EMBO/sd-character-level-ner huggingface dataset:
Figure 3A. Enrichment of H3.3 in the rearranged allele of the Ig light chain (IGVLR) compared with the unrearranged allele (IGVLUR). Each bar represents the mean of 3 independent ChIPs each assayed with 3 repeat qPCRs (**** = p<0.0001, unpaired T-test). B. Igλ mRNA levels in wild type and h3.3 cells determined by qPCR, normalised to EF-1α expression and to WT. n = 3 (ns = not significant, unpaired T-test). C. Surface Ig (sIg) in multiple clones of wild type (blue) and h3.3 (red) cells. Green = unstained control. D. ChIP for H3K4me3 and H3K36me3 in IGVLR and IGVLUR. Each bar represents the mean of 3 ChIPs for H3K4me3 and IgG and 2 for H3K36me3, each assayed with 3 qPCR repeats (statistical analysis as in Panel A). E. 4sUDRB mapping of transcription kinetics through IGVL. Representative locations of qPCR amplicons (blue) are shown relative to the TSS of IGVL (distance is shown in parenthesis). qPCR signals were normalised to the enrichment at time zero (before release). Error bars represent 1SD of qPCR triplicates.
Quantification of disease-specific single metabolites in blood A Relative values of single metabolites and creatine/creatinine ratios in blood of primary MD, IBM and NMD patients, and MIRAS carriers compared to controls.Figure 3A Relative values of single metabolites and creatine/creatinine ratios in blood of primary MD, IBM and NMD patients, and MIRAS carriers compared to controls. Data information: All data represent mean ± SD. For individual metabolites: *P < 0.05, **P < 0.01, ***P < 0.001 (two sample T-test). For creatine/creatinine (cr/crn) ratio: *P = 0.022, **P = 0.005, ***P = 0.0001 (Kruskal-Wallis test with Dunn´s multiple comparisons test or Mann-Whitney test). B Relative values of single metabolites in blood of adult IOSCA (marked "IOSCA") patients and one IOSCA child patient compared to controls. Data information: All data represent mean ± SD.
Figure 5(A) MEF expressing CRISPR/Cas9 targeting luciferase (wt) or single clones generated from MEF expressing CRISPR/Cas9 targeting the spata2 gene (ko) were treated with mTNF (10 ng/ml) as indicated. The blots were probed with antibodies recognizing P-IkBαIkBα, P-p38, p38, P-JNK, JNK, P-p65, p65 and tubulin. (B) 15P-1 Sertoli cells expressing CRISPR/Cas9 targeting luciferase (as control) or single clones generated from 15P-1 Sertoli cells expressing CRISPR/Cas9 targeting the spata2 gene were treated with mTNF (10 ng/ml) as indicated. The blots were probed with antibodies recognizing P-IkBα, IkBα, P-p38, p38, P-JNK, JNK, P-p65, p65 and tubulin. (C) TAK1-/-MEF were infected to express a CRISPR/Cas9 system targeting luciferase or the spata2 gene, from which single clones were generated. The cells were treated with mTNF (10 ng/ml) or mTNF along with Necrostatin-1 (Nec-1) for 1-3 h as indicated. The blot was sequentially probed with antibodies recognizing PARP, cleaved caspase-3 and tubulin. (D). The same cells as used in (C) were treated as before with TNF (10 ng/ml) or TNF along with Necrostatin-1 (Nec-1) for one and 2 h as indicated. Caspase activity was determined using the fluorogenic substrate DEVD-AMC. (E) Cells from the experiment as shown in (D) were analyzed for apoptosis by Annexin V staining after 2 h. In (D) and (E), a representative experiment is shown with error bars referring to technical replicates. (F) Cells as described before were treated with mTNF (10 ng/ml) for the indicated time and lysates were subjected to immunoprecipitation with an anti-FADD antibody. The immunoprecipitates were subjected to Western blotting and probed for RIPK1, FADD and actin.
Dyn2 inhibition leads to enlarged autolysosomal structures and prevents the autophagy of lipid droplets.Figure 3.Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and co-stained with antibodies specific for LAMP1 (red) and LC3 (green). After Dyn2 knockdown, a juxtanuclear aggregation and enlargement of the LAMP1-positive compartment is observed (C and D, arrows). Increased labeling of LC3 is also detectable after knockdown of Dyn2. (E) Western blotting of Hep3B lysates after a 3-d treatment with either the control or Dyn2-targeted siRNA and further treatment with or without 50 µM leupeptin. Densitometry-based analysis of six independent experiments is shown at the bottom of the figure. (F) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore, in the presence or absence of 50 µM leupeptin. Quantitation of LC3-II levels relative to control are shown below the blots. The data are represented as mean ± SE; *, P ≤ 0.05.
Figure 5a-b) Skinbow mice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day. Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase. a) Photomicrographs show optical sections from a confocal image stack acquired on a whole mounted skin preparation immunostained with anti-BrdU antibody (red). White dots represent hair follicles at different chase time points. a-b) Skinbowmice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day. Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase. b) At 5 weeks of chase, BrdU staining (green) in skinbowskin revealed single cells distant from hair follicles. White dashed circles: hair follicles. White line: small clones. White arrows: BrdU+ cells. (scale bar = 20 µm). c-d) In Skinbowmice induced at P42-46, TPA was applied for one day, 3 weeks post-induction and animals were sacrificed at 48hours following a short pulse of BrdU (2h). c) Photomicrographs show optical sections from a confocal image stack acquired on a 0.5 mm back skin section immunostained with anti-ki67 antibody. Ki67+ cells are labelled with Alexa 647 (red). Back skin from treated and untreated areas was collected 48hrs after TPA topical application. c-d) In Skinbowmice induced at P42-46, TPA was applied for one day, 3 weeks post-induction and animals were sacrificed at 48hours following a short pulse of BrdU (2h). d) Multiple optical section from a Z stack acquisition of whole mount preparation of skin treated with TPA and stained with anti-BrdU (green). Small clones not attached to HF (2 examples figured by dashed lines) can be tracked from the corneocyte layer down to the basement membrane where BrdU staining demonstrates their proliferation. e) (i to iv) Representative individual sections from Z stacks, show small clones (lines) identified in back skin treated (i and ii) or not with TPA (iii and iv). (scale bar = 100 µm). (v to viii) Maximal intensity projection from stack acquired on whole mounted skin. A contribution of the infundibulum and the bulge to the IFE is observed in back skin treated with TPA (v to vi) compared to skin treated with vehicle (vii and viii). (scale bars = 100 µm). f) Reduction in small clone frequency in the areas treated with TPA. Histogram chart showing the clone size distribution and relative frequency of small clones not attached to HF in areas treated with TPA and not treated with TPA in a same mouse. Mann Whitney test, ** p=0.0079. Data pooled from 3 mice represented as median with interquartile range. The number of clones analyzed is indicated on the graph. * p=0.0272. Data are represented as mean ± s.e.m.
Generation of CoA and its utilization in the apicoplast Pantothenate is metabolized into dephospho-CoA through a series of enzymes within the cytosol (PanK1/2, PPCS1/2, PPCDC, and PPAT).Figure 1Pantothenate is metabolized into dephospho-CoA through a series of enzymes within the cytosol (PanK1/2, PPCS1/2, PPCDC, and PPAT). The final step involves the generation of CoA by DPCK and is believed to occur within the apicoplast. Within the apicoplast, CoA is used to generate acetyl-CoA and malonyl-CoA and is used by ACPS to carry out the 4-phosphopantetheine modification of ACP to generate holo-ACP. The CoA produced within the apicoplast is also believed to be exported into the cytosol for utilization throughout the parasite cell. PanK1/2: pantothenate kinase 1 or 2; PPCS1/2: phosphopantothenoylcysteine synthase 1 or 2; PPCDC: phosphopantothenoylcysteine decarboxylase; PPAT: phosphopantetheine adenylyltransferase; DPCK: dephospho-CoA kinase; ACP: acyl carrier protein; ACPS: acyl carrier protein synthase; ACC: acetyl-CoA carboxylase; PDH: pyruvate dehydrogenase.
Increased GABA levels activate Tor in starvation conditions and inhibit pexophagy and mitophagy by acting through Sch9WT and sch9? strains were grown, as described earlier, for pexophagy or mitophagy assays with or without GABA.Figure 4A,B WT cells were cultured under pexophagy (A) or mitophagy (B) conditions with or without GABA and rapamycin. S6 phosphorylation at the indicated time points was analyzed by immunoblotting with a loading control. C Samples were analyzed for Pot1 degradation by immunoblotting (45 kD). D GFP production during mitophagy was analyzed by immunoblotting. E Proposed model for the regulation of pexophagy and mitophagy by GABA. Elevated GABA activates Tor1 in starvation conditions and inhibits pexophagy and mitophagy by activating Sch9.
Figure 5Increase of Vegfr2 expression in the vasculature of TfebEC-/- mice. Representative immunostaining images of TfebEC-/- embryonic vessels (E10.5) stained with anti-endomucin and anti-Vegfr2 Abs (scale bars: 50 µm). Bar graph indicates the Vegfr2 mean intensity only in endomucin+ vessel areas (embryos n=6, mean±SEM; ***p< 0.0001 versus control embryos by Student's t-test). Increase of Vegfr2 expression in the vasculature of TfebiEC-/- mice. Representative immunostaining images (i) and detail (ii) of the vascular front and vascular plexus of the retina (p5) of control and TfebiEC-/- mice with anti-iB4 and anti-Vegfr2 Abs (scale bars: 50 µm). Bar graph indicates the Vegfr2 mean intensity only in iB4+ vessel areas Increase of Vegfr2 expression in the vasculature of TfebiEC-/- mice. Representative immunostaining images of the glomerulus (p17) of control and TfebiEC-/- mice with anti-podocin, anti-CD31 and anti-Vegfr2 Abs (scale bars: 50 µm). Bar graphs indicate the Vegfr2 mean intensity in CD31+ vessel areas VEGFR2 expression is regulated by TFEB. qPCR of VEGFR2 in lung ECs obtained from control and TfebiEC-/- mice (D) Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP VEGFR2 expression is regulated by TFEB. qPCR of VEGFR2 in lung ECs obtained from control and in human sh-TFEB Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP VEGFR2 expression is regulated by TFEB. Immunoblots of total lysates from scr-shRNA and sh-TFEB ECs probed with anti-VEGFR2 and α-tubulin Abs. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α-tubulin Analysis of TFEB binding to the VEGFR2 promoter in human ECs. ChIP was performed using digested chromatin from human control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as "+IgG") or with Ab anti-TFEB (indicated in the bar graph as "+Ab anti-TFEB"), followed by qPCR for VEGFR2.
Loss of caldendrin decreases the number of synaptopodin-positive spines, which can be rescued by caldendrin overexpression, but not by the F-actin stabilizationA Representative confocal images of mouse primary hippocampal neurons stained against homer1 and synaptopodin.Figure 7A Representative confocal images of mouse primary hippocampal neurons stained against homer1 and synaptopodin. The groups compared are wild-type (cald WT), caldendrin knock-out (cald KO), caldendrin knock-out expressing caldendrin-GFP (cald KO cald), and caldendrin knock-out treated with jasplakinolide (cald KO JPK). Scale bar = 5 µm. B - C Quantification of spine density, synaptopodin clusters and synaptopodin-positive spines as shown in A (mean ± SEM. Kruskal-Wallis test with Dunn's multiple comparisons post-test. *** p < 0.0001. cald wt n = 34 cells, 2 experiments; cald KO n = 33 cells, 3 experiments; cald KO cald n = 10 cells, 1 experiment; cald KO JPK n = 14 cells, 1 experiment). (B) The total number of synaptopodin clusters is unaffected by the loss of caldendrin. (C) The number of synaptopodin-positive spines is reduced in cald KO compared to the WT neurons. This phenotype could be rescued by over-expression of caldendrin (cald KO + cald), but not by treatment with JPK (cald KO + JPK). D Model depicting the suggested mechanism of spine apparatus formation. Increased activity results in increased ER visits to the spine, which is mediated by calmodulin-associated myoV. In a subset of spines that are enriched with caldendrin, calcium-dependent myoV-caldendrin interaction transforms the processive motor into an F-actin tether, thereby stabilizing the ER in the spine. Subsequent accumulation of synaptopodin results in the formation of the spine apparatus.
Detection of physical association between AKT and Rab25 by protein complementation assay (PCA).Figure 5Ovarian cancer HEY cells (upper panel) were co-transfected with AKT-IFPN and Rab25-IFPC or with AKT-IFPC and Rab25-IFPN. Cells become fluorescent due to an interaction between AKT and Rab25 bringing the two halves of the fluorescent protein into proximity creating a stable complex and restoring fluorescence. Hela cells stably expressing AKT-IFPN (lower panel) were transfected with Rab25-IFPC or PDK-IFPC as control. A brightfield image corresponding to the fluorescent image is shown and transfected cells indicated by an arrow. Detection of Rab25 and AKT interaction by immunoprecipitation. Ovarian cells expressing HA-tagged Rab25 were IP with anti-HA antibody. The resultant complex was separated by gel electrophoresis and detected by WB using anti-AKT antibody. Generation of Rab25 deletion mutants. Expression of the deletion mutants was detected by immunofluorescence staining (upper panel) and WB (middle panel) using anti-GFP antibody which interacts with YFP. Diagrammatic representation of the structure of the mutants is shown in the lower panel. Rab25 deletion mutants do not alter cellular glycogen and ATP levels. Ovarian cancer cell lysates were collected 24 h post transfection. (a) p < 0.05 versus empty vector transfected cells.
Figure 5(A) Control and/or patient (index case, IC PSMC3) fibroblasts were subjected to a 24-h transfection with pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3 prior to RIPA-mediated protein extraction and subsequent western-blotting using antibodies specific for ubiquitin, PSMC3 (i.e. Rpt5) and α-Tubulin (loading control). (B) Densitometry analysis showing the relative ubiquitin contents detected by western blotting in patient fibroblasts exposed to either pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3, as indicated. The y-axis represents the percent changes in densitometry measurements (of pixel intensities using Image J) which are set as 100 % for cells transfected with the pcDNA3.1/empty vector (mock) at 24 post-transfection (n=4, *: p<0.05, t-test). Bars show the mean +/- SEM. (C) Patient (index case, IC PSMC3) fibroblasts transfected with either pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3 were exposed to a 30-nM treatment of carfilzomib or left untreated (as a negative control). After 16 hours, cells were collected and subjected to RIPA-mediated protein extraction prior to SDS-PAGE and subsequent western-blotting using antibodies specific for ubiquitin, TCF11/Nrf1, Rpt1 (PSMC2), Rpt3 (PSMC4), Rpt5 (PSMC3), Rpt6 (PSMC5), β1, β2, β5, β5i, PA28-α and α-Tubulin (loading control), as indicated. For the TCF11/Nrf1 staining, two exposure times are shown.
Figure 2(A) Immunofluorescence analysis of DSBs associated foci as marked by γH2AX in lung adenocarcinoma (A549) cells, cultured either in low glucose (5.5mM, left panel) or high glucose (30mM, right panel) for 5 days, then treated with etoposide (5µM for 60 minutes). The resolution of DNA-DSBs foci, as marked by γH2AX was monitored over 24 hours after drug treatment, CML (marked in green) served as induction control (scale 10µm). (B) Mean percentage of DSBs positive nuclei as evidenced by γH2AX positivity after pre-treatment of different reducing carbohydrates at 0, 3 or 24 hours. More than 400 cells were analyzed for each bar (mean ± SD, : p<0.01;: p<0.001). (C) Still images of A549 cells showing the live recruitment of hPARP-mCherry and hXRCC4-GFP, at the site of laser-induced DNA-DSBs from the cells pre-treated with the indicated reducing sugars as described in methods. (D) Quantitative analysis of the efficiency of NHEJ-repair in a reporter cell line, as described in Methods. Cells were cultured in low glucose (5.5mM) or high glucose (30mM) for 5 days, fructose (30mM), ribose (20mM), for 3 days (graph bar 4). I-SceI, untransfected cells served as a negative control. Shown is the average from three independent experiments (mean ± SEM, : p<0.01 : p<0.001). (E) Quantitative analysis of the NAD+/NADH ratio in A549 cells, cultured in either low (5.5mM, LG), or high glucose (30mM, HG) for 5 days, fructose (30mM, FR), ribose (20mM, RI), for 3 days (graph bar 4). Quantification was performed after homogenization in extraction buffer (Data represent mean ± SD, : p<0.01;: p<0.001; N=3). (F) A549 cells were cultured in either low (5.5mM; lane 1) or high glucose (30mM; lane 2) for 5 days, fructose (30mM; lane 3), ribose (20mM; lane 4), for 3 days. The cell extracts were then for immunoprecipitation by using anti-PARP, or a non-specific species control antibody. The PARP, or its interacting partner DBC1 was then detected using PARP, or a DBC1 specific antibody.
Figure 5A ChIP analysis was performed on wild type (untagged) and abo1-GFP cells and the resulting DNA analysed by qPCR for centromeric (dh, dg and imr) repeat sequences. Data are the mean of four independent biological repeats and error bars represent ±SEM. P-values calculated using a two-tailed unpaired t-test indicates that all loci are significantly enriched (P < 0.05) relative the untagged control. B The indicated strains expressing GFP-Swi6 were subjected to ChIP analysis. The level of centromeric (dg) repeat sequences relative to the euchromatic control locus, adh1+ was determined by qPCR and scaled to a clr4Δ (- H3 K9me control) mutant. Data are the mean of two independent ChIP experiments and error bars represent the range of the data. C ChIP analysis of histone H3 levels at centromeric (dg) repeat sequences was determined by qPCR. Data are the mean of three independent ChIP experiments and error bars are ±SEM. P-values were calculated using a two-tailed unpaired t-test. D. ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences regions in wild type and pob3Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant difference (P > 0.05) for wild type and pob3Δ cells. E ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences in wild type and pob3Δ cells. Data are the mean of duplicate experiments and error bars represent the range of the data. F ChIP analysis of Abo1-GFP over the dh and imr regions in wild type and swi6Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant differences (P > 0.05) for wild type and swi6Δ cells.
alx1 function in Zebrafish.Figure 6A. Dissected flatmount wild type and alx1-/- zebrafish larvae craniofacial cartilages after Alcian blue staining, the anterior points to the left of the page in all images. The ventral cartilages appear normal, but the alx1-/- anterior neurocranium (ANC) appears narrow, with the midline element that is derived from the frontonasal NCC being absent. The Meckel's cartilage (arrow, MC) is also diminutive. Scale bar: 200 µm. B. Zebrafish alx1 mutants (blue) show reduced detectable expression of alx1 but increased expression of alx3, alx4a compared to wild type controls (green). alx4b expression levels are similar between wild type and alx1-/- lines. Data is represented as the mean of all pooled embryos from three different clutches. The RT-qPCR relative expression values were normalized to elfa and 18S expression using the ΔΔCT method. Data from each clutch were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. Statistical significance denoted by *; p<0.0001 between WT zebrafish and alx1-/- at all measured time points; at 10 ss, 24 hpf and 36 hpf for alx3; and at 24 hpf and 36 hpf for alx4a. Refer to Table EV2 for p-values. C. Dissected flatmount of zebrafish embryos injected with Alx1DN, after Alcian blue staining. The embryos developed an absence of the frontonasal derived median portion of the anterior neurocranium (ANC) and a profound hypoplasia of the Meckel's and ventral cartilages. In the most severely affected zebrafish, a nearly abrogated ANC was observed. Scale bar: 200 µm. D. Lineage tracing experiments in control and Alx1DN mutant embryos revealed aberrant migration of anterior cranial NCC when alx1 is disrupted. In the control animal, the anterior cranial NCC always migrate to contribute to the median portion of the ANC. In contrast, the anterior cranial NCC labeled in the Alx1DN animals fail to migrate to the median ANC, where the ANC structure is narrower and the labeled cranial NCC are found in an anterior and lateral ectopic location (white asterisks). Scale bar: 250µm.
Figure 3Sequence alignment of miR-183 and miR-96 and their target sites in the 3'UTR of FoxO1 (A) or PDK4 (B) from various species. Relative expression of miR-183 and miR-96 in C2C12 myocytes transfected with miR-183 or miR-96 agomir (left) (n=3). Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ago-183 or Ago-96 (right) (n=3). Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ago-183 or Ago-96. Relative expression of miR-183 and miR-96 in C2C12 myocytes transfected with miR-183 or miR-96 antagomir (left) (n=3). Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ant-183 or Ant-96 (right) (n=3). Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ant-183 or Ant-96. Sequence alignment of miR-183 (G) and miR-96 (H) and their target sites in the 3'UTR of ATGL from various species. Relative mRNA expression (I, left) and western blot analysis (J, top) of ATGL in C2C12 cells transfected with agomir for miR-183 (Ago-183) or miR-96 (Ago-96). Relative mRNA expression (I, right) and western blot analysis (J, bottom) of ATGL in C2C12 cells transfected with antagomir for miR-183 (Ant-183) or miR-96 (Ant-96). (n=5 per group for RT-PCR analysis) Relative mRNA expression of ATGL and western blot analysis of FoxO1 and ATGL in C2C12 cells transfected with Ago-183/96 and FoxO1 as indicated (n=3).
Figure 3A The stronger attenuating effect of light in diurnal NHPs than in nocturnal rodents leads to the interspecies variation observed in the response to PF-670. B The simulated PRCs of diurnal NHPs have a higher ratio of maximal magnitudes between advance and delay zones (A/D=1.25) than that of the experimentally measured nocturnal mouse PRC (A/D=0.41 Here, the red line and red range represent the mean±SD of the simulated 12 h light PRCs of the NHP model with different gating and adaptation Similarly, the light PRC of diurnal humans has a higher A/D ratio than that of nocturnal mice D The effect of PF-670 can be heterogeneous due to interindividual variability in photosensitivity. E Model N with a narrow high photosensitivity zone (inset) simulates a smaller magnitude of the advance zone of 12 h light PRC than model W with a wide high photosensitivity zone. F Thus, model N simulates a larger constant stable phase delay than model W when a single daily 25 mpk dose is given at ZT8 under LD 12:12.