EMBO/sd-character-level-ner huggingface dataset:
Figure 6A, Endo-GFP-ORP2 cells were treated with control or OCRL siRNAs for 2 days, stained with PI(4,5) P2 antibody and imaged by confocal microscopy. B, Cells stably expressing NPC1-mCherry were treated with control or OCRL siRNAs for 2 days, or transfected with GFP-ORP2 or -ORP2-mHHK (PI(4,5) P2 binding-deficient mutant) for 1 day, stained with PI(4,5) P2 antibody and imaged by confocal microscopy. For FAK inhibition, GFP-ORP2 transfected cells were treated with 10 μM PF228 for 4 h. Dashed lines indicate cell edges. Arrowheads indicate colocalization of PI(4,5) P2 and NPC1. C, Quantification of B. Values from Ctrl siRNA and Vector control were pooled and are shown as Ctrl siRNA/Vector ctrl. Mean ± SD, n = 20-43 cells pooled from 2 independent experiments. Student's t-test. D, E, Cells stably expressing NPC1-mCherry were transfected with GFP-ORP2 or GFP-ORP2-mHHK for 1 day and were indicated, treated with 10 μM PF228 for 4 h. For degron ORP2 cells, NPC1-mCherry was transiently transfected for 2 days and cells incubated without (-IAA; ORP2 present) or with IAA (+IAA; ORP2 depleted) for 1 h. Arrowheads indicate tubular NPC1 organelles. Live cell images were acquired by widefield epifluorescence miroscopy and the longest NPC1 tubule per cell was measured. Mean ± SD, n = 102-122 cells. Students's t-test. Please see also ctrl (MovieEV4) and PF228 treated cell (MovieEV5), both videos 1 min recordings with 1 s frame rate.
Figure 3Dose-response curves representing the rate of mCherry expression from the MDM2 (pink dots) and CDKN1A (purple dots) promoters as a function of total p53 levels. Values of expression levels were averaged across all cells in the first 15-h response to the long duration Nutlin-3 dosing regimen. Solid black lines represent the best fit to a Hill function model of promoter activation. Dashed lines indicate the half-maximal threshold of p53 levels for promoter activation and the half maximal promoter activity for each promoter.
Coq9Q95X mice showed moderate CoQ deficiency.Figure 2A-F CoQ9 levels in tissue homogenates from brain (A), cerebellum (B), heart (C), kidney (D), extensor (E) and triceps surae (F) of male and female Coq9+/+ and Coq9Q95Xmice at 6 and 12 months of age. Data are expressed as mean ± SD. Statistical analysis was performed on 6-month-old Coq9+/+mice versus 6-month-old Coq9Q95Xmice and 12-month-old Coq9+/+mice versus 12-month-old Coq9Q95Xmice. **P < 0.01; ***P < 0.001. Student's t-test (n = 8 for each group).
a) Alignment of the Beclin 1 CC domain sequences from various species.f1(a) Alignment of the Beclin 1 CC domain sequences from various species. The coloured segments and the italic abcdefg on top of the sequence indicate the heptad repeats typical of CC structure. The coloured spheres below the sequence indicate the residues at a (orange) and d (blue) position of each heptad repeat. (b) The antiparallel dimeric CC structure of the Beclin 1 CC domain. Each monomer helix is coloured in gradient varying from blue at N-terminal to red at C-terminal. The italic letters N and C indicate the N- and C-terminal ends. The dot in the centre marks the two-fold symmetry in the CC domain structure. (c) Helical wheel presentation of the 13 heptad repeats in Beclin 1 CC domain. Only 7 heptad repeats are mapped, representing the unique half of the antiparallel symmetric dimer. (d) Close-up view of the canonical and imperfect a-d' pairings. Each residue is illustrated in ball-and-stick model. Main-chain atoms are coloured blue and side-chain atoms are coloured pink for canonical pairings or golden for imperfect pairings. The a-d' packing is illustrated by van der Waals spheres depicting the side-chain atoms. (e) The distribution of electrostatic charges on Beclin 1 CC domain surface. Colouring scheme is according to GRASP. The oval circle highlights the E224a-D221e' repulsive pair.
Figure 6HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted of FUNDC1 (FUNDC1 KD) were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies. GFP-Parkin stably expressing HeLa cells with mock treatment (Mock) or depleted of FUNDC1 (FUNDC1 KD) were incubated with 20 μM CCCP for 2 h and immunostained for Tom20. Scale bar, 5 μm. 293T cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h, immunoprecipitated with anti-FLAG M2 beads and then analyzed by IB using the indicated antibodies. Three percent of lysates was analyzed as input. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1. GFP-Parkin stably expressing HeLa cells were treated and then subjected to PLA using antibodies against FLAG and PGAM5. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1. Scale bar, 5 μm. Values are means ± SEM (n = 3). *P<0.05 and **P<0.01 as compared with Mock (+CCCP) by paired Student's t-test.
The AMPK-ULK1 interaction is important for ULK1-mediated autophagy.Figure 3(A) 293T cells were transfected with empty vector or HA-tagged wild type (WT) or deletion mutant (Δ654-828) ULK1 and subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with the indicated antibodies. (B) U-2OS cells stably expressing GFP-LC3 were infected with lentivirus expressing human Ulk1 shRNA (shhUlk1) or scrambled shRNA (shScr) and subjected to selection with 1 µg/ml puromycin for 5 days. The cells were then transfected with wild type mouse Ulk1 (mUlk1), mUlk1 Δ654-828 or control empty vector for 24 h, incubated in serum-free DMEM overnight, and cultured in complete medium with or without 10 mM metformin for 20 h. The images were obtained using a fluorescence microscope. The scale bars represent 10 µm. (C) The number of GFP-LC3 dots per GFP-positive cell in (B) was quantified (mean ± s.d.; n = 81). (D) U-2OS cells stably expressing GFP-LC3 were transfected with 0, 2 or 4 µg of pcDNA3 encoding HA-tagged ULK1 deletion mutant (554-828). The total amount of plasmid DNA used for each transfection was normalized at 4 µg with empty vector. After 24 h transfection, the cells were starved in nutrient-free medium for 1.5 h, fixed in 4% paraformaldehyde, and analyzed by fluorescence microscopy. The number of GFP-LC3 dots per GFP-positive cell was quantified (mean ± s.d.; n = 100). Statistical significance was determined by Student's t-test and the asterisks indicate P<0.0001.
P5 promotes hair growth ex vivo.Figure 3A Ex vivo cultured human HF treated with vehicle, P5, or APN for 6 days; scale bar: 1 mm; dotted gray bar: the length of pigmented hair shaft. B The net length of elongated hair shaft is measured and compared to the control group (n = 38 HFs from 3 independent donors / group); Two-way ANOVA with Dunnett's test compared to the control group. C IF staining (p-AMPK) of the HF bulb area. Yellow arrow indicates p-AMPK signal; scale bar: 100 µm. D IF staining (Ki-67) of the HF bulb area; scale bar: 100 µm. E The numbers of Ki-67-positive cells normalized to DAPI-stained cells (n = 8 HFs / group); One-way ANOVA with Dunnett's test compared to the control group.
a) Histological analysis of paraffin sections at 14 h and 24 h RPF shows intact salivary glands present in dUTX1 compared with control at 24 h RPF.f3(a) Histology analysis of paraffin sections at 14 h and 24 h RPF shows intact salivary glands present in dUTX1 compared with control at 24 h RPF. Ovals indicate the position of salivary glands and fragments. Scale bar represent 50 μm. Quantification of the salivary gland phenotypic data was at 24 h RPF. (b,c) Caspase staining and activity are reduced in dUTX1 salivary glands at 14 h RPF. (b) Cleaved caspase-3 antibody (green) and Lamin antibody (red) staining are shown in control and dUTX1 salivary glands at 14 h RPF. Scale bar represents 50 μm. (c) Caspase activity was measured from 14 h RPF control and dUTX1 salivary glands using DEVD-AMC as a substrate. Data are mean from three independent experiments each using at least 20 salivary glands from control and dUTX1 for preparing cell extracts. The error bars represent s.e.m. *P-value0.05 (Student's t-test). (d) Autophagy puncta examined using mCherry-Atg8a (red) and nuclei stained with Hoechst (blue). Scale bar represents 100 μm. The quantitation of the mCherry-Atg8a puncta as the mean fluorescent pixel intensity per cell (control n=242, dUTX1 n=224), with error bars representing s.e.m. *P-value0.05 (Student's t-test).
Figure 5(A) Schematic illustration of the treatment regimen. From an average age of 5 weeks RDEB mice were given daily injections of 0.1 or 1.0 mg/kg body weight Ang-(1-7) or an equal volume of PBS. (B) Kaplan Meier survival curve of mice treated as in A, P values as indicated, the data were tested with log-rank test - PBS treated vs. 0.1 mg/kg Ang-(1-7) treated and PBS treated vs. 1.0 mg/kg Ang-(1-7) treated; n = 12-15 mice as indicated in the figure. (C) Photos of left and right forepaws of RDEB mice before and after 7 weeks of daily injections with 0.1, 1.0 mg/kg Ang-(1-7) or PBS. (D) Plot of numbers of toes lost during the 7-week observation period, data tested with Kruskal-Wallis test, P values indicated, n= 20-24 paws as indicated in the figure. Median and range are shown. ( (E) Toe length measurement of forepaws expressed as the percentage of original length. Mice were treated as in A, values for treatment start (0) and 2, 4 and 7 weeks of treatment are shown. The data were analyzed by one-way ANOVA with Tukey's correction. P values < 0.05 are considered significant and shown; n = 20-24 paws. Individual data points, mean ± S.E.M are shown.
Figure 5(A-C) Representative Southern blots showing DSB repair products (top), percentage of BIR among repair products (bottom left) and viability (bottom right) of WT and sir2∆ in the H-150 assay (A), H--150 no-gap assay (B) or native mating type switching H-1400 assay (C). (mean ± SD; n = 3). Welch's unpaired t-test was used to determine the p-value in panels A-C.
Figure 4(A) Western blots of Nedd4-2, total cofilin, phospho (p)-cofilin and GAPDH from Nedd4-2 WT or Nedd4-2 cKO forebrains at postnatal day 14. Representative western blots and quantification of total cofilin relative to GAPDH and p-cofilin relative to total cofilin (n = 6 for Nedd4-2 WT and Nedd4-2 cKO brains) were shown. (B) Immunocytochemistry of cofilin, p-cofilin and dendritic marker MAP2 from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons at DIV 16. Representative dendrites of Nedd4-2 WT and Nedd4-2 cKO neurons and quantification of total cofilin relative to MAP2 and p-cofilin relative to total cofilin are shown (n = 25 and 26 for Nedd4-2 WT and Nedd4-2 cKO neurons, respectively). Scale bar: 5 µm. (C) Immunocytochemistry showing cofilin, p-cofilin, and dendritic marker MAP2 from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons induced with or without cLTP at DIV 16. Representative dendrites and quantification from Nedd4-2 WT or Nedd4-2 cKO cultures are shown (n = 36-39 neurons). Scale bar: 5 µm. For quantification, data from cLTP induction were normalized to data from control (CTL) condition.
HS induces sustained H3K4me3 and H3K4me2 methylation at HSP22.Figure 1Dynamics of histone modifications after an acclimatizing HS (ACC) or no HS (NHS) in Col-0 wild type at HSP22.0 (A) and HSP70 (B). Seedlings were subjected to ACC or a control treatment (NHS) 4 d after germination. At the indicated time points after the treatments, ChIP-qPCR was performed with antibodies against H3K9ac, H3K4me3, H3K4me2 and H3. Schematics show positions of regions analyzed. Amplicon positions relative to TSS are HSP22.0: 1, -2570 bp; 2, +235 bp. HSP70: 1, 4192 bp downstream of the 3'UTR; 2, +47 bp. Data shown are averages over three biological replicates. Amplification values were normalized to input, H3 and 4 h NHS region 2. The bottom panel shows the H3 signal normalized to input and 4 h NHS region 2. Squares and triangles within bars mark significant differences (p<0.01 and p<0.05, respectively, Student´s t-test) between ACC and NHS samples of the same time point. Error bars indicate SE.
The redistribution of the MICOS core components Mic27 and Mic60 precedes the release of cytochrome c in apoptotic Drp1 knockdown cells.Figure 5Drp1 knockdown cells were treated with DMSO (control) or with actinomycin D for 14 h to induce apoptosis and were decorated with antisera against Bax, cytochrome c, and Mic27 or Mic60 (see Appendix Figure S4). Three-color images were taken, whereby Mic27 or Mic60 were recorded in the STED mode. Relying on the Bax and the cytochrome c signals, we discriminated between healthy and apoptotic mitochondria before and after cytochrome c release. The distributions of Mic27 or Mic60 were analyzed by determining the normalized variance of the fluorescence intensity, which is a sensitive measure for the distribution of the labelled protein. The numbers within the columns represent the numbers of images analyzed and the error bars represent the standard error of the mean.
Figure 1a-b, Representative images of cilia (a) and dot plots (b) showing primary cilia length variation at different time points after serum starvation (SS). Markers in (a) show ARL13B (cilia) and PCNT (Basal body). The graph shows individual length of cilia from 3 biological replicates (n>150 cilia per sample and experiment). Scale bar: 1µm. Data shown in include mean ±s.d. are calculated by two-tailed unpaired student t-test. c, Percentage of ciliated RPE1 cells after SS from a. 3 biological replicates, n>100 cells per sample and experiment repetition. Data shown include mean ±s.d. are calculated by two-tailed unpaired student t-test. d, Representative images show cilia segmentation based on acetylated tubulin (Ac tub), polyglutamylated tubulin (Glu tub), ARL13B and PCNT. The cartoon on the left indicates proximal segments (PS, Glu tub rich-region) and distal segments (DS). DS were determined by calculating the difference between PS and total cilia length based on ARL13B staining as depicted by the dashed lines on the images. Scale bar: 1.5µm. e, Quantification of d. Comparative analysis of average cilia length based on Ac tubulin and ARL13B (cilia) staining shown for specified cilia length groups (2<, 2-3, 3-4 and >4µm). Three biological replicates, n=400 cilia per sample and repetition. The numbers above the cartoons show the ratio of the calculated Ac tub/whole cilia length. Data shown include mean ±s.d. are calculated by two-tailed unpaired student t-test. f, Average length of PS and DS for the cilia length groups depicted. Representative images (d) and quantifications of PS (Glu tub) and whole cilia (ARL13B) length are shown. The numbers above the cartoons show the ratio of the calculated DS/whole cilia length. Three biological replicates, n>650 cilia per sample and repetition. Data shown include mean ±s.d. are calculated by two-tailed unpaired student t-test. g, Percentage of cilia with depicted DS length after 8h and 48h SS. Three biological replicates, n>150 cilia per sample and repetition. Data shown include mean ±s.d. and P values of are calculated by two-tailed unpaired student t-test.
Conceptual relationships between endogenous HMGB1 and autophagy.Figure 8.Conceptual relationships between endogenous HMGB1 and autophagy. ROS trigger HMGB1 translocation to the cytosol in the setting of starvation-mediated autophagy. Cytosolic HMGB1 then binds Beclin1, which requires C23/45. This results in dissociation of Beclin1-Bcl-2 and subsequent induction of autophagy. C106 mutation (C106S) in HMGB1 impairs its nuclear localization and promotes autophagy. Inhibition of HMGB1 translocation by ethyl pyruvate (EP) blocks autophagy. Additionally, HMGB1 promotes phosphorylation and activation of the ERK1/2 (p-ERK1/2) pathway, which is an important autophagy-dependent signal pathway.
Figure 1A) Quantification of the effect of individual BCL-2-family proteins on HCT AKO cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n>30 cells per condition per experiment. *** p<0,001 and * p<0,05 with respect to GFP condition. B) Representative confocal immunofluorescence and Bright Field (BF) images of an apoptotic HCT AKO cell expressing tBID-GFP (green), showing cyt c release (magenta), formation of pyknotic nuclei (cyan) and plasma membrane blebs. Scale bar 10 µm. C) Western blot analysis of SMAC and cyt c release in GFP-BAX, tBID-GFP and GFP-BCLXL expressing cells at 12 h, 16 h and 24 h after transfection in HCT AKO. SN (supernatant, cytosolic fraction), P (pellet, membrane fraction) and PS (Ponceau Staining). D) Quantification of caspase activity in HCT AKO cells expressing GFP-BAX, GFP-BAK, tBID-GFP, GFP-BCLXL and GFP at different time points after transfection. n=3 independent experiments. *** p<0,001, **p<0,025 and * p<0,05 with respect to GFP condition. Error bars represent S.D. E) Representative western blot analysis of PARP1 cleavage upon tBID-GFP expression in HCT AKO cells. n=3 independent experiments. F) Kinetics of the expression of tBID-GFP (blue) and PI intake upon tBID expression (purple) and unstransfected HCT116 AKO cells (in grey). Plots show the average fluorescence intensity (dark lines) and its standard deviation (shaded areas) from 3 technical replicates and 3 independent experiments. Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min. G) Quantification of PI+ cells from total transfected cells with GFP-BAX, tBID-GFP, GFP-BCLXL and GFP. n=3 independent experiments with >10000 cells per condition per experiment. *** p<0,001 with respect to untransfected condition. H) Cell death induced by tBID-GFP in HCT AKO cells in the absence and presence of ZVAD, NSA and Ferrostatin-1. Data correspond to three independent experiments, n>30 cells per condition per experiment. *** p<0,001 with respect to MOCK condition.
Figure 4(A) Images of Drosophila eyes expressing human ATXN1(82Q) with either knockdown or overexpression of the Drosophila RSK3 homolog Sk6II. Scale bar: 100 μm (top) and 20 μm (bottom). (B) Improvement of motor performance by the knockdown of Sk6II in Drosophila SCA1 model expressing ATXN1(82Q) in the central nervous system. Mean ± SEM, *P < 0.05, Linear mixed-effect model ANOVA, n = 10/genotype, four times of measurements. (C) A representative image of a mouse brain 4 weeks after AAV injection of shRsk3 into 12 weeks old SCA1 mice (Atxn1154Q/2Q) cerebellum. Scale bar: 2 mm. (D) Western blot analysis of wild-type (2Q) and mutant Atxn1 (154Q) in the cerebella of 16 weeks old SCA1 mice dissected 4 weeks after the stereotaxic injection of AAV9 harboring shControl (n = 3), shRsk3 (n = 3), or shAtxn1 (n = 2). Mean ± SD, *P<0.05, ***P<0.001 ****P<0.0001, one-way ANOVA. (E) Western blot analysis of wild-type (2Q) and mutant Atxn1 (154Q) in the brainstem (BS) and cerebella (CBM) of 8 weeks old Atxn1154Q/2Q (n = 6) and Atxn1154Q/2Q; Rsk3+/- mice (n = 7). Mean ± SD, *P<0.05, **P<0.01, two-tailed t-test. (F) Rotarod motor performance test result of Atxn1154Q/2Q; Rsk3+/- mice at 11-12 weeks old. Mean ± SEM, *P<0.05, two-way ANOVA, n = 35, 31, 34, 38 for WT, Rsk3+/-, Atxn1154Q/2Q, Atxn1154Q/2Q; Rsk3+/- genotype, respectively.
Figure 6A, B Representative oil red staining after 7 days of adipogenic differentiation in Lepr-Cre+ subsets (A) and qPCR analysis of Adipoq and Pparg expression (B) (n=3 independent experiments). Scale bars were 100 µm. C, D In vitro chondrogenic differentiation of sorted periosteal cells. Representative alcian blue staining after chondrogenic differentiation for 21 days and cryosection (C). Scale bars were 100 µm. Chondrogenic efficiencies were quantified by qPCR analysis of Acan and Col2a1 expression (D) (n=3 independent experiments). E, F In vitro osteogenic differentiation of sorted periosteal cells. Representative alizarin red staining after osteogenic differentiation for 14 days. Scale bars were 200 µm. Osteogenic efficiencies were quantified by qPCR analysis of Sp7 and Bglap expression (F) (n=3 independent experiments).
Figure 2A Comparison of differentially processed transcripts (RNAprof) and differential AS genes (AtRTD2-SUPPA) with Differentially Expressed Genes (DEG). B Number of genes containing at least one differential RNA processing event (as defined by RNAprof p.adj < 0.001) in CDS, introns, 5'UTR and 3'UTR between RNAi-ASCO1 and WT. Up and Down fractions correspond to increase or decrease, respectively, of RNA-seq coverage in RNAi-ASCO1 for each specified gene feature. C Proportion of DAS events identified by AtRTD2-SUPPA in RNAi-ASCO1 compared to WT; Alternative 3' site (A3'), Alternative 5' site (A5'), Intron Retention (IR), Exon skipping (ES). D Comparison of differentially processed transcripts (RNAprof) and differentially AS genes (AtRTD2-SUPPA) with genes showing significant isoform switch events (Isoswitch). E Summary of the predicted consequence of the isoform switch events as shown by the feature acquired by the upregulated isoform. ncRNA stands for noncoding RNA, and NMD stands for nonsense mediated decay. F Comparison of differentially processed transcripts (RNAprof), differentially AS genes (AtRTD2-SUPPA) and differentially expressed genes (DEG) with genes significantly upregulated in the upf1upf3 mutant [56], indicating genes potentially regulated by NMD.
Mitochondrial Gasz interacts with Armi and Piwi bound with piRNA precursor.Figure 2Armi and Piwi co-immunoprecipitated with Flag-Gasz (F-Gasz) from the mitochondrial fraction (mito fraction) of OSCs (input). F-Gasz was ectopically expressed in the cells by transfection prior to immunoprecipitation. *: Heavy chains of antibodies used for immunoprecipitation. n.i.: Non-immune IgG used as a negative control. Co-immunoprecipitation from whole-cell lysate of OSCs before (control) and after (Zuc KD) Zuc depletion and subsequent western blotting show that the levels of Piwi and Armi in the Flag-Gasz (F-Gasz) complex increased upon Zuc depletion (Zuc KD). Northern blotting shows that the flam-piRNA precursors in the Flag-Gasz complex (Flag) accumulated upon Zuc depletion (Zuc KD). Upper: The flam-piRNA precursors associated with Piwi disappeared upon Armi depletion (Armi KD) in Zuc-lacking OSCs (Zuc KD). Lower: The abundance of immunopurified Piwi is shown by western blotting.
Figure 4Transcript levels in RPKM (Reads Per Kilobase transcriptome per Million mapped reads) of GalNAc-Ts in two individual clones of isogenic HepG2 lines as indicated. Apart from minor changes in the GALNT1 and GALNT2 transcript levels all transcript levels were similar. Bars represent mean values and error bars SD.