EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 5a - c. Pseudo-colored, optical longitudinal cross sections of 5 DAG roots expressing (a) PIN2-GFP (PIN2::PIN2-GFP, P2wt) (b) PIN2S439D-GFP (PIN2::PIN2S439D-GFP, P2D) and (c) PIN2S439A (PIN2::PIN2S439A-GFP, P2A) - all in eir1-4 background - 12 HAT to ammonium or nitrate supplemented media. "e" denotes epidermis and "c" cortex, respectively. Color code represents GFP intensity from low (blue) to high (white) values. Scale bar=50 µm. d. Box plots display the distribution of the cell membrane derived PIN2-GFP fluorescence intensity (FI) values (in arbitrary units, a.u.) in roots transferred to ammonium ((grey, epidermis (ep) and red, cortex (co) and to nitrate (blue, epidermis (ep) and green, cortex (co)). 5 cells per roots were analyzed in at least 9 roots per genotype per treatment. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR. e and f. Microscopic images of 5 DAG old roots expressing (e) PIN2::PIN2S439D-GFP and (f) PIN2::PIN2S439A-GFP 12 HAT to ammonium or nitrate amended media. "e" denotes epidermis and "c" cortex, respectively. White arrows point to PIN2-GFP protein localization on the lateral membranes. g. Box plots display lateralization index (fluorescent signal detected on apical/basal membranes vs inner/outer membranes) of P2wt, P2D and P2A roots transferred to ammonium (grey) or nitrate (red) supplemented medium. At least 24 cells from 5 roots were analyzed per genotype per treatment. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.
Figure 4(A) Consensus replacement. Heatmap depicting the mean expression fold changes compared to wild-type expressions after replacing with motif consensus sequences derived by XXmotif. Constitutive and developmental promoters are highlighted in red and green, respectively. (B) Heatmap depicting the mean expression fold changes compared to wild-type expressions after replacing consensus insertion into motif-less core promoters. (C) Boxplots depicting log expression change and significance level upon inserting consensus motifs of INR, INR3 and Ohler7 motifs (columns in A) into the core promoters (rows in A). Left panel: INR into CG15674 (two-sample t-test **p = 0.0033); middle panel: INR2 into CG10915 and CG15674 (Wilcoxon rank-sum test ***p = 0.00018); right panel: Ohler7 into Geminin, CG10915 and CG15674 (Wilcoxon rank-sum test ****p = 3.4×10-5). The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements. (D) Heatmap depicting the mean expression fold changes compared to wild-type expressions for motif knockout and substitution with positionally or functionally equivalent motifs from other architectures. Constitutive and developmental promoters are highlighted in red and green, respectively. (E) Boxplot depicting the effects of INR being substituted by INR2 in cas and CG8157 (all measurements in these two core promoter constructs were pooled together; Wilcoxon rank-sum test **p = 0.0051 for comparing substitution with knockout (significant) and p = 0.17 for comparing substitution with wild-type (not significant). The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. 3-4 biological replicate measurements.
Figure 6(A) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT, tor1Η, and gad8Η strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-tubulin IB is shown as a control for loading. The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. P-Taf12 to Taf2 ratios were calculated from 3 independent experiments and individually plotted in a graph below the IBs, together with the mean and SE. Averaged values from all WT controls grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 3). (B) P-Taf12 was followed by anti-FLAG IB of protein extracts from sty1Η cells (left panel) or ssp2Η cells (right panel), grown in rich conditions (R) or starved for 45 minutes (S). An anti-tubulin IB is shown as a control for loading. The star (*) symbols label an unspecific band detected by the anti-FLAG antibody in S. pombe. Shown are IBs that are representative of 2 independent experiments. (C) TAP-tagged Gad8 was immuno-precipitated using IgG-sepharose beads (IP) from strains containing FLAG-tagged Taf12, grown either in rich medium (left panels) or starved for 45 minutes (right panels). TAP-tagged Gad8 was eluted from beads using the TEV protease, releasing a shorter form of Gad8 (CBP-Gad8). Eluates were loaded and immuno-blotted (IB) using anti-FLAG or anti-CBP antibodies, together with 1% of whole cell extracts (WCE), to detect Taf12 co-precipitation with Gad8. A strain which does not contain any TAP tag is shown as a negative control for the IP. Shown is an IB representative of two independent experiments. (A,C) The star (*) symbols label unspecific bands detected by the FLAG or the CBP antibodies in S. pombe. (D) Endogenous Gad8-HA was affinity purified from starved cells, in order to activate its kinase activity (left and middle panels), and mixed with recombinant, purified GST-Taf12, GST-Taf12-5A, or GST-Fkh2 proteins (right panels) in a kinase assay buffer. Each assay was then analyzed by IB and probed with an anti-phospho-AKT substrate antibody.
Blood vessel dysmorphia is associated with macrophage polarization in progressive grades of human glioma.Figure 4A. Glut1immunohistochemistry on human grade II and grade IV glioma samples (50µm depth stack). Blood vessel diameter increases and network complexity decreases with increasing grade. Statistical analysis: A.C.D. one-way ANOVA followed by multiple comparisons Tukey's test; Scale bars: A-B: 100µm. B. CD68, MHCII and MRC1 immunohistochemistry on human grade II and grade IV glioma samples (50µm depth stack). Statistical analysis: A.C.D. one-way ANOVA followed by multiple comparisons Tukey's test; Scale bars: A-B: 100µm. C. Macrophage polarization quantification. Low-grade glioma is characterized by high number of M1 polarized macrophages. High-grade glioma is characterized by high number of M2 polarized macrophages. Statistical analysis: A.C.D. one-way ANOVA followed by multiple comparisons Tukey's test; D. Correlation analysis of M2 like tumor accumulation (MRC1 positive) with vessel dysmorphia depending on the tumor grade. Statistical analysis: A.C.D. one-way ANOVA followed by multiple comparisons Tukey's test; E. Quantification of macrophage distance to the nearest blood vessel. Macrophages relocate close to blood vessels in late stage gliomas. For n of patients per group refer to TableEV1. Statistical analysis: E. Spearman correlation test. Error bars: meanSD. Scale bars: A-B: 100µm.
Figure 5A. Analysis of Fn colonization in mouse xenograft tumors by IHC. An inoculum of 5×106 HCT116 cells was injected subcutaneously and bilaterally into the nude mice. Once the tumors became visible (after 3-4 days), approximately 5×106 CFU of Fn 12230 washed in PBS or 1 mg/ml Congo Red (CR) were injected into the tumors. The tumors were extracted one week later, and the formalin-fixed and embedded (FFPE) tissues were stained with mAb 7H7. Note the brown stains in tumor injected with PBS-washed tumor, but not in CR-washed tumor. Scale bar = 20 μM. B. Effects of secreted amyloid-like FadA on tumor growth. HCT116 cells were injected into the nude mice as described above, followed by injection of 5×106 CFU of Fn 12230 (Fn, n=7), lam (n=3) or US1 (∆fadA, n=4). The tumor volumes were measured immediately before bacteria injection (Day 0, designated as 100%), and at 3 days after injection (Day 3). The horizontal lines represent averages of tumor volumes (left panel). The individual tumors are shown (right panel). *p < 0.05, **p < 0.01, ***p< 0.001 (t-test). C. Effect of anti-amyloid antibody OC on FadAc-mediated tumor growth. HCT116 cells were injected into the nude mice (n=6) as described above, followed by injection of 4 μl of 2 mg/ml FadAc mixed with 6 μl OC antibody or rabbit IgG control (rIgG) into tumors on the opposite sides. The tumor volumes were measured on Day 0 and Day 5 as described above. The horizontal lines represent the averages (left panel). The individual tumor pairs are shown (right panel, n=6). *p < 0.05, **p < 0.01 (t-test). D. Effect of detergent-resistant pellets prepared from Fn 12230 (Fn) and US1 (∆fadA) on tumor growth. HCT116 cells were inoculated into the nude mice as described above. In one group of mice (n=3), sarkosyl-resistant pellets prepared from Fn 12230 and US1 (∆fadA) were injected into the tumors on opposite sides. In another group (n=5), sarkosyl-resistant pellets prepared from Fn 12230 were mixed with OC or rabbit IgG control (rIgG) before injecting into the tumors on opposite sides. The tumor volumes were measured described above. The vertical lines represent the standard deviations. The individual tumor pairs are shown (right panel). *p < 0.05, **p < 0.01, ***p< 0.001 [compared to US1 (∆fadA), t-test].
a) FLAG–AMBRA1 was transfected into HEK293 cells together with ULK1WT or ULK1K46I Myc-tagged proteins.figf1(a) FLAG-AMBRA1 was transfected into HEK293 cells together with ULK1WT or ULK1K46I Myc-tagged proteins. Protein extracts were immunoprecipitated (IP) using anti-FLAG antibody. (b) HEK293 cells were grown either in normal or in EBS medium for 1 h; protein extracts were immunoprecipitated using anti-ULK1 antibody or rabbit immunoglobulin as a negative control (IgG). Autophagy induction is shown by p62 degradation. (c) HEK293 cells were treated as in b, in the presence of leupeptin and stained by anti-AMBRA1 (red) and anti-ULK1 (green) antibodies. The merge of the two signals and DAPI (blue) for nuclear staining is shown in the right panels. Scale bar, 6 μm. The outlined areas are shown at a higher magnification in the lower panels. White arrows point to the co-localization puncta. Measurements by Metamorph indicate a co-localization ratio of 46% between ULK1 and AMBRA1 on autophagy induction. (d,e) ULK1 and AMBRA1 constructs are illustrated. Asterisks mark nonspecific bands corresponding to immunoglobulins. (d) HEK293 cells were co-transfected with vectors encoding AMBRA1 together with ULK1-Myc FL (full length) or its deletion mutants encoding the kinase and the proline-rich domains (Δ829-1,051), and the ULK1 carboxy-terminal domain (Δ1-828). Protein extracts were immunoprecipitated using anti-Myc antibody. (d,e) ULK1 and AMBRA1 constructs are illustrated. Asterisks mark nonspecific bands corresponding to immunoglobulins. (e) HEK293 cells were co-transfected with vectors encoding ULK1-HA together with β-galactosidase-Myc (βgal), AMBRA1 full-length (FL), and F1, F2 or F3 Myc-tagged proteins. Protein extracts were immunoprecipitated using anti-HA antibody. The relative amounts of co-immunoprecipitatedULK1 and F1 or F3, respectively, are the same (see upper and lower left panel). (f) HEK293 cells were co-transfected with vectors encoding AMBRA1, ULK1-Myc and ATG13-FLAG. Protein extracts were immunoprecipitated using anti-FLAG antibody. (g) HEK293 cells were transfected with scramble siRNA or ATG13 siRNA. Protein extracts were immunoprecipitated using ULK1 antibody or IgG. The band density ratio of immunoprecipitated AMBRA1 relative to immunoprecipitated ULK1 is analysed, with the control ratio arbitrarily defined as 1.00. n = 3 extracts prepared from independent experiments; data are presented as means±s.d. and significance is P = 0.04. Uncropped images of blots/gels are shown in Supplementary Fig. S6. Source data of statistical analysis are shown in Supplementary Table S1.
Figure 5A Western blot analysis of PPARγ protein levels in control and TRAF4-depleted cells and quantification of protein levels normalized to β-actin expression. B Western blot analysis of PPARγ protein levels in control and TRAF4 overexpressing cells and quantification of protein levels normalized to β-actin expression. C, D Western blot analysis of PPARγ protein levels in control and TRAF4 overexpressing cells in the absence or presence of MG132 or 3MA and quantification of protein levels normalized to β-actin expression. E Ubiquitination of endogenous PPARγ in control and TRAF4 overexpressing cells were differentiated by treatment with DMI. Cells were treated with MG132 for 6 h.
a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT with or without MSO.figf4(a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT with or without MSO. After 24 h, the cells were starved of serum, amino acids and glucose in D-PBS containing the indicated inhibitors and stimulated with amino acids for 10 min. Cell lysates were analysed for protein levels of phospho-S6K (Thr 389) and actin. Shown are representative blots of three independent experiments. (b,c) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and MSO (0.25 M) for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy. (b) Shown are representative pictures (n=4). Scale bars, 100 μm. (c) Quantification of co-localization between mTOR and LAMP2 as seen in b. Depicted are the mean ±  s.e.m. of the percentage of co-localized pixels from four experiments. *P0.05, **P0.01. (d) DLD1 cells expressing FOXO3(A3)-ER were treated with or without 4-OHT, MSO and BafA1 (200 nM). After 24 h, cell lysates were analysed for protein levels of LC3 and tubulin. Shown are representative blots (n=4). (e) DLD1 cells expressing FOXO3(A3)-ER were transfected with glutamine synthetase (GS) siRNA or a non-targeting siRNA and stimulated with 4-OHT in glutamine-free DMEM containing 0.1% FBS. After 24 h, cell lysates were analysed for protein levels of glutamine synthetase, LC3 and actin. Shown are representative blots (n=2) (f) DLD1 cells expressing FOXO3(A3)-ER were transfected with glutamine synthetasesiRNA or non-template (NT) control siRNA and stimulated with 4-OHT for 16 h. Cells were lysed and equal amounts of proteins were analysed for levels of p62 or glutamine synthetase. Shown are representative blots (n=2). (g) DLD1 cells were stimulated with LY294002 or PKB inhibitor VIII for 24 h. Cell lysates were analysed for protein levels of LC3 and actin. Shown are representative blots (n=4). Uncropped images of blots are shown in Supplementary Figs S7 and S8.
Core circadian clock transcript distributions show area dependence and distinct gene-to-gene correlation structures.Figure 2(A) Log conditional mean mRNA count for a given area as a function of the log area. Each cell is sorted by area by placing it in one of 20 bins that range from the 5th to the 95th percentile. The conditional mean given the area E[m|A] is then the mean mRNA count of each bin. The area is normalised such that the average area is equal to one. Solid lines represent a least-squares linear fit to the data. (B) Top image: Dual-channel smFISH targeting Bmal1 and Cry1 simultaneously, taken at 17h after Dex synchronisation. Blue, nuclei stained with DAPI; green dots, Bmal1 transcripts; red, Cry1 transcripts. Bottom image: Dual-channel smFISH targeting Nr1d1 and Cry1 simultaneously, taken at 25h after Dex synchronisation. Blue, nuclei stained with DAPI; green dots, Nr1d1 transcripts; red, Cry1 transcripts. Scale bar: 20μm. (C) Bivariate distributions of mRNA counts per cell for dual-channel smFISH targeting either Bmal1 and Cry1 or Nr1d1 and Cry1. Each dot corresponds to a single cell, and the contours represent KDE estimates of the density. Time represents the number of hours after Dex synchronisation, and R represents the Pearson correlation coefficient.
Figure 5BMPR1A is highly expressed in HCC tumor tissues provided by Wang's cohort (GSE14520). Kaplan-Meier analyses of liver cancer outcomes in the cancer genome atlas (TCGA). P value for Kaplan-Meier curves was determined using a Mantel-Cox log-rank test. Representative immunohistochemistry staining of BMPR1A was detected in HCC samples. Scale bar, 100 μm. BMPR1A overexpression rescues sphere formation ability reduced by HAND2-AS1 depletion in HCC samples. Representative sphere formation is shown on the left panel. Scale bar, 100 μm. Percentages of sphere-forming cells were calculated as means ± SD (right panel). n=4. BMPR1A depletion reduces tumor-initiating capacity. n=12 mice per group. Representative whole-body imaging of Huh7-Luc cells transduced with scramble (Ctrl) or BMPR1A shRNAs. Quantification for tumor numbers of 7 weeks after tumor cells orthotopically implanted to B-NSG mice. Calculated data are shown as means ± SD (n = 5 mice per group). Macroscopic appearance of livers from 10-month-old mice treated by DEN or vehicle. Scale bar, 1 cm. Black arrows indicates tumors. Representative H&E and immunohistological staining with Ki67 antibody in liver sections of 10-month-old DEN-treated WT and Bmpr1a-/- mice. Scale bar, 100 μm. Right: Quantitation of Ki67-positive cells in DEN-treated mice. Data are shown as means ± SD. n=5. BMP signaling inhibitor noggin decreased tumor sphere formation. Scale bar, 100 μm. Right: Data are shown as means ± SD (n=4).
The malate-aspartate shuttle supports the increase of both glutamine and fatty acid metabolism induced by MPC inhibition.Figure 4(A) Schematic representation of proposed mechanism by which MPC inhibition engages the malate-aspartate shuttle (MASh). GOT1/2, glutamic-oxaloacetic transaminase; OGC1, oxoglutarate carrier 1; Aralar, mitochondrial aspartate-glutamate carrier; CPT, Carnitine Palmitoyltransferase. The coordinated work of OGC1 and Aralar provide a mechanism to allow fatty acid oxidation in the absence of pyruvate. Fatty acid oxidation generates acetyl-CoA which condenses with oxaloacetate to generate citrate. In the absence of pyruvate, oxaloacetate can be generated by glutamine metabolism. (B) Effect of transaminase inhibition on UK5099-induced energy expenditure. Brown adipocytes were pre-treated with vehicle (DMSO), 50-100 nM UK5099, 1 mM aminooxyacetic acid (AOA), or a combination of UK5099 and AOA for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of the tested compounds. Data show quantification of non-stimulated OCR from n=5 individual experiments. (C-E) Effect OGC1 downregulation on UK5099-induced energy expenditure. Primary brown adipocytes were transduced with Scramble RNA (Scramble) or shOGC1 (OGC1 KD) adenovirus. Cells were pre-treated for 2 h with vehicle (DMSO) or 100 nM UK5099 before OCR measurements. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM,) oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (C) Representative OCR traces averaging 4 technical replicates. (D) Quantification of non-stimulated OCR as measured in (C) from n=6 individual experiments. (E) Quantification of Eto-sensitive OCR as measured in (C) from n=6 individual experiments. Data were normalized to vehicle for each experiment. (F-H) Effect of Aralar1 downregulation on UK5099-induced energy expenditure. Primary brown adipocytes were transfected with Scramble RNA (Scramble) or siRNA for Aralar1 (Aralar1 KD). Cells were pre-treated for 2 h with vehicle (DMSO) or 100 nM UK5099 before OCR measurements. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM), oligomycin a (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin a (AA; 4 µM) were injected where indicated. (F) Representative OCR traces averaging 4 technical replicates. (G) Quantification of non-stimulated OCR as measured (G) from n=4 individual experiments. Data were normalized to vehicle for each experiment. (H) Quantification of Eto-sensitive OCR as measured in (F) from n=4 individual experiments. (I) Brown adipocytes transfected with Scramble RNA or Aralar1 siRNA were treated for 24 hours with vehicle (DMSO) or 10 µM UK5099. Data shows quantification of the ratio of aspartate to glutamate abundance as measured by GC-MS from n=3 individual experiments.
Figure 4(A) CD86 and CD40 expression was quantified on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) DCs in non‑draining (ndLN) and draining (dLN) popliteal lymph nodes of WT and Irf3‑/‑Irf7‑/‑ mice injected with poly(I:C) or PBS. Data are pooled data from 2 independent experiments. (B) Experimental set-up. Irf3‑/‑Irf7‑/‑ recipient mice were lethally irradiated and reconstituted with WT (GFP+) and Irf3‑/‑Irf7‑/‑ (GFP‑) bone marrow cells (BMCs) mixed at different ratios. Chimeras were injected in the footpad 4 weeks later with poly(I:C) and WT and Irf3‑/‑Irf7‑/‑ DCs from non‑draining and draining popliteal lymph nodes were analyzed 24h after poly(I:C) injection by flow cytometry. (C) Microscopy images of sliced draining lymph nodes in CD11c‑eYFP:Irf3‑/‑Irf7‑/‑ chimeras with 90:10 (left) and 5:95 (right) ratios. Type 1 IFN‑competent cells appear in green. Mosaic images were generated by acquisition of overlapping imaging fields. Scale bars represent 25 µm. (D-E) CD86 and CD40 expression on WT (blue dots) and Irf3-/-Irf7-/- (orange dots) DCs from draining lymph nodes of mixed‑bone marrow chimeras with indicated WT:Irf3‑/‑Irf7‑/‑ ratios. (D) Representative dot plots of CD86 and CD40 expression on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) DCs of chimeras injected with PBS or poly(I:C). (E) Flow cytometric quantification of CD86 and CD40 expression on WT and Irf3‑/‑Irf7‑/‑ DCs in chimeras injected with PBS or poly(I:C). Results from 5:95 and 10:90 chimeras were similar and pooled. Data are pooled from 3 independent experiments.
Fig.Figure 10A, B Time-lapse series of single-cell wound healing in a larva expressing mCherry-Atg8a (magenta) (A58>mCherry-Atg8a) and endogenously tagged FasIII (GFP gene trap; green), a transmembrane component of septate junctions. (B) Higher magnification of the post-wounding time points in (A). Images from Movie EV18; see also Movies EV19 and EV20. Each frame is a merge of 68 planes spaced 0.28 μm apart. A, n=11 larvae C Transmission electron microscopy of larval epidermis overexpressing Atg16B shows autophagosomes in direct contact with plasma membrane (magenta arrows) and at the sites of cell-cell junctions (orange arrows). The lower panel shows a higher magnification of Atg16B image in Fig 3D.