EMBO/SourceData huggingface dataset:
Figure 5Mice were placed in the centre of the Y-maze and allowed to explore freely for 10 minutes. The percentage of spontaneous alternations in the Y-maze were measured to assess working memory (n=10 mice per group). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; *P<0.05, **P<0.01A one-hour open field test was performed and path length measured as an indicator of hyperactivity (n=10 mice per group). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P<0.001. Symbols above bars are versus WT
Figure 6Representative images of the hippocampus and cortex (layers II-V) from control and treated mice stained with anti-mouse glial fibrillary acidic protein (GFAP; red)/DAPI (nuclear, blue) to identify activated astrocytes, 20x, scale bar: 50μm Representative images of the hippocampus and cortex (layers II-V) from control and treated mice stained with isolectin B4 (ILB4) to identify activated microglia, 20x, scale bar: 50μThe average number of GFAP positive astrocyte cells over three fields of view per mouse of the hippocampus (C) and the cortex (D) were counted (n=4 mice per group, average of 3 fields of view per mouse). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; **P<0.01, ***P<0.001. Symbols above bars are versus WTThe average number of ILB4+ positive microglial cells over three fields of view per mouse of the hippocampus (C) and the cortex (D) were counted (n=4 mice per group, average of 3 fields of view per mouse). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P<0.001. Symbols above bars are versus WT
Figure 1Colocalization of AcGFP-Siwi, AcGFP-BmAgo3 and mCherry-BmVasa in BmN4 cells. Line scans (white line in the enlarged region) show that Siwi-D670A mutant but not BmAgo3-D697A mutant dissociates from BmVasa foci. Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel. Scale bar, 8 μm.Colocalization of AcGFP-Siwi and mCherry-BmDcp2 in BmN4 cells. Line scans (white line in the enlarged region) show that Siwi-D670A mutant but not BmAgo3-D697A mutant overlaps with BmDcp2 foci. Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel. Scale bar, 8 μm.
Figure 2(Left) Effect of 1,6-Hexanediol treatment on AcGFP-Siwi and AcGFP-Siwi-D670A foci. Z-stacks were taken prior to the addition of 1,6-Hexanediol or medium and after 30 minutes incubation at RT. Z-projections of the middle 6 μm stacks are then normalized and pseudo-colored with Fire LUT to visualize the Siwi foci. Scale bar 10 μm. Scale bars represent pixel intensity in arbitrary units (A.U.) (Right) High granule/cell intensity ratio of Siwi-D670A persists despite 1,6-Hexanediol treatment. Average intensity ratio from each of the independent Z-stacks (n=30 per set) is quantified (see Materials and Methods) and depicted as box plots. Representative data from N = 3 independent experiments are shown. P-values were calculated by Asymptotic Wilcoxon rank sum test.Co-expression of Siwi-D670A increases granule-to-cell intensity ratio of BmSpnE and BmQin foci. (Upper) Representative Z-projections (maximum intensity) of mCherry-BmSpnE and mCherry-BmQin foci. AcGFP signal from FITC channel is not shown. Scale bar 8 μm. (Bottom) Average intensity ratio from each of the independent Z-stacks (n=6 per set) is quantified (see Materials and Methods) and depicted as box plots. Representative data from N ≥ 3 independent experiments are shown. P-values were calculated by Asymptotic Wilcoxon rank sum test.Depletion of BmSpnE or BmQin results in segregation of Siwi-D670A from BmDcp2-containing P-bodies. (Top-left) Representative Z-projections (Maximum intensity). Scale bar 8 μm. (Top-right) Enlarged area from the white box. Scale bar 2 μm. (Bottom) Box plot showing that depletion of BmQin (n=32 cells) or BmSpnE (n=32 cells) reduces colocalization ratio between Siwi-D670A and BmDcp2, compared to control (dsRLuc, n=32 cells). Representative data from N = 3 independent experiments are shown. Bonferroni-corrected P-values were calculated by Asymptotic Wilcoxon rank sum test. See also Figure EV3.
Figure 4Co-expression of BmVasa-E339Q/Siwi-D670A results in aggregates with larger sizes. Representative Z-projections (Maximum intensity) of mutant aggregates. Scale bar 1 μm.
Figure 5Split violin plots of piRNA expression fold change between AcGFP (control) and piRNA factor(s) overexpressed libraries. Overexpression of Siwi-D670A, BmVasa-E339Q and both mutants increase non-TE-derived piRNA (red, 626 genes) while TE-derived piRNA (blue, 818 genes) does not change. Bonferroni-corrected P-values and the effect sizes (r) were calculated by Asymptotic Wilcoxon rank sum test.
Figure 1Fig 1: Urinary triclosan levels are elevated in TC mothers following 6 months of exposure. Summaries are provided as the interquartile range with median of triclosan molecules in a picogram/microliter (pg/L). Urinary triclosan measurements are available for 38 mothers (17 TC, 21 nTC) and 33 infants (15 TC, 18 nTC). The p-value for the mothers in Figure 1 (represented by ***) is p=5.07e-5 (Mann-Whitney U test).
Figure 2(A) PCoA of Bray-Curtis dissimilarity for all (n=221) samples shows that gut communities cluster by mothers and infants.(B) PCoA separated by time and treatment.
Figure 3Fig 3: TC randomization does not decrease gut microbial diversity in infants or mothers. Shannon diversity measures are plotted as the interquartile range with median for each TC exposure class and time point grouping for infants and mothers.
Figure 4(A) Differentially abundant taxa between nTC and TC households. Values left of the grey line indicate an enrichment in nTC household and values to the right indicate an enrichment in TC households. (A) Analyses are separated by mothers and infants for all samples across the 3 time points (FDR adjusted p-value < 0.01).Differentially abundant taxa are displayed for (B) infantsat per visit (FDR adjusted p-value < 0.05).Differentially abundant taxa are displayed for(C) mothers at per visit (FDR adjusted p-value < 0.05).
Figure 1(A) Adult muscles were transfected with YFP‐LC3 and with pDsRed2‐Mito. Animals were starved for 24 h or denervated for 7 days. Muscles were exposed and observed in situ using confocal in vivo microscopy. At least five animals per condition have been studied. Scale bar, 20 μm.(B) Adult muscles were transfected with YFP‐LC3 and with pDsRed2‐Mito. Lysosomes were inhibited by treating mice with 50 mg/kg of chloroquine for 7 days. Chloroquine treatment started at the day of denervation and lasted for 7 days, whereas for fasting experiments at the 6th day of chloroquine injection food was removed for 24 h. Muscles were exposed and observed in situ using confocal in vivo microscopy. At least five animals per condition have been studied. Scale bar, 20 μm.
Figure 2(A) Imaging of FoxO3‐mediated modification of the mitochondrial network in soleus muscle of living mice. Adult soleus muscles were transfected with pDsRed2‐Mito and either c.a.FoxO3 or mock vector (control). Two weeks later, muscles were exposed and observed in situ using two‐photon microscopy. At least five animals per condition were studied.(B) Overexpression of c.a.FoxO3 induces mitochondrial changes and removal through autophagy. Muscle fibres were transfected by electroporation with YFP‐LC3, pDsRed2‐Mito and either c.a.FoxO3 or mock vector. Two weeks later, fluorescent fibres were analysed for mitochondrial distribution by confocal microscopy. A higher magnification of the square is depicted on the right panels. White arrows point autophagosomes engulfing the mitochondria.(C) Overexpression of c.a.FoxO3 induces reduction in myofibre size and decrease in SDH staining. Left image: immunostaining for FoxO3 shows atrophic fibres, which over‐expressed FoxO3 (red) and are labelled by asterisks. Nuclei are stained by Hoechst (blue). Right image: histochemistry for succinate dehydrogenase (SDH) on serial section. Note that fibres positive for FoxO3 are negative for SDH staining (asterisks).(D) Electron micrograph of a control and c.a.FoxO3‐expressing fibres. Muscles were processed with standard fixation‐embedding procedure. Black and white arrows indicate subsarcolemmal and intermyofibrillar mitochondria, respectively. Note that subsarcolemmal mitochondria are reduced in FoxO3 overexpressing fibres, whereas intermyofibrillar mitochondria are smaller and paler than control ones. Red arrowheads show a vesicle containing mitochondria. The image is representative of the FoxO3‐transfected fibres.
Figure 3(A) RNAi‐mediated knockdown of Fis1 showed by western blot assay. MEF cells were transfected with vectors expressing four different Fis shRNAs. For in vivo experiments Fis (4) was used. Efficiency of protein knockdown is indicated in Supplementary Table S2.(B) Blockade of Fis1 and of Bnip3 by RNAi reduces muscle loss during fasting. Adult skeletal muscles were transfected with specific shRNAs for mouse Fis1 (oligo 4) and mouse Bnip3 or co‐transfected and 8 days later mice were starved for 48 h. Cross‐sectional area (CSA) of transfected fibres, identified by GFP, was measured as described earlier (*P0.01) n=450.(C) RNAi of Fis1 and Bnip3 significantly prevents MuRF‐1 and atrogin‐1 promoters activation during starvation. TA muscles were transfected with either 5 kb of the MuRF‐1 promoter (left panel) or 3.5 kb of atrogin‐1 promoter (right panel) with or without shRNAs vectors against Fis and Bnip3. In fasting experiments, after 7 days from transfection mice were starved for 24 h. A renilla‐luciferase construct (pRL‐TK) was co‐transfected to normalized for transfection efficiency. The activation of MuRF‐1 and atrogin‐1 promoters was determined by firefly activity and normalize for the renilla. Each condition represents the average of at least three independent experiments±s.e.m. (*P0.05).(D) Muscle atrophy induced by FoxO3 is reduced by d.n.DRP1. Adult skeletal muscles were transfected with HA‐tagged c.a.FoxO3 with or without d.n.DRP1 and examined after 2 weeks. CSA of transfected fibres, identified by anti‐HA immunofluorescence, was measured as described earlier (*P0.001) n=400.(E) Blockade of the fission machinery by d.n.DRP1 or Bnip3 inhibition by RNAi reduces FoxO3‐mediated autophagosome formation. Animals were transfected with YFP‐LC3, c.a.FoxO3 in presence or absence of d.n.DRP1 or shRNAs against Bnip3. Twelve days later, muscles were collected and analysed for fluorescent vesicles formation (*P0.001) n=450.(F) FoxO3‐dependent activation of MuRF‐1 and atrogin‐1 is significantly reduced by inhibition of the fission machinery. MuRF‐1 promoter (left panel) or atrogin‐1 promoter (right panel) was co‐transfected into adult TA muscle with or without c.a.FoxO3 and in the presence or absence of d.n.DRP1. A renilla‐luciferase vector was co‐transfected to normalize for transfection efficiency. Eight days later, firefly and renilla‐luciferase activity was determined. Each condition represents the average of at least three independent experiments±s.e.m. (*P0.05).
Figure 4Overexpression of DRP1/Fis1 and Bnip3 proteins induces changes in the mitochondrial network in vivo. In vivo imaging of the mitochondrial network was performed in muscles expressing Bnip3, an atrophy‐related gene under FoxO3 control, and DRP1/Fis1 proteins. Adult muscles were transfected with plasmids encoding mitochondrially targeted yellow fluorescent protein (mtM13‐YFP) and either mock vector, Bnip3, HA‐DRP1, Fis1 or DRP1/Fis1. Seven days later (for Bnip3) or 12 days later (for DRP1 and Fis1), muscles were observed in vivo with confocal microscopy. TMRM was injected into the muscles to monitor mitochondria, which retain membrane potential. Muscles transfected with mtM13‐YFP and mock vector showed the orderly pattern of intermyofibrillar and subsarcolemmal mitochondria (upper panel‐IMF). Scale bar: 20 μm.
Figure 5(A) Adult TA muscles were transfected with either DRP1, Fis1, DRP1 and Fis1 or mock vector. Muscles were collected 12 or 21 days after transfection and cross‐sectional area of transfected fibres, identified by immunofluorescence, was measured as described earlier (*P0.001) n=400.(B) Bnip3 or Bnip3 l expression for 7 days causes a 20% reduction of the cross‐sectional area. TA muscles were transfected with Bnip3, Bnip3l or mock vector. Cross‐sectional area of transfected fibres, identified by immunofluorescence, was analysed (*P0.001) n=500.(C) Quantification of LC3‐positive autophagic vesicles in adult muscles co‐tranfected with YFP‐LC3 and DRP1, Fis1 or DRP1/Fis1 expression plasmids or mock vector.(D) Mutations in BH3 domain of Bnip3 (Bnip3BH3mut) do not affect BNIP3‐mediated autophagy. Adult muscles were co‐transfected with YFP‐LC3 and Bnip3 or Bnip3BH3mut expression plasmids and LC3‐positive vesicles were quantified.(E) Bnip3‐mediated atrophy is not modified by mutating the BH3 domain (Bnip3BH3mut). Adult TA muscles were transfected with Bnip3 or Bnip3BH3mut expression plasmids and cross‐sectional area was quantified 7 days later and plotted as % of controls (*P0.001) n=440.
Figure 6(A)In vivo imaging of the mitochondrial network was performed in muscles expressing Fis1K148R. Adult muscles were transfected with plasmids encoding mitochondrially targeted yellow fluorescent protein (mtM13‐YFP) and Fis1K148R. Twelve days later muscles were observed in vivo with confocal microscopy. TMRM was injected into the muscles to monitor mitochondria, which retain membrane potential. Mitochondrial network was greatly altered but mitochondrial membrane potential was conserved.(B) FDB muscle fibres were transfected by electroporation in vivo. Eight days later adult fibres were isolated and placed in cell culture. Adult fibres were loaded with TMRM (5 nM) for 30 min at 37°C. TMRM accumulates in the mitochondria that maintain mitochondrial membrane potential. Oligomycin (Olm, 5 μM) and the protonophore FCCP (4 μM) were added at the indicated time points. TMRM staining were monitored in at least 10 fibres per construct (*P0.001).(C) hFisK148R‐transfected muscles were collected 12 and 21 days after transfection. Cross‐sectional area of transfected fibres, identified by anti‐myc immunofluorescence, was quantified (n=550).(D) Fis1‐dependent activation of MuRF‐1. MuRF‐1 promoter was co‐transfected into adult TA muscle with or without Fis1 or Fis1K148R. A renilla‐luciferase vector was co‐transfected to normalize for transfection efficiency. Eight days later, firefly and renilla‐luciferase activity was determined. Each condition represents the average of at least five independent experiments±s.e.m. (*P0.05).
Figure 7(A) Left panel: mitochondrial fission activates AMPK. HEK293 cells were transfected with a mock vector (control), Bnip3, DRP1, Fis1 or co‐transfected with DRP1 and Fis1. Cells were collected 24 h later and lysates were analysed by immunoblotting. Right panel: immunoblotting analysis shows AMPK phosphorylation in EDL and soleus muscles during muscle atrophy. P‐AMPK/AMPK ratio analysed by densitometric analysis is indicated below the panels. (B-E) Inhibition of AMPK blocks Bnip3‐ and DRP1/Fis1‐mediated atrophy.(B) Adult TA muscles were transfected with Bnip3 together with d.n.AMPK or mock vector and cross‐sectional area was measured (*P0.001) n=480.(C) Adult TA muscles were transfected with DRP1 and Fis1 together with d.n.AMPK or mock vector. Twelve days later muscles were collected and cross‐sectional area was quantified (*P0.001) n=400.(D, E) RNAi‐mediated knockdown of AMPK α 1 and α 2 inhibits Bnip3‐ and DRP1/Fis1‐dependent atrophy. Mouse TA muscles were co‐transfected with Bnip3 or DRP1 and Fis1 and pSuper vectors expressing shRNAs against lacZ (control) or AMPK. Twelve days later, TA muscles were collected and the cross‐sectional area was quantified (*P0.001) n=450.
Figure 8(A) Quantitative PCR analysis was performed in triplicates using specific oligonucleotides (see Supplementary Table 1) (*P0.01).(B) Immunoblotting analysis shows that AICAR treatment causes phosphorylation of AMPK and the downstream target ACC but does not affect phosphorylation of AKT and FoxO3.(C) FoxO3 binding to atrogin‐1 and MuRF‐1 promoters, as determined by ChIP, is induced by AICAR treatment.(D) RNAi‐mediated knockdown of FoxO3 inhibits DRP1/Fis1‐dependent atrophy. Mouse TA muscles were co‐transfected with DRP1 and Fis1 and pSuper vectors expressing shRNAs against either lacZ (control) or FoxO3. Twelve days later, muscles were collected and the cross‐sectional area was quantified (*P0.001) n=450.
Figure 1A Purified TAP-tagged Rik1-containing complexes analyzed by SDS-PAGE and silver staining. mock: a mock purification from an untagged strain. Proteins identified by LC-MS/MS are indicated at right, with the number of identified unique peptides in parentheses.B In vitro ubiquitylation assays using biotinylated ubiquitin, purified CLRC, and recombinant S. pombe histone H3 as the substrate. Proteins were analyzed by Western blotting (WB) using the indicated antibodies. Asterisks indicate ubiquitylated histone H3 species.C Ubiquitylation assays using recombinant histone H3, the N-terminal tail of histone H3 (residues 1-36) fused with GST (H3N-GST), or GST alone as the substrate. Both fission yeast (sp) and human (hs) full-length H3 proteins and H3N-GST fusion proteins were examined. The proteins were analyzed by SDS-PAGE followed by either silver staining or Western blotting with an anti-biotin antibody. Asterisks indicate ubiquitylated histone H3 species.
Figure 2A Ubiquitylation assay using H3N-GST. The components included in the assay and the ubiquitylated H3N-GST species are indicated at right.B MS/MS spectrum of the histone H3 peptide corresponding to residues 10-17. The observed y and b ions and fragment map are shown.C Ubiquitylation assay using biotinylated ubiquitin and recombinant wild-type H3N-GST (WT) and arginine-substituted H3N-GST mutants as substrates. Proteins were analyzed by Western blotting and silver staining. mock: reaction without substrate. Asterisks indicate ubiquitylated H3N-GST proteins.
Figure 3A Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was analyzed by SDS-PAGE and silver staining. The input sample and mock precipitation using total mouse IgG are also shown. The gel slices indicated by boxes were excised and subjected to LC-MS/MS analysis.MS/MS spectrum of the histone H3 peptide corresponding to residues 9-17. The observed y and b ions and fragment map are shown. C Summary of the ubiquitylated peptides identified by LC-MS/MS. D Relative abundance of ubiquitylated lysine residues in chromatin-associated histone H3 fractions. Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was subjected to LC-MS/MS analysis, and the abundance of ubiquitylated lysine residues in each fraction is shown Since short peptides cleaved at unmodified lysine residues could not be efficiently detected in the LC-MS/MS analysis, these data do not accurately reflect the abundance including unmodified lysines.
Figure 4B Heterochromatic silencing assays of wild-type and mutant Hht1-FH strains. Silencing at the mating-type Kint2::ura4+ was evaluated. Ten-fold serial dilutions of the indicated strains were spotted onto non-selective medium (N/S), medium lacking uracil (-Ura), and medium containing 5-FOA (5-FOA).C ChIP analysis of H3K9me2 levels associated with the mating-type loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates.D ChIP analysis of Rik1-myc levels associated with the mating-type cenH and centromeric dg loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates.E Heterochromatic silencing assays comparing the effects of H3 lysine substitution mutants. Silencing at the mating-type kint2::ura4+ was evaluated
Figure 5In vitro HMTase assays. Recombinant H3N-GST (WT or K9R) pre-ubiquitylated by the CLRC was purified and used in the HMTase assay with 6×His-tagged recombinant Clr4 (His-Clr4). Proteins were analyzed by SDS-PAGE and Coomassie Brilliant Blue (CBB) staining (left) and autoradiography (right).C,D In vitro HMTase assay using disulfide-linked ubiquitylated histone H3. H3K14CUb and control histone H3 (H3C110A) were used as substrates in the HMTase assay, and analyzed The 3H-methyl group incorporation was measured using a liquid scintillation counter (D). Error bars indicate the standard deviations calculated from three independent experiments.
Figure 6B Recombinant proteins were resolved by 10% SDS-PAGE and visualized by CBB staining.C In vitro HMTase assay using disulfide-linked ubiquitylated histone H3. H3K14CUb and control histone H3 (H3C110A) were used as substrates in the HMTase assay, and analyzedD In vitro pulldown assays of GST-Clr4 proteins with biotinylated ubiquitin. Bound ubiquitin was analyzed by Western blotting with HRP-conjugated streptavidin (left). Precipitated GST proteins were analyzed by SDS-PAGE and CBB staining (right).F Whole cell lysates prepared from control cells, cells expressing FLAG-tagged wild-type Clr4 (F-clr4) or mutant Clr4 (F-clr4∆63-127), or clr4∆ cells were subjected to immunoblotting using the indicated antibodies.G Heterochromatic silencing assays of wild-type and clr4 mutant strains. Silencing at the mating-type Kint2::ura4+ was evaluated.H ChIP analysis of H3K9me2 levels associated with the silencing marker gene inserted in the mating-type loci (Kint2::ura4+) and the mating-type cenH and centromeric dg loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates.I The levels of Kint2::ura4+, mating-type cenH, and centromeric dg transcripts were quantified by RT-qPCR and calculated as fold change to the wild-type expression. Error bars indicate standard errors from three technical replicates.
Figure 1B. Protein interaction analysis by Nano LC-MS. Unique peptide counts and protein sequence coverage, as well as signal intensities (PSI) for Tailor (bait) and CG16940/dmDis3l2 in control IP (FLAG-GFP) and FLAG-TailorIP are indicated. A.u., arbitrary units.D. Co-immunoprecipitation of FLAG-dmDis3l2 (bait) and GFP-Tailor full-length and truncations expressed in Drosophila S2cells. GFP-Nibbler (Nbr) served as negative control. Domain architecture of GFP-Tailor truncations are indicated.E- G. Recombinant protein interaction analysis using the indicated epitope tags and purification methods. Domain architecture of bait and prey are indicated.E- G. Recombinant protein interaction analysis using the indicated epitope tags and purification methods. Domain architecture of bait and prey are indicated.E- G. Recombinant protein interaction analysis using the indicated epitope tags and purification methods. Domain architecture of bait and prey are indicated.H and I. Immunostaining and imaging of Myc-Tailor and GFP-dmDis3l2 (H) or endogenous dmDis3l2 (I) in S2 cells. Single color channel images show total inversions. Scale bar = 5 µm.