2way ANOVA analysis of variance. For bar graph, p values are two tails unpaired Student's T-tests (B, E), or 1way ANOVA with Bonferroni (C) or Dunnett's multiple comparison posttests (D and F). *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001. (B,C) eEF2K -/- MEFs show a decrease susceptibly to NFR compared to eEF2K +/+ controls (B) whereas eEF2K reconstitution restores NFR sensitivity (C). B-F Dose response curves for cell viability after 48h NFR measured using MTS assay. Curves and bar graph for EC50 are mean ± s.e.m. of 3 independent experiments performed in triplicate. For curves, p values are 2way ANOVA analysis of variance. For bar graph, p values are two tails unpaired Student's T-tests (B, E), or 1way ANOVA with Bonferroni (C) or Dunnett's multiple comparison posttests (D and F). *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001. (D-F), HeLa (D), A549 (E) and MCF7 (F) clones with CRISPR-Cas9 generated eEF2K deficiency (CrEEF2K) show a decrease susceptibly to NFR compare to control cells (CrLuci). G NFR-mediated toxicity assessed using AnnexinV/PI staining and FACS analysis after 24h treatment. Histogram shows the percentage of dead cells (AV+/PI+). Data are mean ± s.e.m. of 3 independent experiments. P values are 2way ANOVA with Bonferroni posttest. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001.
a) Increased tumour initiation in KRas;Atg5fl/fl mice as compared with KRas;Atg5fl/+ littermates.f3(a) Increased tumour initiation in KRas;Atg5fl/fl mice as compared with KRas;Atg5fl/+ littermates. Representative histological sections at 2 weeks after AdCre inhalation are shown. Insets show typical lung lesions. Haematoxylin and eosin staining. Scale bars, 2 mm for whole lung section; 50 μm for insets. (b) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) in KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation. n=6 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link). (c) Tumour growth curves based on mean tumour-to-lung area ratios (±s.e.m.) determined in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates at the indicated time points. At least three different planes from each lung were analysed. n≥6 per cohort for each time point. *P0.05, **P0.01 (unpaired, two-sided Wicoxon test). (d) MicroCT analysis of lung tumours of KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice 18 weeks after AdCre inhalation. Representative data from individual mice are shown. Scale bars, 5 mm
Figure 4Immunoblot analysis of SNC1 (A-B), SUMM2-3HA (C), RPS4HA (D), RPP4myc (E), RPS2HA (F) and RPM1myc (G) protein levels in the indicated genotypes. For all panels, equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.
Figure 8Huh-7 cells were first serum-starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493/503, TRAPPC9, Rab18 and γ-COP.A. In the merged images, bodipy 493/503 was pseudocolored in green, TRAPPC9 in red, and ADRP in blue. In the colocalization between TRAPPC9 and ADRP on the right side, the ADRP signal was re-colored to green for easy visualization. Bar = 10μm. Huh-7 cells were first serum-starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493/503, TRAPPC9, Rab18 and γ-COP.B. Bodipy 493/503 was pseudocolored in green, Rab18 in red, and γ-COP in blue. In the colocalization between Rab18 and γ-COP on the right side, the γ-COP signal was re-colored to green for easy visualization. Bar = 10μm.
Coherent feed-forward networks coordinate the regulation of FT by the circadian clock.Figure 7Coherent feed-forward networks coordinate the regulation of FT by the circadian clockAs highlighted in the discussion, the flowering system is based on a combination of coherent feed-forward pathways, allowing a single component to play multiple reinforcing roles in the system (Mangan Alon,2003). This is highlighted here in the case of the EC and the PRRs.The EC can repress FT expression through at least three partially redundant pathways, involving coordinated control of CDF1, FKF1 and GI levels.Similarly, the PRRs can activate FT expression through at least three pathways. Rectangles denote protein species, and parallelograms denote transcript species. Solid and dashed lines indicate direct and indirect forms of regulation, respectively.
Figure 1Immunofluorescent detection of Nanog and Esrrb protein in wild-type E14Tg2a ESCs cultured for three days in GMEMβ/FCS/LIF. (A) Widefield images of a representative colony. : Immunofluorescent detection of Nanog and Esrrb protein in wild-type E14Tg2a ESCs cultured for three days in GMEMβ/FCS/LIF. Quantification of the mean Nanog and Esrrb fluorescence levels measured in nuclei identified by automatic segmentation of single optical plane images obtained by confocal microscopy. Values are expressed in Arbitrary Units (AU). Oct4 negative cells, also identified by immunostaining, were excluded from the analysis. The red line represents the moving average of the data distribution. Representative of three independent experiments each including at least 1000 nuclei. C: Colonies of TNG E-tdT ESCs showing GFP expression from Nanog and Esrrb-tdTomato fusion protein expression from Esrrb after three days in GMEMβ/FCS/LIF. D: Flow cytometric analysis of Esrrb and Nanog fluorescent reporter expression in SSEA1+ TNG E-tdT ESCs cultured for three days in GMEMβ/FCS/LIF with or without puromycin. E: Immunofluorescent detection and quantification of total Nanog and Esrrb protein in E14Tg2a ESCs overexpressing Nanog (left) or Esrrb (right) cultured for three days in GMEMβ/FCS/LIF. Parental E14Tg2as are shown as a reference. The red lines indicate the negative thresholds defined by staining ESCs differentiatied for 3 days in the absence of LIF and presence of retinoic acid. F: Flow cytometric analysis of Esrrb-GFP and Nanog-mCherry fusion protein expression in SSEA1+ NER ESCs cultured for three days in GMEMβ/FCS/LIF. G: Histograms showing expression levels of Esrrb-GFP and Nanog-mCherry from the respective targeted endogenous alleles in SSEA1+ NER ESCs overexpressing Nanog (left) or Esrrb (right) cultured for three days in GMEMβ/FCS/LIF. Parental NER ESCs are shown as a reference, and wild-type E14Tg2a set the negative thresholds. H: Comparative flow cytometric analysis of Esrrb-2a-tdTomato and Nanog:GFP expression in undifferentiated SSEA-1+ TNG E-2a-tdT or Nanog-null ES∆N E-2a-tdT ESCs cultured for three days in GMEMβ/FCS/LIF. Data are shown as dot plots (top) and histogram (bottom).
MYC controls TSC1 expression through transcriptional and miR-15a-mediated regulation.Figure 5Accumulation of EU-labeled (Click-It, Invitrogen) TSC1-mRNA over 3 h in high MYC (-Tet) versus low MYC (+Tet) P493-6 cells (mean ± st. dev., n=3 technical replicates). Drawing of the TSC1 promoter and luciferase-reporter constructs with the predicted E-box (CACGTG, pos. -317/-322) indicated. Relative luciferase units (RLU) of co-transfection experiments using the indicated TSC1-promoter-reporters with MYC normalized to empty pcDNA3 expression vector in HeLa cells (mean ± st. dev., n=3). Immunoblot shows MYC overexpression and α-tubulin as loading control. TSC1 mRNA turnover determined by pulse and chase (Click-It, Invitrogen) over 8 h in P493-6 cells, either treated with tetracycline to repress MYC or left untreated (mean ± st.dev., n=3 technical replicates). miR-15a expression determined by qRT-PCR in P493-6 cells treated with tetracycline for 3 days (+Tet) to suppress MYC expression or in untreated (-Tet) cells (mean ± st. dev., n=3 technical replicates). Immunoblots showing expression levels of TSC1, S6K/P-S6K and α-tubulin in P493-6 and HEK293T cells overexpressing miR-15a or a control miRNA. At the right miR-15a overexpression was determined by qRT-PCR in P493-6 and HEK293T cells (mean ± st. dev., n=3 technical replicates). Immunoblot showing TSC1 expression, S6K/P-S6K and α‑tubulin in MCF-7 cells transfected with LNA modified anti-sense miR-15a oligos or control LNAs. At the right, successful miR-15a knockdown determined by qRT-PCR in MCF-7 cells is shown (mean ± st. dev., n=3 technical replicates). Relative luciferase activities (RLU) derived from reporter constructs containing TSC1-3´UTR wild type sequences or with mutated miR-15a seed sequence binding site on co-transfection with miR-15a in MCF-7 cells (mean ± st. dev., n=3 biological replicates). The drawing at the left shows the TSC1-3'UTR with the position of the miR-15a/16-1 seed sequence and used mutation indicated. Rate of oxygen consumption in P493-6 (-Tet) cells ectopically expressing miR-15a or control-miRNA vector, under basal conditions and in response to 10 μM DNP or 10 μM oligomycin where indicated (mean ± st. dev., n=8 biological replicates).
Figure 3(A) Quantification of major acetylation sites on histones H3 and H4 in young and midlife flies. Midlife flies display a transformed histone acetylation profile including elevation (H4K12, H3K9, H3K9/14 and H3K23) and reduction (H4K8 and H3K18). Data were normalized to 1-week-old (young) flies. N=15-22 (histone 4) and 12-19 (histone 3) per group. Error bars indicate the S.E.M. (B) Male flies show a gradual increased lifespan and prolonged pre-mortality plateau phase at various length of exposure to 18º C (cold) compared to the 25º C (control) ambient temperature. Survival 90% control=28 days, cold=58 days. Median survival; Control=38 days, cold=80 days. N=295 (control) and 238 (cold). Log Rank Test, χ2 =527.1, p<0.0001. (C) Comparison of the activity of key metabolic enzymes between midlife flies grown at 18ºC and 25ºC. Midlife flies grown at 25ºC have lower CS and ATPCL activity to midlife flies at 25ºC. CS, citrate synthase; ATPCL, ATPcitrate lyase. N=9 midlife vs 6 cold midlife for CS and 7 midlife vs 6 cold midlife for ATPCL. (D) Metabolite levels show reduced levels citrate and ATP in midlife flies at 18ºC. The levels of acetyl-CoA are not significantly different despite a trend of reduced levels in 18ºC flies. N=6 midlife vs 5 cold midlife. (E) Midlife flies grown at 18ºC show a general reduction of histone acetylation states including acetylation states which are increased upon aging (Figure 3A - H4K12, H3K9, H3K9/14 and H3K23). Of note, H4K8 is increased in midlife when flies are kept at 18ºC. N=16-21 (histone 4) and 13-16 (histone 3) per group. Data for the midlife flies were normalized to young flies as shown in panel A. (*P<0.05, **P<0.01, ***P<0.001). Error bars indicate the S.E.M. in all the graphs. Unpaired two tailed t-tests were used for calculating the p-values in A, C-E. All male flies were collected from a mixed male/female population.