EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A) GAPC enzymatic activity was measured in wild-type crude extracts treated with H2O (mock), and 100 µM of Tyr-Asp, Tyr, Asp, and Ile-Glu. (B-D) The enzymatic activities of GAPC (B), GAPA/B (C), and GAPN (D) were measured in the wild type and double k.o. mutant gapc1gapc2 treated with H2O (mock), and 100 µM of Tyr-Asp. Data information: Data are mean ± SEM of n=4 (A-D; four technical replicates) , significance was assessed using unpaired two-tailed Student t-test. (E) Schematic representation of glycolysis and pentose phosphate pathways. G6P: glucose 6-phosphate; F6P: fructose 6-phosphate; FBP: fructose 1,6-bisphosphate; DHAP: dihydroxyacetone-phosphate; G3P: glyceraldehyde 3-phosphate; 1,3bisPGA: 1,3bis-phosphoglycerate; 3PGA: 3-phosphoglycerate; PPP: pentose phosphate pathway; R5P: ribose 5-phospate. (F) Glucose-labeled C6 and C1 flux experiment in 10-day-old Arabidopsis seedlings. C6/C1 ratio for control (water treated) and Tyr-Asp (100 µM) treated wild type seedlings after 35, 70 and 145 min is shown. Data information: Data are mean ± SEM of , n=3 (F; three independent flasks) significance was assessed using unpaired two-tailed Student t-test. (G) NADPH/NADP+ ratio. (H) NADH/NAD+ ratio. Ratios were calculated from the NAD+, NADH, NADP+, and NADPH measurements. Data information: Data are mean ± SEM of n=5-6 (G-H; five to six independent seedlings flasks). significance was assessed using two-way ANOVA (p≤0.05; letters show significance associated with the Tyr-Asp treatment). C stands for control; n.s.: not significant.
Figure 1Confocal micrographs show CK7 (green) and vimentin (red) in patient-derived HGSC cells cultured in 3D collagen for 7 d. Scale bars: 50 μm. Confocal micrographs of EphA2 (red) in HGSC cells cultured in 3D collagen for 7 d and treated for 72 h with 10 μM cisplatin (5 μM for OCKI_p01). The intensity of EphA2 is comparable only between mock and treatment conditions for each patient. Scale bars: 20 μm. Chart illustrates EphA2 fold change after treatment. Mock is set to one. N = 3.
Effect of a dual expression of actins on cell viability and cytoskeletal organization.Figure 6A Schematic of the experiment performed in a diploid yeast background. B Doubling times of diploid yeast strains cultures, grown at 25ºC in YPD medium. N2/Ca and Ca/N2 cells express the same actins but markers used for selection are exchanged. Data are presented as mean +/- SD (n = 3 for all conditions; technical replicates). (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). C Phalloidin staining depicting F-actin organization. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm. D In vivo actin network deviation indexes. Data are presented as mean +/- SD (n = 30 for all conditions). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). E Polarity Indexes. Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Kruskal-Wallis test, with multiple comparisons). F Effect of CK-666 (75 µM) on the organization of the actin cytoskeleton. Cells were stained with phalloidin after 30 min incubation with CK-666. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm. G In vivo actin network deviation indexes of cells treated with DMSO or CK-666. Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).
Figure 1A) Condensates formed by purified TDP-43 at 150 mM salt observed by brightfield microscopy at different time points. B) TDP-43 condensates without RNA control or in the presence of A(GU)6 (8 μM), A(GU)18 (2.6 μM), or A(CA)6 (8 μM) (Supplementary Table 1) at 150 mM NaCl (4 μM TDP-43). C) RNA binding-deficient TDP-43 mutants generated by site-directed substitutions in RRM1 (F2L) or in both RRM domains (F4L) and substitution of RRM1 and RRM2 with monomeric GFP (N-GFP-C). Phase separation of the mutants without added RNA (control) or in the presence A(GU)6 (8 μM), A(GU)18 (2.6 μM), or A(CA)6 (8 μM) at 150 mM NaCl (4 µM protein).
Figure 4Immunoblots showing the levels of PIF4 and PIF5 proteins at 22°C in 4-d-old Col, cbf1-1, cbf1-2, and CBF1-myc seedlings grown in darkness (D) or continuous W or R light. Anti-RPN6 was used as a sample loading control. numbers below the immunoblots indicate the relative band intensities of PIF4 or PIF5 normalized to those of RPN6, respectively. Phenotypes and hypocotyl lengths of 4-d-old Col, cbf1-1, pif4 pif5, cbf1 pif4 pif5, 35S:PIF4 and 35S:PIF4 cbf1 seedlings grown at 22°C in continuous W or R light. Error bars represent SD from 20 seedlings. **P < 0.01 and ***P < 0.001 (two-tailed t-test) for the indicated pairs of seedlings. Scale bar = 1 mm. NS, not significant. Immunoblots showing the PIF4 protein levels in 4-d-old Col, 35S:PIF4, and 35S:PIF4 cbf1 seedlings grown at 22°C in D or continuous W or R light. Anti-RPN6 was used as a sample loading control. numbers below the immunoblots indicate the relative band intensities of PIF4 normalized to those of RPN6, respectively.
High resolution morphology of DAPk family protein-induced cell death.Figure 3.(A) Transmission electron micrographs of 293 cells transfected with luciferase (1) p55/TNFR1 (2a-2c), DRP-1 Δ73 (3a and 3b; part of fig. 3b is magnified on its right), and DAPk ΔCaM (4). The different stages of autophagic development are depicted as follows: immature autophagic vesicles (double arrow), mature autophagic vesicles and autolysosomes (black arrow), and residual bodies (dashed arrow). Empty vacuoles (white arrow) are also shown. Arrowheads show condensed and fragmented chromatin. m, mitochondria; g, Golgi apparatus. Bar, 1 μm. (B) Transmission electron micrographs of MCF-7 cells transfected with luciferase (1), p55/TNFR1 (2a-2c), DRP-1 Δ73 (3a and 3b), and DAPk ΔCaM (4). Immature autophagic vesicles (double arrow), autophagic vesicles (black arrow), and residual bodies (dashed arrow) are shown. Insets in B, 2b and 2c, correspond to empty vacuoles, whereas those in B, 3a and 4, correspond to autophagic vesicles. Arrowheads show condensed and fragmented chromatin. m, mitochondria; dm, darkened mitochondria; n, nucleus; g, Golgi apparatus; v, vacuoles; ly, lysosomes; er, endoplasmic reticulum.
TNC expression, abundance and localization of CD8 TIL in breast tumors and survival of human breast cancer patients(A-C) Adjacent human breast cancer tissue microarray (TMA) sections were stained with the indicated antibodies, scale bar, 50 μm, arrows point at CD8 TIL (A) and, CD8 TIL were counted per field (B), or in the stroma and nest, respectively (C), N = 103 TNC+ tumors, N = 116 TNC- tumors.Figure 7(A-C) Adjacent human breast cancer tissue microarray (TMA) sections were stained with the indicated antibodies, scale bar, 50 μm, arrows point at CD8 TIL (A) and, CD8 TIL were counted per field (B), or in the stroma and nest, respectively (C), N = 103 TNC+ tumors, N = 116 TNC- tumors.. (B) ** p = 0.0055, Fisher's exact test. The central band represents the median, the ends of the box the first and the third quartiles, and the whiskers reach from each quartile to the minimum or maximum. (C) ** p = 0,0055, Mann-Whitney test. (D-G) Kaplan Meier analysis to address correlation of TNC expression (D), total numbers of CD8 TIL (E), combined high (above median) CD8 TIL and high or low (above/below median) TNC (F) and stromal CD8 cells (G) with metastasis-free survival (MFS) of breast cancer patients. Stromal CD8- or CD8+ is defined as below or above median stromal CD8 TIL per area. Log-rank test. (H, I) Correlation of TNC and CD8a levels with overall survival (OS) using survival analysis on breast cancer patient cohort GSE 19783-GPL6580. Hazard ratio (HR) and p values are indicated in Appendix Fig. S5N. Log-rank test.
Figure 8A. C-terminal amino acid sequence of KV10.1, indicating the predicted importin-α recognition sequence (bold) and the deleted fragment n KV10.1ΔNLS (red). B. Deletion of the CLS does not preclude functional expression at the plasma membrane. Two electrode-voltage clamp recordings from Xenopus oocytes injected with cRNA for a KV10.1 channel lacking the CLS at the C-terminus revealed voltage-gated currents after stimulation using the potential depicted in the inset.C. The Conductance/voltage relationship of the deleted channel was similar to that of wild-type (shown for comparison as a solid line). D. In the absence of serum, the C-terminus of KV10.1 reduced the number of cilia (maintained cells deciliated) also after deletion of the CLS (data for controls is reproduced from Figure 5A)The CLS is required to induce deciliation. When hTERT-RPE1 and 3T3 cells transfected with KV10.1 or KV10.1ΔCLS were serum starved, and serum was reintroduced after 24 hours for 4 h, KV10.1ΔCLS failed to reduce the fraction of ciliated cells. E. The CLS is required for KV10.1-induced tumorigenesis. CHO cells were transfected with the indicated constructs and implanted into the flank of nude mice. KV10.1ΔCLS transfected cells did not induce larger tumors than the control.
Figure 4(A and B) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then (A) with LLOMe or (B) starved in EBSS. (C) IL-1β levels in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe. (D) Immunoblot analyses of p62 levels in THP-1 cells subjected to knockdowns as indicated and starved for 3 h. (E) mRFP-GFP overlapping areas of tandem HeLa cells reconstituted with flag-pro-IL-1β and myc-Caspase 1 and starved for 3 h. Mask overlay, iDev software-defined objects (primary objects, cell outlines; internal secondary objects, GFP (top), mRFP (middle), or their overlapping area (bottom)). Bars, 10 μm. (F) IL-1β levels in supernatants of starved tandem HeLa cells reconstituted with flag-pro-IL-1β and myc-Caspase 1 as in E.
Figure 1A. Upregulation of Rabl2 during multiciliogenesis. Cultured mTECs were induced to undergo multicilia formation by growing at an air-liquid interface (ALI) for the indicated time and subjected to immunoblotting. Ift52, Ift139, Arl6, and acetylated tubulin (Ac-tub) served as ciliary markers. Gapdh served as the loading control. B. Rabl2 levels in different tissues from 8-week-old mice. C. Rabl2 knockout strategy and representative genotyping PCR results using the indicated primers (P1-P4). FRT, flippase recognition target; LacZ, β-galactosidase gene; Neo, neomycin-resistant gene; IRES, internal ribosome entry site; LoxP, Cre recombinase recognition site. D. Complete depletion of Rabl2 in tissues (from 8-week-old mice) upon its gene knockout. Normal ciliary formation in representative bright-field micrographs of the indicated tissues (from 8-week-old mice) or cells. Cultured mEPCs were fixed at day 7 post serum starvation. Ac-tub served as ciliary marker. DAPI labeled nuclear DNA. Typical cilia or flagella are indicated by arrows, in which yellow arrows denote primary cilia. Normal ciliary formation in representative , scanning electron micrographs of the indicated tissues (from 8-week-old mice) or cells. Cultured mEPCs were fixed at day 7 post serum starvation. Ac-tub served as ciliary marker. DAPI labeled nuclear DNA. Typical cilia or flagella are indicated by arrows, in which yellow arrows denote primary cilia. Normal ciliary formation in representative scanning electron , or immunofluorescent micrographs of the indicated tissues (from 8-week-old mice) or cells. Cultured mEPCs were fixed at day 7 post serum starvation. Ac-tub served as ciliary marker. DAPI labeled nuclear DNA. Typical cilia or flagella are indicated by arrows, in which yellow arrows denote primary cilia. I, J. Normal ciliary formation of Rabl2-/- MEFs. MEFs derived from E12.5 mouse embryos were serum-starved for 48 h to induce primary cilia (I). Statistical results (J) were from three independent experiments. Ciliogenesis of at least 200 cells was scored in each experiment and condition. Ciliary lengths of 80 cells were measured in each experiment and condition and the results of the same genotypes were pooled together.
Figure 4A) U2OS and PALB2 cell lines were transfected with UNC (Negative Control) or PALB2 siRNA and 24 hours later Doxycycline was added to the PALB2 cell lines. Left panel, cells were treated the following day with IR (5 Gy) and 2 hours later Nocodazole was added for 6 hours, or treated with only Nocodazole for 6 hours. Right panel, cells were treated with IR (5 Gy) and left to recover for 16 hours. Cells were fixed and stained with the pMPM2 antibody to detect the mitotic cells by flow cytometry. The percentages of mitotic cells in the IR+Nocodazole samples were normalized to the Nocodazole samples (left panel, n=3, error bars=SEM). For the samples with the 16 hour recovery after IR the percentage of mitotic cells in the WT-PALB2 expressing cells was set to one (right panel). (n=3, **p<0.005, *p<0.05, unpaired Student's t-test, error bars=SEM). B) The relative intensity of phosphorylated H2AX (pS139) was examined in the total population of WT- and TMA-PALB2 expressing cell lines 18 hours post exposure to IR (5 Gy). Cell lines were prepared as in above, fixed at the indicated time point, and stained for pH2AX. Cells were imaged with a 20X air objective on a Scan^R workstation (Olympus), mean relative pH2AX intensity was calculated from background subtracted images using the Scan^R analysis software. (* p<0.0001, unpaired Student's t-test). C) Number of 53BP1 nuclear in TMA-PALB2 expressing G1 daughter cells. Cell lines were prepared as above and left untreated (NT) or exposed to IR (3 Gy), and 16 hours later Cytochalasin B was added for 8 hours to block cytokinesis. The irradiated bi-nucleated cells were scored for 53BP1 nuclear bodies. Scale bar = 10μm.
LC3B immunostaining.Fig 6A) Effect of bortezomib treatment on LC3B immunostaining of Cal-78 and SW-1353 cells. Double immunolabeling with DAPI (blue) and anti-LC3B (green) antibodies was performed as described in the methods section. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II (bar: 20 μm). B) Cal-78 and SW-1353 cells were treated either with bortezomib alone or in combination with the lysosomal protease inhibitors E64d and pepstatin A (Inh; striated bars) and cell viability was analysed by the MTS assay. Untreated cells were measured as controls (light grey; n = 8, mean ± S.D.).
a, Top: representative micrographs of autophagosomes and autophagolysosomes in hepatocytes from RagA+/+ neonates fasted for 1 h.f3a, Top: representative micrographs of autophagosomes and autophagolysosomes in hepatocytes from RagA+/+ neonates fasted for 1 h. Typical autophagosome with a double limiting membrane (arrows); autophagosome and several autolysosomes (arrowheads). Scale bars, 5 µm. Bottom: frequency of the two types of organelle (early: autophagosomes; late: autophagolysosomes) detected in cell profiles of hepatocytes and skeletal myocytes from RagA+/+ and RagAGTP/GTPneonates. b, Protein extracts from livers of neonates at Caesarean section (0 h) and fasted for 10 h were immunoblotted for autophagy markers LC3B and p62. c, Protein extracts from skeletal muscle and heart from neonates at Caesarean section (0 h), fasted for 1 and 2 h, were immunoblotted for indicated proteins. d, Triggering of autophagy by amino-acid withdrawal in MEFs. MEFs were deprived of amino acids for the indicated time points, whole-cell protein extracts were obtained and mTORC1 activity and autophagic activity determined by immunoblotting. e, Recombinant LC3B-GFP was expressed in RagA+/+ and RagAGTP/GTPMEFs and LC3B localization, in the presence and absence of amino acids, monitored by fluorescence microscopy. LC3B-GFP (green fluorescent protein) clustering, indicative of autophagy, was observed in amino acid-starved RagA+/+ but not RagAGTP/GTPMEFs. Scale bar, 10 µm. f, Localization of recombinant TFEB-GFP was determined in MEFs as in e. Nuclear (active) TFEB was observed in RagA+/+ MEFs upon amino-acid withdrawal.
Figure 8A and B Blood glucose levels and serum insulin levels in 2-h GTT 12 weeks after HFD. Inset graphs in (A) and (B) depict the respective analysis of the area under the curve (AUC). Data shown are from one experiment (n = 6 for each genotype), representative of a total of two independent experiments. Results are means ± SEM. *P = 0.0287, **P = 0.0173, ***P = 0.0332, ****P = 0.0453, #P = 0.0077, ##P = 0.0344, ###P = 0.0412, ####P = 0.0495, ^P = 0.0435, ^^P = 0.0304 (Student's t-test). C ITT 12 weeks after HFD. Data shown are from one experiment (n = 6 for each genotype), representative of a total of two independent experiments. Results are means ± SEM. *P = 0.0127, **P = 0.0232, ***P = 0.0373, ****P = 0.0385 (Student's t-test). D Insulin-stimulated Akt phosphorylation (Ser473) in WAT, liver, and skeletal muscle of control and ad-FADD mice. The experiments were performed in triplicate. E Adiponectin mRNA was measured in isolated epididymal adipocytes by RT-PCR. Data are expressed as mean ± SEM from three independent experiments. *P = 0.0044 (Student's t-test). F-H Total adiponectin levels (F), HMW adiponectin (G), and the ratio of HMW to total adiponectin (H). n = 5 for each genotype. Data are expressed as mean ± SEM. *P = 0.0217, **P = 0.0116, ***P = 0.0175 (Student's t-test). F-H Total adiponectin levels (F), HMW adiponectin (G), and the ratio of HMW to total adiponectin (H). n = 5 for each genotype. Data are expressed as mean ± SEM. *P = 0.0217, **P = 0.0116, ***P = 0.0175 (Student's t-test). I Basal and insulin-stimulated glucose uptake in control and FADD deficient primary isolated adipocytes. Data shown are from one experiment (n = 4 for each genotype), representative of a total of two independent experiments. Results are means ± SEM. *P = 0.0073 (Student's t-test). J Basal and insulin-stimulated glucose uptake in 3T3L1 cells differentiated to adipocytes and infected with lentivirus containing FADD shRNA or scrambled shRNA control. Data are expressed as mean ± SEM from three independent experiments. *P = 0.0092 (Student's t-test). K Basal and insulin-stimulated glucose uptake in control and FADD deficient primary isolated adipocytes treated with DMSO or 5 μM MK886. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0135 (one-way ANOVA). NS, not statistically significant.
A) After 4 h of starvation in 1% K‐acetate medium in the presence of PMSF, autophagic vesicles can be visualized in vacuoles of wild‐type and pep4Δ prb1Δ cells using Normaski optics.Figure 2(A) After 4 h of starvation in 1% K‐acetate medium in the presence of PMSF, autophagic vesicles can be visualized in vacuoles of wild‐type and pep4prb1 cells using Normaski optics. In aut2Δ and aut7Δ cells no autophagic vesicles can be seen. Bar represents 20 μm. (B) Total protein breakdown of cells starved on nitrogen‐free medium is significantly reduced in aut2Δ and aut7Δ cells compared with wild‐type cells. pep4Δ (pra1Δ) cells are almost completely impaired in starvation‐induced vacuolar protein breakdown. Cells were pulse‐labeled with [35S]methionine and chased on non‐radioactive starvation medium. Aliquots were taken at the indicated times and acid soluble small peptides generated by proteolysis were determined according to Straub et al. (1997). (C) Survival during starvation. Like pep4Δ (pra1Δ) cells, which are almost completely impaired in vacuolar proteolysis, aut2Δ and aut7Δ cells exhibit a significantly reduced survival rate during starvation on nitrogen‐free medium as compared with wild‐type cells. Aliquots of cells incubated in 1% K‐acetate were plated and growing colonies were determined according to Straub et al. (1997).
Figure 2A. Colony forming assays show increased proliferative potential of cells cultured in 3C compared to freshly isolated epidermal cells (Epi d0; control) (mean ±SEM; n=5; *p ≤ 0.05, Mann-Whitney U-test). 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2. B. Full thickness skin reconstitution assay with freshly isolated epidermal cells (Epi d0) or 3C cultures shows that cells cultured in 3C retain their self-renewal capacity and multipotency. A representative recipient of 4 mice/condition is shown. Right panel shows quantification of hair follicle density. Note that 3C cultures give rise to more hair compared to controls (mean ±SEM; n=4; *p ≤ 0.05, Mann-Whitney U test). C. Long-term culture of cells in 3C was performed as shown in the left panel. 3C cultures maintain a stable population of CD34+α6+ long term (data are shown using a Box-and-Whisker plot: box indicates 25th and 75th percentiles; error bars represent 10th and 90th percentiles; middle line is the median; n=3-5 independent experiments). p: passage. D. Full thickness skin reconstitution assay using 3C cells from passage 1 (p1) and passage 15 (p15) show that cells cultured in 3C maintain their multipotency and self-renewal capacity in long-term cultures. A representative recipient of 4 mice/condition is shown. See also Appendix figure S2.
Figure 7A PBL27 does not interact with MAPKKK5-KD:C in a yeast two-hybrid assay. B Addition of CERK1 to an in vitro kinase assay enhances phosphorylation of MAPKKK5-KDK375M:C by PBL27. Asterisks indicate artificial bands derived from CERK1:IC. C CERK1 does not phosphorylate the C-terminal domain of MAPKKK5 in vitro. The in vitro kinase assay was performed with recombinant proteins of GST-PBL27, MAPKKK5-C and the intracellular kinase domain of CERK1 (GST-CERK1:IC). The protein loading control was shown by staining with Coomassie Brilliant Blue. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography. Asterisks indicate artificial bands. D The alanine substitution mutations at the residues conserved in the activation loop of reduces the phosphorylation of PBL27 by CERK1:IC. The in vitro kinase assay was carried out with recombinant proteins of GST-PBL27K112E , GST-PBL27K112E, 3xA and CERK1-IC. E The kinase activity of GST-PBL273xA was analyzed by the in vitro kinase assay using [32P]γ-ATP.
a) Dvl2 underwent autophagy-mediated degradation.figf2(a) Dvl2 underwent autophagy-mediated degradation. E-cadherin was used as a loading control for membrane fraction, and tubulin as a loading control for cytosolic proteins. The asterisk indicates the phosphorylated form of Dvl2. (b) The half-life of Dvl2 was prolonged in Atg5−/− cells. MEFs were subjected to starvation (starv.) for the indicated durations and harvested for immunoblotting with the indicated antibodies. Dvl2 levels were quantified (right panel). Autophagy induction is indicated by the levels of LC3 II and p62 degradation. (c) Reintroduction of Atg5 in Atg5−/− MEFs restored starvation-induced Dvl2 degradation. MEFs infected with recombinant adenovirus expressing GFP or Atg5 were starved for the indicated durations, and the cell lysates were subjected to immunoblotting with the indicated antibodies. (d) Dvl2 degradation was slowed down in Atg7−/− MEFs. MEFs were subjected to starvation for the indicated durations and harvested for immunoblotting with the indicated antibodies. Dvl2 levels were quantified (right panel). (e) The continued degradation of Dvl2 in Atg5−/− cells is proteasome-dependent. MEFs were starved for 2, 4 or 8 h with MG132 (1 μM) or BFA1 (0.1 μM) for different durations, followed by anti-Dvl2 immunoblotting to detect endogenous Dvl2 protein levels. Tubulin served as a loading control. Uncropped images of blots are shown in Supplementary Information, Fig. S8.