EMBO/sd-character-level-ner huggingface dataset:
Expression of R3hdml increases during skeletal muscle regeneration, and R3hdml is important both for skeletal muscle regeneration and function.Figure 5A Seven days after CTX injection, skeletal muscles were dissected, fixed, and embedded in paraffin. Two consecutive sections were utilized, and the first section was subjected to in situ hybridization using an R3hdml sequence-specific RNA probe to detect Rh3dml, while the second section was used for IHC using the anti-MyoD antibody. MyoD protein was visualized with DAB stain (brown). R3hdml expression is highly detected in MyoD-positive cells. Scale bar in low magnification images = 100 µm; in high magnification images = 25µm. B One hundred microliters of 10 µM CTX was intramuscularly injected into the forearm muscle of anesthetized R3hdml KO ([b], n = 10) and wild type control ([a], n = 10) mice. Subsequently, hand grip was evaluated 0, 3, 6, and 14 days after CTX injection. R3hdml genes were overexpressed in the forearm muscle of anesthetized R3hdml KO mice ([c], n = 10) and hand grip was also evaluated 0, 3, 6, and 14 days after CTX injection. Data are expressed as the means ± standard error of the mean (SEM). ** p < 0.01; **** p < 0.0001 vs. wild type control; # p < 0.05; ## p < 0.01 vs. R3hdml KO mice; two-way ANOVA followed by a Bonferroni post hoc comparison of the individual time points. C Myofibers were freshly dissected from 8-12-week-old R3hdml KO (n = 4) and control mice (n = 4) 35 days after CTX injection, and the number of satellite cells was evaluated by Pax-7 staining. Arrowheads indicate Pax-7-positive cells. The number of Pax-7-positive cells per myofiber was counted. At least 50 myofibers in each animal were evaluated. Closed columns, wild type controls; open columns, R3hdml KO mice. Data are expressed as the means ± standard error of the mean (SEM). * p < 0.05; Student's t-test.
Figure 1Normalised Wnt/STOP signalling activity by quantification of the protein expression of two Wnt/STOP target proteins, β-catenin and c-Myc, in mitotic (M) and asynchronised cells (A) after silencing 47 Wnt signalling regulators. ELISA validation of selected Wnt/STOP signalling regulators (n=3). Immunoblotting and (D) quantification of the stability of four STOP target proteins in both G1S and natural mitotic cells treated with BCL9 siGenome pools and CHX (n=3). Representative pictures (left panel) and the normalised biosensor intensity based on live cell imaging of the cells with a GSK3-GFP biosensor following different treatments before and after metaphase (n=10). Scale bars represent 10 μm. Venn diagram analysis of the number of Axin1, APC, and Wnt3a signalling and/or BCL9-regulated transcriptional targets. Myc and β-catenin target gene enrichment analysis based on the transcriptome targets regulated by BCL9. The representative xenograft tumours (top panel) and tumour growth analysis (bottom panel) of scramble and BCL9 stable knockdown cells. LF: left flank of mouse, RF: right flank. n=6 for shS1 vs. shB1, n=5 for shS1 vs. shB2. Immunohistochemical staining of the xenograft; the red arrows indicate the representative mitotic cells. Scale bars represent 200 μm.
Figure 1Nucleosomal array oligomers are globular.A Nucleosomal array oligomers were stained with DAPI and examined using FM (fluorescence microscopy) as described in the Materials and Methods section. Shown are representative images obtained in 4.5 mM and 10mM MgCl2.B Control FM images obtained in 0, 1 and 2.5 mM MgCl2. C Nucleosomal array oligomers were negatively stained and visualized by TEM as described under Materials and Methods. Shown in the left panels are representative images obtained in 4.5 mM, and 10 mM MgCl2. Shown in the right panels are images of the interior of the oligomers (white arrows, left panels) after cropping and re-scaling.
Yip1A interacts with pIRE1 at ERES.Fig 2(A) Representative immunoblots showing co-immunoprecipitation with pIRE1. After 5 hr of Tm treatment, immunoprecipitation was performed on HeLa cell lysates with an anti-pIRE1 antibody (lanes labeled 'pIRE1') or control anti-rabbit IgG (lanes labeled 'IgG'), and the immunoprecipitates were analyzed by Western blotting with a panel of antibodies against molecules involved in the ER-Golgi vesicular transport pathways or against ER- (Sec61α, HSP47, and calnexin), ERGIC- (ERGIC53) and cis-Golgi- (GM130) resident proteins. (B) Representative immunoblot showing the co-immunoprecipitation of pIRE1 with Yip1A. Following 5 hr of Tm treatment, immunoprecipitation was performed on HeLa cell lysates with an anti-Yip1A antibody (lane labeled 'Yip1A') or control anti-rabbit IgG (lane labeled 'IgG'), and the immunoprecipitates were analyzed by Western blotting with an anti-pIRE1 antibody. (C) Representative confocal micrographs of HeLa cells double-stained for pIRE1 (green) and Yip1A (blue) after 5 hr of Tm treatment. Insets are magnifications of the boxed areas on the main image. Co-localized proteins were detected as large foci. Scale bars are 10 μm. (D) Representative confocal micrographs of HeLa cells triple-stained for pIRE1 (green), Sec31 (red), and Yip1A (blue) after 5 hr of Tm treatment. Insets are magnifications of the boxed areas on the main image. Co-localized proteins were identified as large, bright foci (arrows). Scale bars are 10 μm.
Figure 4(A) The mRNA expression level of human cathelicidin (CAMP) in skin lesions from healthy individuals (n = 10) and patients with rosacea (n = 15). Data (B) Co-immunostaining of cathelicidin and pS6 on skin sections from HS and rosacea. Scale bar, 50 μm. Epi, epidermis. (C) Immunostaining of mouse cathelicidin (CRAMP) in skin sections from WT and Raptor cKO mice injected with LL37 or control vehicle (n = 6 for each group). Bottom right panels, magnified images of dotted line areas. Epi, epidermis; Der, dermis; Scale bar: 50 μm. (D) Quantification of relative fluorescence intensity for CRAMP in epidermis (n = 6). Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test (D) was used. (E) Immunoblotting of cathelicidin in cell lysates from primary human keratinocytes treated with different doses of LL37 (0 - 8 μM) and scramble LL37 (8 μM) for 24 h. Cathelicidin protein levels were analyzed. (F) Immunoblot analysis of cathelicidin and pS6 in cell lysates from primary human keratinocytes treated with LL37 (4 μM) ± RAPA for 24 h. Cathelicidin protein levels were analyzed. (G) Immunostaining of cathelicidin in HaCaT keratinocytes treated with LL37 (4 μM) ± RAPA for 24 h. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. All results are representative of at least 3 independent experiments. Data represent the mean ± SEM.
a) Overexpression of Cdk5/p35 enhances EndoB1 dimerization in 293T cells.figf4(a) Overexpression of Cdk5/p35 enhances EndoB1 dimerization in 293T cells. Samples were run on the same gel and spliced together to create the image shown (b) Thr 145 phosphorylation of EndoB1 facilitates its dimerization in 293T cells. (c) Diminished interaction between EndoB1 and UVRAG in Cdk5−/− mouse brain lysates. (d,e) Thr 145 phosphorylation of EndoB1 enhances its interaction with UVRAG (d) in 293T cells. (d,e) Thr 145 phosphorylation of EndoB1 enhances its interaction with UVRAG (d) and Beclin 1 (e) in 293T cells. (f,g) Gel-filtration analysis on the membrane fraction of mouse brain lysates. Fractions obtained were subjected to immunoblot analysis (f), or immunoprecipitation followed by immunoblotting (g). Uncropped images of blots are shown in Supplementary Fig. S8.
a) Time-lapse images of MCF10A cell-in-cell structures with an internalized cell that undergoes apoptosis.figf4(a) Time-lapse images of MCF10A cell-in-cell structures with an internalized cell that undergoes apoptosis. The arrows mark the recruitment of LC3 around apoptotic fragments (see Supplementary Movie S7). Scale bar, 10 μm. (b) Quantification of LC3 recruitment to apoptotic internalized cells in the presence of ATG5 or FIP200 siRNA; n, number of cells analysed; ***P0.0001, chi-squared test. (c) Apoptotic U937 cells expressing H2B-mCherry phagocytized by J774 macrophages expressing GFP-LC3. Confocal images show LC3 recruitment to an engulfed corpse (arrow; see Supplementary Movie S8). Scale bar, 15 μm. (d) Quantification of LC3 recruitment to apoptotic phagosomes in control and Atg5-shRNA-expressing cells; ***P0.0001, chi-squared test. (e) Correlative light-electron microscopy of an apoptotic phagosome with GFP-LC3 recruitment (top left image, white arrow). The LC3-loaded phagosome consists of a single membrane (black arrow in right image, which is a higher magnification of the area outlined in the bottom left image; EM, electron microscopy; AP, apoptotic cell nucleus). (f) Representative images of apoptotic cell phagocytosis and degradation in control (n=21) and Atg5 shRNA (n=21) J774 GFP-LC3 cells; ***P0.0001, chi-squared test. Scale bar, 10 μm. See also Supplementary Fig. S3.
Figure 7A) Effect of GTx-024 and AR-42 monotherapies on cachexia-related differentially regulated genes (DEGs) from RNA-seq analyses of Study 1 gastrocnemius muscles. All three panels consist of individual genes plotted with respect to their log2 fold change and -log10 Benjamini-Hochburg adjusted p-values from the comparison of cachexia vs tumor-free controls. Colors of the points reflect the DEG status of each gene for the given comparison. B) Results from transcription factor pathway-focused GSEA of tumor-bearing (cachexia) versus tumor-free control transcriptomes. STAT3 and ATF-1 gene sets used for subsequent combined analyses are hatched. C) Significance values from GSEA using combined STAT3 gene sets identified in B. Each treatment group is compared to tumor-bearing control (cachexia) transcriptomes. D) Heat map of DEGs within the combined STAT3 gene sets representing mean z scores calculated from normalized RNAseq count data. Tumor-free control (black checkered), GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green). E) Results of GSEA using combined ATF-1 gene sets identified in B across treatment groups versus tumor-bearing control (cachexia) transcriptomes. Tumor-free control (black checkered), GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green). F) Heat map of DEGs within the combined ATF-1 gene sets (mean z score). Treatment groups are as in D. G) mRNA expression of mediators of IL-6 signaling upstream of STAT3. Data presented as mean±SD of per animal log-transformed fold change (log2FC) values versus tumor-free controls. Groups: Tumor-free control (n=6), tumor-free GTx-024 treated (n=5), tumor-bearing receiving vehicle (n=4), GTx-024 (n=5), AR-42 (n=5) or Combination (n=6).
a, A gene-trap vector containing a splice acceptor followed by the &bgrgeo (&bgr-galactosidase/neomycin resistance fusion cassette) exon disrupts the Atg16l1 gene locus by insertions within the intronic regions after exons 6 and 10 for the ATG16L1HM1 and ATG16L1HM2 lines, respectively.figf1a, A gene-trap vector containing a splice acceptor followed by the bgeo (b-galactosidase/neomycin resistance fusion cassette) exon disrupts the Atg16l1 gene locus by insertions within the intronic regions after exons 6 and 10 for the ATG16L1HM1 and ATG16L1HM2 lines, respectively. WT, wild type. b, MEFs were generated from day 12.5embryos of Atg16l1 mutant mice, and western blot analysis was used to detect the a and b isoforms of ATG16L1 from whole-cell lysates c, d, Atg5-/- and ATG16L1HM MEFs were grown in the presence of the chemical inducer of autophagy rapamycin (Rap, 50 µg ml-1) and cyclohexamide (CHX, 5 µg ml-1) for 0, 4, 8 and 12 h. Cell lysates were analysed by western blot for loss of P62 expression (c). P62 levels were quantified by densitometry normalized to actin (n = 3; d). *P 0.04; **P 0.008; ***P = 0.0003 e, f, Western blot analysis of LC3 expression in MEFs grown in the presence of rapamycin (50 µg ml-1) or dimethylsulphoxide (DMSO) control for 4 h (e). LC3-II expression in the presence of rapamycin was quantified and normalized to actin (n = 6; f). g, Detection of ATG16L1, LC3 and P62 by western blot of ileal lysates from ATG16L1HM mice. ATG16L1 can be detected in both mutant lines on longer exposures. h, i, Quantification of ATG16L1 (h) and P62 (i) normalized to actin detected in ileal lysates (n = 3). P values were calculated using two-tailed Student's t-test. Error bars, s.e.m.
Characterization of PIWI genes in arthropod species.Figure 1A Phylogenetic analyses of PIWI genes in 174 arthropod genomes. A maximum-likelihood phylogenetic tree was generated on the basis of the protein sequences of PIWI genes using FastTree software version 2.1.10. The reliability of each tree branch was evaluated using the SH branch support test with 1000 resamples. The red dots on a branch indicate genes with an SH test result >0.5. The different colors outside of the phylogenetic tree represent different arthropod orders. B Protein distance matrix for Piwi, Aub, and Ago3 in Drosophila (Dm) and L. migratoria (Lm). C The numbers of Piwi-like genes in the 174 arthropod species. D Distributions of pairwise Ka/Ks (nonsynonymous to synonymous nucleotide substitution rates) ratios between PIWI duplicates in arthropods (n = 17 in Drosophila and n = 37 in others, Mann-Whitney U test, *P<0.05). The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value.
Figure 3A. Western blot detection of PEX13 in HeLa/Parkin cells transfected with indicated siRNA and siRNA-resistant PEX13 plasmid. To achieve similar PEX13 protein expression levels, 0.75 μg PEX13 I326T and PEX13 W313G plasmids were transfected compared to 0.25 μg WT PEX13. Total plasmid level was adjusted using empty vector. Asterisk denotes non-specific band. B. Representative images of Parkin-mediated clearance of TOMM20 in HeLa/Parkin cells treated with PEX13 siRNA and transfected with indicated PEX13 siRNA-resistant plasmid and then treated with CCCP (10 μM, 16 h). Scale bars, 20 μm. See Fig EV3B for representative images of mitochondrial morphology (TOMM20 staining) in control cells treated with DMSO. C. Quantification of experiment shown in (B). Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. *p<0.05, **p<0.01,***p<0.001; two-tailed unpaired t-test. D. Western blot detection of PEX13 expression in wild-type (WT) and PEX13 W313G (W313G) mutant primary human fibroblasts. E. Representative images of Parkin-mediated clearance of TOMM20 in wild-type (WT) and PEX13 W313G (W313G) mutant primary human fibroblasts treated with Oligomycin (2.5 μM) + Antimycin A (250 nM) for 24 h. Scale bars, 20 μm. W313G cells varied in morphology; outlined cell in upper right panel shows a representative cell with larger size and abnormal mitochondrial morphology (see Fig EV3C for higher resolution imaging) and outlined cell in lower right panel shows a representative cell with accumulation of fragmented mitochondria that would be scored as positive in (F).F. Quantification of experiment shown in (E). Results represent mean ± SEM of 5 groups of 10 images of random fields of cells (>350 cells analyzed per sample). Similar results were observed in two independent experiments. ***p<0.0001; two-tailed unpaired t-test.
Figure 8A Single plane confocal microscope image during germband entry from control or atosPBG embryos, or those expressing pths, GR/HPR, and LKR/SDH in macrophages (mac>). Embryos were stained for antibodies against S293-phosphorylated inactivated Pyruvate Dehydrogenase (pPDH, green) or total PDH (magenta) in macrophages (red). Higher pPDH/PDH ratios are consistent with higher ATP/ADP levels. Data information: Scale bars: 10µm B Quantification of normalized pPDH/PDH levels calculated from fluorescence intensities in macrophages from the genotypes in (A) during initial germband invasion. The pPDH/PDH ratio is significantly increased in atosPBG embryos expressing either pths, GR/HPR, or LKR/SDH in macrophages compared to the atosPBG embryos. This argues that the decreased Atos function in atosPBG macrophages, resulting in lower cellular ATP/ADP ratios, was restored by expressing either its targets or murine orthologs. N=3 independent experiments. Control (n=9 embryos) vs. atos mutant (n=10) p<0.0001. Control vs. atos mutant rescued with mac>pths (n=10) p=0.77; rescued with mac>GRHPR (n=9) p=0.48; rescued with mac>LKRSDH (n=10) p=0.012. For atos mutant vs. atos rescued with mac>pths or mac>GRHPR p<0.0001. vs. atos rescued with mac>LKRSDH p=0.0014. Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. One-way ANOVA with Tukey C-D (C) Confocal images or (D) quantification of the macrophages in germband in Stage 12 embryos from the control, atosPBG, and atosPBG expressing atos itself or GR/HPR, or LKR/SDH in macrophages. Germband edge: dotted white line. For (D) control (n=29 embryos) vs. atos mutant (n=19) p<0.0001. Control vs. atos mutant rescued with mac>atos (n=27 embryos) p=0.99; rescued with mac>GRHPR (n=28) p=0.29; rescued with mac>LKRSDH (n=20) p=0.036. atos mutant vs. atos rescued with mac>atos or mac>GRHPR p<0.0001; rescued with mac>LKRSDH p=0.0134. Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. One-way ANOVA with Tukey Scale bars: 50µm E Single plane confocal microscope images during germband entry from control or atosPBG embryos, or those expressing mammalian orthologs mFAM214A or B (FAMA-B) in macrophages (mac>). Embryos were stained for antibodies against S293-phosphorylated inactivated Pyruvate Dehydrogenase (pPDH, green) or total PDH (magenta) in macrophages (red). Data information: Scale bars: 10µm F Quantification of normalized pPDH/PDH levels measured from fluorescence intensities in macrophages from the genotypes in (E) during initial germband invasion. The pPDH/PDH ratio is significantly increased in atosPBG embryos expressing either mammalian ortholog mFAM214A or B in macrophages compared to the atosPBG embryos. Results from three independent experiments. Control (n=10 embryos) vs. atos mutant (n=11) p<0.0001. Control vs. atos mutant rescued with mac-mFAMA (n=14 embryos) p=0.06; rescued with mac-mFAMB (n=12) p=0.13. atos mutant vs. atos mutant rescued with mac-mFAMA p=0.0025 or mac-mFAMB p=0.0019. Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. One-way ANOVA with Tukey
Figure 7A, Immunoblot of the indicated proteins of BNGE from female scaf1+/+ and scaf1-/- whole zebrafish mitochondria for the indicated diet (representative of n=2). B, Maximum uncoupled (FCCP) oxygen consumption rate in whole zebrafish mitochondria (females Δ1 n=4 and Δ2 n=4, and same number for their respective controls) with glutamate (Glu), malate (Mal) and succinate (Succ). RNAseq data from scaf1+/+ and scaf1-/- skeletal muscle for the indicated diet (standard diet scaf1+/+ and scaf1-/- n=4, double diet scaf1+/+ n=3, scaf1-/- n=4). (C-F) Volcano plots of differentially expressed genes (DEGs). (C) Comparison between scaf1-/- and scaf1+/+ zebrafish in standard diet. (D) Comparison between scaf1-/- and scaf1+/+ zebrafish in double diet. (E) Comparison of scaf1+/+ zebrafish in double diet and standard diet. (F) Comparison of scaf1-/- zebrafish in double diet and standard diet. In blue, significant DEGs (p-adj < 0.05, log2FC > |1|), in grey not significant DEGs, red circles represent non-significant differentially regulated OXPHOS genes, green circles represent significant differentially regulated OXPHOS genes, purple scaf1 (cox7a2l). RNAseq data from scaf1+/+ and scaf1-/- skeletal muscle for the indicated diet (standard diet scaf1+/+ and scaf1-/- n=4, double diet scaf1+/+ n=3, scaf1-/- n=4). (G) Heatmap of metabolic differentially regulated pathways in Gene Set Enrichment Analysis (GSEA) in the indicated comparisons. (H) Heatmap of differentially regulated growth Hallmarks in GSEA in the indicated comparisons.
V600EBRAF-driven lung tumours secrete NPC2.Figure 5NPC2 immunoblots of concentrated whole lung CM (top) and whole lung lysates (bottom) generated from Braf+/+;CreER™ (WT) or BVE (VE) mice. Whole lung lysates were obtained from 3-to 8-week-old mice. NPC2 immunoblots of un-concentrated CM (containing 10% FCS) from fractionated IMC and AT2 cultures. Total protein staining of the membrane by Amido black (bottom) serves as a loading control showing serum albumin at 70 kDa, derived from FCS in the media. Npc2 mRNA expression in the purified IMC and AT2 populations as determined by quantitative RT-PCR. Mean + SD (n = 3) of Npc2 expression normalised to Gapdh is indicated. NPC2 immunoblots of cell lysates from freshly isolated IMCs and AT2 cells. NPC2 immunoblots of cell lysates from freshly isolated or cultured (48 h in serum-free DMEM) IMCs. NPC2 immunoblots of cell lysates from primary IMCs cultured with or without 50 μg/ml bNPC2 for 48 h. CLSM imaging of NPC2-Alexa488 taken up by IMCs. (Left) Freshly isolated IMCs were incubated for 2 h with (+NPC2-Alexa488) or without (-NPC2-Alexa488) 82.5 nM NPC2-Alexa488, chased in NPC2-free media for additional 2 h and live imaged by CLSM. Single-colour (top) and DIC-merged (bottom) fluorescence images are indicated. Scale bar, 20 μm. (Right) Zoomed images (DIC-merged and single-colour) from different fields showing vesicular and reticular distribution of incorporated NPC2-Alexa488. Scale bar, 10 μm. Npc2 mRNA expression in IMCs cultured for 48 h with or without 50 μg/ml bNPC2 as determined by qRT-PCR. Mean ± SD (n = 3) of Npc2 expression normalised to Hprt1 is indicated. The diagram indicates our hypothetical model for the source of NPC2 in IMCs, based on our ex vivo observations.
Fig.Figure 1(a) Paradigms of early life trauma in mice and humans. In mice, early life trauma consists in an exposure to unpredictable maternal separation combined with unpredictable maternal stress (MSUS). In humans, exposure involves paternal loss and maternal separation (PLMS) in early childhood (age 5-11). (b) Scheme illustrating the MSUS model and control mice showing blood collection and breeding to generate an offspring. For MSUS (symbolised by yellow blitz), newborn pups are separated from their mother unpredictably 3h/day from postnatal day (PND) 1 to 14. During separation, the dam is exposed to different stressors unpredictably9. Serum was prepared from blood collected from 3-month old MSUS and control males. (c) Differential pathway enrichment of metabolites in MSUS plasma from adult males and their offspring compared to controls (each group n = 5), and serum (PLMS n = 20, Control n = 14) and saliva (PLMS n = 25, Control n = 14) from PLMS and control children. Asterisk and hashtag represent FDR after multiple testing corrections using Benjamini-Hochberg (BH) test. Columns indicate significance for positive (+) and negative (-) enrichment. (/) symbolizes non-significance. FDR, false discovery rate. ALA/LA, alpha-linolenic acid/linoleic acid. AA, arachidonic acid. HETE, hydroxyeicosatetraenoic acid. (d) Individual metabolites in ALA/LA and AA pathways significantly altered in both MSUS and PLMS. Numbers represent fold change according to a heat scale (right).