EMBO/SourceData huggingface dataset:
Figure 4A) Example genomic region of DNA binding by MSL2 (DNA), and chromatin binding of MSL2 (Chrom.), MOF and H4K16ac MNase-ChIP-seq profiles in early (stage 5-8, orange) and late (stage 13-15, purple) mixed-sex embryos. Called peaks/regions are represented as boxes above the tracks. Graph represents pooled IP/Input signal of 3 biological replicates.B) Size distribution of X-chromosomal MOF and H4K16ac regions, which are present in both early and late embryos. P-value (Wilcoxon rank sum test) is indicated. Total number of regions are 459, 443, 854 and 400, respectively.C) Average composite plots of MOF MNase-ChIP-seq centered at Transcription Start Site (TSS) or Transcription Termination Site (TTS) in early or late embryos for X-chromosomal genes (total=1515). Graph represents average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across sites.D) Average composite plots of H4K16ac MNase-ChIP-seq centered at TSS or TTS in early or late embryos for X-chromosomal genes (total=1515).
Figure 5A) Scatterplot comparing X-chromosomal genic H4K16ac MNase-ChIP-seq levels in early (stage 5-8) and late (stage 13-15) mixed-sex embryos. Genes are grouped by k-means clustering (k=3). The numbers of genes are cluster 1 = 979, cluster 2 = 364 and cluster 3 = 172. B) Log2 Fold Change (log2FC) of RNA-seq between female (F) and male (M) embryos at genes grouped as in A (cluster 1-3). C) Log10 distance between the middle of a gene and its nearest functional MSL2 DNA peak (cluster 1-3). Red line indicates median distance to the nearest peak.
Figure 1A‐F′ Confocal fluorescent images of wild‐type (A, C, E) and mtm‐3(tm4475) (B, D, F) embryos at the 1.5‐fold stage stained by DAPI (A-F) and anti‐SQST‐1 (A′, B′), anti‐SEPA‐1 (C′, D′) or anti‐LGG‐1 (E′, F′) antibodies. Scale bars: 5 μm.G Western blot analysis of LGG‐1‐I and LGG‐1-II (lipidated) in wild type, epg‐6, and mtm‐3.H mtm‐3 and epg‐6 mutant L1 larvae have shortened mean and maximum life span in the absence of food. At least 300 animals were scored each day.
Figure 2A-C Fluorescent images of wild‐type embryos (1.5‐fold stage) expressing GFP::MTM‐3 (A), GFP::MTM‐3(C459S) (B), or GFP::MTM‐3(C459S, del PH‐G) (C).D‐G″ Confocal fluorescent images of wild‐type (D‐D″), mtm‐3 (E‐E″), atg‐2 (F‐F″), and vps‐34 (G‐G″) embryos at the 200‐cell stage expressing GFP::MTM‐3(C459S) and stained by anti‐LGG‐1 antibody. Insets show magnified views. Arrows indicate overlapping GFP and LGG‐1 puncta. Scale bars: 5 μm.H, I Number of GFP::MTM‐3(C459S)‐positive puncta and co‐localization of GFP::MTM‐3(C459S)‐ and LGG‐1‐positive puncta in various strains [mtm‐3(tm4475), atg‐2(bp576), lgg‐1(bp500), vps‐34(h797)]. At least 10 (I) and 15 (H) embryos were scored per strain. Data are shown as mean ± SD. Data from different mutant backgrounds were compared with wild type. **P 0.0001; N.S.: not statistically different.J MTM‐3 but not MTM‐3(C459S) exhibits phosphatase activity toward PtdIns3P and PtdIns(3,5)P2 in vitro.
Figure 3A-K Confocal fluorescent images of 200‐cell‐stage embryos from the indicated strains stained by both anti‐SEPA‐1 and anti‐LGG‐1 antibodies. Insets show magnified views. Arrows indicate overlapping SEPA‐1 and LGG‐1 puncta. The wild type contains no visible SEPA‐1 aggregates at this stage and is therefore not shown. Scale bars: 5 μm.L The percentage of SEPA‐1 puncta co‐localizing with LGG‐1 in each strain. At least 10 embryos were scored per strain. Data are shown as mean ± SD. **P 0.0001; N.S.: not statistically different.
Figure 4A-G Confocal fluorescent images of 1.5‐fold‐stage embryos from the indicated strains expressing both SQST‐1::GFP and NUC‐1::mCHERRY. Insets show magnified views. Arrows indicate overlapping or closely associated GFP and mCHERRY. Scale bars: 5 μm. The wild type contains no visible SQST‐1::GFP puncta at this stage and is therefore not shown.H, I The percentage of SQST‐1::GFP‐positive puncta that overlap with (H) or are close to (I) NUC‐1::mCHERRY‐positive structures in each strain. At least 10 embryos were scored per strain. Data are shown as mean ± SD. **P 0.0001; N.S.: not statistically different.J-L TEM analysis of embryos in wild type (I) and mtm‐3(tm4475) (J, K). Double‐membrane autophagosomes (arrows) were observed in mtm‐3 but not wild‐type embryos. Scale bars: 500 nm.
Figure 5A-H Fluorescent images of embryos at the 4‐fold stage in the indicated strains expressing ATG‐18::GFP (A-D), ATG‐18(FKKG)::GFP (E, F), or ATG‐18(FTTG)::GFP (G, H).J Number of wild‐type or mutated ATG‐18::GFP puncta in each strain. At least 15 embryos were scored per strain (mean ± SD). **P 0.0001; N.S: not statistically different.I Confocal fluorescent images of 4‐fold mtm‐3 embryos expressing ATG‐18::GFP (I′) and stained by DAPI (I) and anti‐LGG‐1 (I″) antibody. Insets show magnified views. Arrows indicate ATG‐18 puncta labeled by anti‐LGG‐1 antibody. Scale bars: 5 μm.
Figure 1(A) Microcomputed tomography (micro-CT) images of the proximal femur from 12-week-old male WT and Sigmar1 gKO mice. Scale bars, 1 mm. (B) Quantification of bone volume per tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp), trabecular thickness (Tb. Th), cortical region BV/TV (Ct. BV/TV) and cortical thickness (Ct. Th, mm) (n = 5 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. Unpaired two-tailed Student's t-test (B was used for statistical analysis.(C) Coronal images of the fifth lumbar spine. Scale Bars, 1 mm. (D) Quantification of trabecular bone parameters of lumbar spine (n = 5 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. Unpaired two-tailed Student's t-test D, was used for statistical analysis.(E) TRAP staining of tibias from male WT and Sigmar1 gKO mice. Scale bars, 50 μm. (F and G) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. Unpaired two-tailed Student's t-test F-G, was used for statistical analysis.(H and I) Representative images and quantitative analysis of calcein double labeling. Scale bars, 20 μm. (n = 6 biological replicates) Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. Unpaired two-tailed Student's t-test , I was used for statistical analysis.
Figure 2(A) Micro-CT images of the proximal femur from female WT or Sigmar1 gKO mice that received sham or ovariectomy surgery for 6 weeks. Scale bars, 1 mm. (B) Quantification of bone volume per tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp) and trabecular thickness (Tb. Th) (n = 5 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test (B were used for statistical analysis.(C) TRAP staining of femur sections from the four groups. Scale Bars, 200 μm. (D and E) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 5 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test , D and E) were used for statistical analysis.(F) TRAP staining to detect osteoclastogenesis of BMMs from WT or Sigmar1 gKO mice. Scale bars, 200 μm. (G and H) Quantification of the size and nuclei numbers of TRAP-positive multinuclear cells (n = 6 biological replicates for G and n = 3 biological replicates for H). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with unpaired two-tailed Student's t-test (G-H were used for statistical analysis.(I and J) Representative images and quantification of the relative pit resorption area of hydroxyapatite-coated plates. WT or Sigamr1 gKO BMMs were seeded on hydroxyapatite-coated plates and treated with 50 ng/mL RANKL (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with unpaired two-tailed Student's t-test J) were used for statistical analysis.
Figure 3(B) Micro-CT images of the proximal femur from sham or OVX mice with different AAV injections. Scale bars, 1 mm.(C) Quantification of bone volume per tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp) and trabecular thickness (Tb. Th) (n = 5 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test (C was used for statistical analysis.(D) TRAP staining of femur sections from the three groups. Scale Bars, 200 μm.(E and F) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 5 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test E-F, was used for statistical analysis.(G) TRAP staining to detect osteoclastogenesis of BMMs treated with different Sigmar1 agonists (10μM) or vector. Scale bars, 200 μm. (H and I) Quantification of the size and nuclei numbers of TRAP-positive multinuclear cells (n = 6 biological replicates for H and n = 3 biological replicates for I). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test , I, was used for statistical analysis.
Figure 4(A) SERCA2 peptides identified through mass spectrometry are shown.(B) HEK-293T cells were transfected with the indicated plasmids and then subjected to immunoprecipitation followed by western blotting to detect exogenous interactions between Sigmar1 and SERCA2. Data information: All results are representative data generated from at least three independent experiments.(C) The lysates from mouse BMMs were incubated with either anti-Sigmar1 antibody or normal rabbit IgG, and the pellets were detected with the indicated antibodies. Data information: All results are representative data generated from at least three independent experiments.(D) Western blots showing SERCA2 expression in HEK-293T cells transfected with different amounts of Sigmar1 plasmids. The cells were transfected with 1 μg SERCA2 plasmid and Sigmar1 plasmid (0.125 μg, 0.25 μg, 0.5 μg, 1 μg). Data information: All results are representative data generated from at least three independent experiments.(E) Upper: schematic representation of various Sigmar1 truncations. Bottom: mapping of Sigmar1 domains critical for SERCA2 binding. HEK-293T cells were transfected with different Sigmar1 truncations, and cell lysates were immunoprecipitated and subjected to western blotting. Data information: All results are representative data generated from at least three independent experiments.(F) Upper: schematic representation of various SERCA2 truncations. Bottom: mapping of SERCA2 domains crucial for Sigmar1 binding. Different truncated SERCA2 plasmids were transfected into HEK-293T cells. After immunoprecipitation, the interaction between truncated SERCA2 and Sigmar1 was detected by western blotting. Red asterisks indicate specific SERCA2 truncation bands. Data information: All results are representative data generated from at least three independent experiments.(H) Western blots showing Q615A mutant truncated SERCA2 expression in HEK-293T cells transfected with different amounts of Sigmar1 plasmids. The cells were transfected with 1 μg Q615A mutant of truncated SERCA2 plasmid and Sigmar1 plasmid (0.125 μg, 0.25 μg, 0.5 μg, 1 μg). Data information: All results are representative data generated from at least three independent experiments.(I) Interactions between Sigmar1 and WT or Q615 mutants of truncated SERCA2 were detected by Co-IP assays. Data information: All results are representative data generated from at least three independent experiments.(J) TRAP staining to detect osteoclastogenesis of BMMs from WT and gKO mice treated with different siRNAs. Scale bars, 200 μm. Data information: All results are representative data generated from at least three independent experiments.
Figure 5(A and B) Western blots showing SERCA2 expression in HEK-293T cells with or without Sigmar1 cotransfection treated with eeyarestatin I or MG132 at the indicated concentration. All inhibitors were applied to cells 8 hours prior to protein collection. Data information: All results are representative data generated from at least three independent experiments.(C) Western blots showing changes in SERCA2 expression in HEK-293T cells in the presence of different doses of flag-tagged Hrd1 plasmids. Data information: All results are representative data generated from at least three independent experiments.(D) Western blots showing alterations of SERCA2 expression in HEK-293T cells pretreated with negative control siRNA (si-NC) or Sel1L siRNA in the presence or absence of Sigmar1. Data information: All results are representative data generated from at least three independent experiments.(E) Truncated myc-tagged SERCA2 ubiquitination levels in HEK-293T cells transfected with empty vector or Sigmar1 were analyzed by immunoprecipitation. Cells were treated with MG132 (10 μM) 8 hours before harvest. Data information: All results are representative data generated from at least three independent experiments.(F) Expression of different truncated SERCA2 lysine mutants in HEK-293T cells transfected with different amounts of Sigmar1 plasmid. Data information: All results are representative data generated from at least three independent experiments.(G) Expression of WT or 2KR (K460A and K541A) mutants of full-length SERCA2 in HEK-293T cells transfected with different amounts of Sigmar1 plasmid. Data information: All results are representative data generated from at least three independent experiments.
Figure 6(B) Micro-CT images of calvaria from mice that received sham or LPS injection with PBS or dimemorfan treatment. Scale bars, 1 mm. (C) Quantification of bone volume/tissue volume (BV/TV) of calvaria from different groups (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test (C, was used for statistical analysis.(D) H&E and TRAP staining of calvaria from the three groups. Scale bars, 100 μm. (E and F) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test E-F, was used for statistical analysis.(H) Micro-CT images of proximal femurs from sham or ovariectomized mice with different treatments. Scale bars, 1 mm.(I) Quantification of bone volume/tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp) and trabecular thickness (Tb. Th) (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test , I, was used for statistical analysis.(J) TRAP staining of femurs from the three groups. Scale bars, 200 μm.(K and L) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test was used for statistical analysis.
Figure 7(A) TRAP staining of hPBMCs treated with human RANKL (50 ng/mL) or dimemorfan for 12 days. Scale bars, 500 μm.(B) Western blot analysis of NFATc1 expression during a 7-day induction of hPBMCs to osteoclasts.(C) hPBMCs were induced to differentiate into osteoclasts for 7 days, and the relative mRNA levels of marker genes were evaluated by RT-qPCR (n=6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test (C) was used for statistical analysis.
Figure 1Purified Drosophila Delta and Serrate NE3 proteins bind to liposomes (D) Binding is reduced in Delta∆β1-2, but not to the Serrate equivalent using liposomes composed of PC:PS:PE-fluoroscein (80:15:5). Notch binding to Drosophila Delta NE3 (E) WT and ∆β1-2 (Delta∆β1-2) variants both bind to Notch, unlike variant with F204A substitution in DSL domain. Comparisons were performed with a two-tailed unpaired t-test. Values are shown as scattered data points with the dark lines representing the means. ns, no significant difference, **P < 0.01; ***P < 0.0001.
Figure 2Adult wings from Dl∆β1-2 flies. No defects are detected in wings from Dl∆β1-2/+ (B), Homozygous Dl∆β1-2/ Dl∆β1-2 have extra vein tissue near L5 and uneven L2 veins (arrowheads; C).Microchaetae are arranged in rows on the thorax of control (yw) flies; these become disordered and more dense in Dl∆β1-2/Dl∆β1-2. White rectangle indicates area scored for E Number of microchaetes per central area (white rectangle in D) in the indicated genotypes.
Figure 4Equatorial region of eye imaginal discs where E(spl)mδ0.5 expression (green) becomes restricted to a single photoreceptor in each cluster (magenta), as detected in control and Dl∆β1-2/+ discs (top panels). In Df(3R)DlFx3 /+ and Dl∆β1-2/Df(3R)DlFx3 discs (bottom panels) E(spl)mδ0.5 expression is reduced (Df(3R)DlFx3 /+) or absent from several clusters (Dl∆β1-2/Df(3R)DlFx3) indicative of reduced Notch signalling. Scale bars correspond to 10 μm.Proportion of photoreceptors clusters that fail to express the E(spl)mδ0.5 reporter in the indicated genotypes. ns, no significant difference, *** P<0.0001 (one-way ANOVA). On the violin plot, dashed line represents the median and the dotted lines show the quartiles.
Figure 5Apical view of wing imaginal disc with homozygous Dl∆β1-2 clones (GFP negative) stained for Dl (red) and Cadherin (blue). (A') Z-projection of apical layers spanning a Dl∆β1-2 clone (GFP negative) located at the DV boundary. No change in apical localisation of Dl (red) and Cadherin (blue) is detected. Panels on the right show the Dl (grayscale) apical localisation on the wing imaginal disc.Uptake assay at t=0. After exposure to extracellular anti-Dl antibody, Dl protein (red) is detected at similar levels apical to Cadherin (blue) in wild-type (GFP) and Dl∆β1-2 mutant tissue (GFP negative). (B') Cross-sectional view of B, Dl protein (red) is present apically relative to Cadherin (Blue) which marks adherens junctions. Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin (Cad) and Delta (Dl), respectively. Uptake assay after 30 minutes, internalised anti-Dl (red) enters the endocytic route and in the cross sectional view (C') can similarly be detected as puncta along the cell axis in wild-type (GFP) and homozygous Dl∆β1-2 (GFP negative) tissue. Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin (Cad) and Delta (Dl), respectively.
Figure 1Average piRNA precursor abundance relative to EV in EV and ints-11 RNAi samples, normalized to the total number of short capped RNA reads mapping to annotated WormBase TSSs (Chen et al., 2013). Error bars represent the standard error of the mean (5 replicates).Distributions of piRNA precursor length in ints-11 RNAi-treated nematodes compared to empty vector-treated nematodes, obtained from deeply sequencing one pair of libraries (inserts sequenced up to 75nt). Distributions of total sequences (E) and total read counts (F) are shown.
Figure 2Co-localization between INTS-6::eGFP and mCherry::PRDE-1 in germ cells. PRDE-1 and INTS-6 co-localize in early stages of meiosis (1,2) and up to mid-pachytene (3), where an additional INTS-6 focus appears (3). When PRDE-1 foci are about to disappear, co-localization is lost (4), and the additional INTS-6 focus remains present well after the loss of PRDE-1 foci (5).
Figure 3Length distributions of nucleoplasmic and chromatin-bound piRNA precursors in N2 empty vector, N2 ints-11 RNAi, tfiis empty vector, and tfiis ints-11 RNAi nematodes. Length shifts in nascent RNA in tfiis mutants are indicated with dashed vertical lines and arrows. The length distributions were calculated from two merged replicates (see Appendix Figure S2 for the length distributions in each replicate).
Figure 4Signal of 5′ P small RNA 5′ ends mapping to piRNA loci as a function of the distance to piRNA TSSs, after removal of reads corresponding to mature 21U-RNAs (see Methods). The signal is normalized to counts per million of non-structural mapped reads. A peak of 5′ P ends centered at +38 from piRNA TSSs is observed.Number of loci with detected cleavage fragments in N2 empty vector nematodes across percentile bins of increasing termination signal strength (top panel). Distributions of fragment read counts per locus for detected loci across percentile bins of increasing termination signal strength (bottom panel). Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range. The data shown here corresponds to one of two N2 EV replicates, see Appendix Figure S6 for this analysis across replicates and genotypes.
Figure 5Distributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif-dependent piRNAs upon ints-11 RNAi knockdown in an N2 and a tfiis mutant background calculated from two merged replicates. N2 empty vector was used as a baseline for fold change calculation. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range.Average normalized CAGE signal (transcripts per million) 2 nt upstream of motif-dependent and motif-independent piRNAs (excluding undetected loci). Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile rangeDistributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif-independent piRNAs upon ints-11 RNAi knockdown in an N2 and a tfiis mutant background. N2 empty vector was used as a baseline for fold change calculation. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range