EMBO/soda-nel huggingface dataset:
Dynamic subcellular compartmentalization ensures fidelity of piRNA biogenesis in silkworms (2021)
Figure 2

Fig 2. Siwi-D670A specifically accumulates with BmSpnE and BmQin at piP-bodies

Fluorescence Recovery After Photobleaching (FRAP) experiment on AcGFP-Siwi and AcGFP-Siwi-D670A. Red dotted lines represent recovery traces of Siwi-D670A foci (n=36) and black dotted lines represent recovery traces of Siwi foci (n=36). Solid lines represent locally estimated scatterplot smoothing (LOESS) curve and the grey areas represent 95% confidence level interval. Fluorescence intensity is quantified with the softWoRx software and normalized to minimum and maximum in a 0-1 scaling.

(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.

Data information: (B-D) Box plots: Centre line, median; box limits, lower (Q1) and upper (Q3) quartiles; whiskers, 1.5 × interquartile range (IQR); points, outliers.
Dynamic subcellular compartmentalization ensures fidelity of piRNA biogenesis in silkworms (2021)
Figure 3

Fig 3. Nuage proteins are required for proper piP-body assembly

All panels in Figure 3 have the following common legend: (Top-left) Representative Z-projections (Maximum intensity). Scale bar 8 μm. (Top-right) Enlarged area from the white box. Scale bar 2 μm. For all panels in Figure 3 except otherwise specified, (Bottom) Box plot with P-value calculated by Asymptotic Wilcoxon rank sum test. See also Figure EV3.

Depletion of Siwi results in segregation of BmQin from BmDcp2-containing P-bodies. Box plot showing that depletion of Siwi (dsSiwi, n=47 cells) reduces colocalization ratio between BmQin and BmDcp2, compared to control (dsRLuc, n=55 cells). Representative data from N = 4 independent experiments are shown.

Depletion of Siwi results in segregation of BmSpnE from BmDcp2-containing P-bodies. Box plot showing that depletion of Siwi (dsSiwi, n=55 cells) reduces colocalization ratio between BmSpnE and BmDcp2, compared to control (dsRLuc, n=55 cells). Representative data from N = 6 independent experiments are shown.

Depletion of Siwi results in colocalization of BmSpnE and BmVasa. Box plot showing that depletion of Siwi (dsSiwi, n=18 cells) significantly up-regulates colocalization ratio between BmSpnE and BmVasa, compared to control (dsRLuc, n=18 cells). Representative data from N = 3 independent experiments are shown.

Depletion of BmVasa results in segregation of Siwi from BmAgo3 foci. Box plot showing that depletion of BmVasa (dsBmVasa, n=12 cells) down-regulates colocalization ratio between Siwi and BmAgo3, compared to control (dsRLuc, n=12 cells). Data points collected from N = 2 independent experiments are shown (statistical test is not performed for N < 3 data).

Depletion of BmVasa results in entanglement of Siwi at piP-bodies. Interaction interfaces are indicated by white arrows. Box plot showing that depletion of BmVasa (dsBmVasa, n=45 cells) mildly up-regulates colocalization ratio between Siwi and BmDcp2, compared to control (dsRLuc, n=49 cells). Representative data from N = 5 independent experiments are shown.

Data information: (A-E) Box plots: Centre line, median; box limits, lower (Q1) and upper (Q3) quartiles; whiskers, 1.5 × interquartile range (IQR); points, outliers.
Dynamic subcellular compartmentalization ensures fidelity of piRNA biogenesis in silkworms (2021)
Figure 4

Fig 4. BmVasa ATPase activity regulates nuage/piP-body partitioning

Localization of AcGFP-BmVasa and mCherry-BmDcp2 in BmN4 cells. Line scans (white line in the enlarged region) show that both wild type and ATPase mutant BmVasa-E339Q do not colocalize with BmDcp2. Line scans of fluorescence intensity are normalized to the highest value and depicted at the right panel. Scale bar 4 μm.

Localization of AcGFP- and mCherry-BmVasa-E339Q with wild-type BmVasa as the nuage marker. Z-projections (Maximum intensity) show that while wild-type BmVasa localizes in perinuclear region, BmVasa-E339Q does not necessarily colocalize with its wild-type counterpart but mostly mis-localizes to distal cytoplasmic granules. Scale bar 8 μm.

Colocalization of BmVasa-E339Q and Siwi-D670A in enormous cytoplasmic aggregates. Wild-type Siwi with an active slicer does not cause aggregation of comparable sizes. Scale bar 8 μm.

Co-expression of BmVasa-E339Q/Siwi-D670A results in aggregates with larger sizes. Representative Z-projections (Maximum intensity) of mutant aggregates. Scale bar 1 μm.

Cumulative proportion of the BmVasa-E339Q/Siwi-D670A aggregate area between 0.4 and 20 square microns. When both mutants are expressed, more than 60% of the counted foci have increased sizes, where some reach 10 square microns which is rare when only one mutant (BmVasa-E339Q) is expressed.

Schematic diagram of the Siwi shuttling model between nuage and piP-bodies. At steady state, wild-type Siwi is required for the piP-bodies assembly and physically interacts with the BmSpnE/BmQin heterodimer. At the same time, Siwi is enriched in nuage, colocalizing with BmVasa. This suggests that Siwi is likely to shuttle between nuage and piP-bodies in a transient manner. Catalytic mutant Siwi-D670A is instead retained in piP-bodies and seldom localizes in nuage, suggesting that slicer activity of Siwi is required for this shuttling. Co-expressing Siwi-D670A and BmVasa-E339Q result in enormous cytoplasmic aggregates, suggesting that BmVasa-E339Q functions as a dominant-negative mutant that masks the ATP-dependent RNPase activity of wild-type BmVasa. Therefore, both activities of BmVasa ATPase and Siwi slicer are required for Siwi shuttling.
Dynamic subcellular compartmentalization ensures fidelity of piRNA biogenesis in silkworms (2021)
Figure 5

Fig 5. Disrupted nuage/piP-body partitioning causes mis-production of mRNA-derived piRNAs

Length distribution of total small RNAs from BmN4 cells overexpressed with AcGFP, mCherry-Siwi or mCherry-Siwi-D670A. Two peaks (20-nt and 27-nt) correspond to siRNA/miRNA and piRNA respectively.

MA plot of differential piRNA expression analysis between Siwi-D670A and Siwi overexpressed libraries. Each dot represents the mapped piRNA (> 25-nt) on a predicted silkworm gene. Predicted CDSs with mean RPM > 2 between two independent naive libraries were categorized into non-TE genes (red), potential-TE genes (orange) and TEs (blue) (see also Figure EV4A and Materials and Methods). Siwi-D670A overexpression up-regulates non-TE gene-derived piRNAs while TE-derived piRNAs are not affected. See also Figures EV4 and EV5.

Split 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.

Normalized coverage per million (CPM) of mapped piRNA reads (> 25-nt) on the CDS region of 4 representative genes. Colored areas represent reads from Siwi-D670A-overexpressed library and the grey areas represent reads from wild-type Siwi-overexpressed library. (Top) piRNA reads mapped to both sense and antisense directions on a TE gene (putative reverse transcriptase) and a potential TE gene (uncharacterized protein) with similar RPM between the two libraries. (Bottom) piRNA reads mapped to the sense direction on non-TE genes (ribosomal protein S9 and eIF4E-1) with elevated RPM in the Siwi-D670A library. Elevated peaks appear to spread around the pre-existing peaks on these genes in the wild-type library.

Fold changes of piRNA expression levels in UTRs and CDS between Siwi-D670A and Siwi overexpressed libraries. Non-TE-derived piRNAs are similarly up-regulated across 5′ UTR (non-TE: 105 genes, TE: 274 genes), CDS (non-TE: 628 genes, TE: 811 genes), and 3′ UTR (non-TE: 126 genes, TE: 257 genes). Bonferroni-corrected P-values were calculated by Asymptotic Wilcoxon rank sum test.

Fold changes of piRNA expression levels in introns (15,065 annotated regions), intergenic regions (11,124 annotated regions), and exons (10,177 annotated regions) between Siwi-D670A and Siwi overexpressed libraries. Only exon-derived piRNAs are up-regulated by Siwi-D670A overexpression, suggesting that the process is strictly cytoplasmic. Bonferroni-corrected P-values and the effect sizes (r) were calculated by Asymptotic Wilcoxon rank sum test.

Data information: (C, E, F) Box plots: Centre line, median; box limits, lower (Q1) and upper (Q3) quartiles; whiskers, 1.5 × interquartile range (IQR); points, outliers.
Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone-responsive myopathy (2018)
Figure 2

Fig.2 Phospholipidome in HSA-Cre mice muscles

(A) Phosphatidic Acid (PA) levels in HSA-Cre and control GC muscles. (B) Phospholipidome in HSA-Cre and control GC muscles. Proportions of indicated phospholipid classes in total phospholipids are represented. PC; Phosphatidylcholine, PE; Phosphatidylethanolamine, PI; Phosphatidylinositol, PS; Phoshpatidylserine, PA; Phosphatidic Acid, PG; Phosphatidylglycerol. (C) Amounts of indicated phospholipid classes normalized by protein content in HSA-Cre and control GC muscles. (D) Total phospholipid (PL) amount normalized by protein content in HSA-Cre and control GC muscles. (E) Distribution of the total carbon number in acyl chains of PC in HSA-Cre and control GC muscles. (F) Distribution of the number of double bonds in acyl chains of PC in HSA-Cre and control GC muscles. (G) Distribution of the total carbon number in acyl chains of PE in HSA-Cre and control GC muscles. (H) Distribution of the number of double bonds in acyl chains of PE in HSA-Cre and control GC muscles. Data for all experiments presented in the figure are mean ± SEM (n=3; *P<0.05, **P<0.01, ***P<0.001)
Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone-responsive myopathy (2018)
Figure 5

Fig. 5 Metabolomics analysis of HSA-Cre muscles

(A) Metabolomics profile of GC muscle of 4-month-old HSA-Cre and control mice presented as heatmap visualization and hierarchical clustering analysis of compounds with p≤0.05, Student T test (see methods for details). Rows: metabolites; columns: samples; color key indicates metabolite expression value (blue: lowest; red: highest) (n=7-4).

(B) Fold change of phospholipid precursors in GC muscles of 4-month-old HSA-Cre and control mice measured by liquid-chromatography (LC) tandem mass spectrometry (MS/MS) (n=7-4, *P<0.05).

(C) Fold change of Lactate/Pyruvate, Pyruvate/AcetylCoA, Pyruvate/Citrate, ATP/AMP, and ATP/ADP ratios in GC muscles of 4 month-old HSA-Cre and control mice (n=7-4, *P<0.05).
Paternal age affects offspring via an epigenetic mechanism involving REST/NRSF (2020)
Figure 4

Figure 4 | Comprehensive transcriptome analysis also identified REST/NRSF target genes in up-regulated genes in offspring derived from aged fathers.

A. Volcano plot for DEGs in the embryonic brain of YFO and AFO at E14.5 (n=3 in each group). Green lines indicate a twofold change and p=0.01. Orange dots indicate REST/NRSF target genes.

B. GO gene sets in AFO up-regulated genes.

C. GO gene sets in AFO down-regulated genes.

D. Motif gene sets in AFO up-regulated genes.

E. GSEA plots of DEGs in NRSF/REST target gene sets.

F. GSEA plots of DEGs for early-fetal gene sets.

G. GSEA plots of DEGs for late-fetal gene sets.

H. GSEA plots of DEGs for SFARI-related gene sets.

I. GSEA plots of DEGs for DNA hypo-methylation neighboring gene sets.

J. GSEA plots of DEGs for DNA hypo-methylation neighboring gene sets with REST/NRSF binding motifs.

Data information: Data are presented in the volcano plot or line chart format. *p<0.05, ** p<0.01, ***p<0.001, determined by nominal GSEA p-value. NES, normalized enrichment score.
Paternal age affects offspring via an epigenetic mechanism involving REST/NRSF (2020)
Figure 5

Figure 5 | REST/NRSF target genes were not enriched at E11.5 in offspring embryonic brains derived from aged fathers.

A. Volcano plot for DEGs in the embryonic brain of YFO and AFO at E11.5 (n=3 in each group). Green lines indicate a twofold change and p=0.01. Orange dots indicate REST/NRSF target genes.

B. GO gene sets in AFO up-regulated genes.

C. GO gene sets in AFO down-regulated genes.

D. Motif gene sets in AFO up-regulated genes.

E. GSEA plots of DEGs for REST/NRSF target gene sets.

F. GSEA plots of DEGs for early-fetal gene sets.

G. GSEA plots of DEGs for late-fetal gene sets.

H. GSEA plots of DEGs for DNA hypo-methylation neighboring gene sets.

I. GSEA plots of DEGs for DNA hypo-methylation neighboring gene sets with REST/NRSF binding motifs.

Data information: Data are presented in the volcano plot or line chart format. *p<0.05, ** p<0.01, ***p<0.001, determined by volcano or nominal GSEA P-value. NES, normalized enrichment score.
Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20 (2021)
Figure 1

Figure 1 - A3A deaminates HBV cccDNA dependent on transcription but is not sufficient to induce its degradation.

A dHepaRG cells were infected with HBV at a multiplicity of infection (MOI) of 100 virions/cell and transfected with siRNA against A3A (siA3A) or control siRNA (-) on day 8 post infection (p.i.). Cells were treated with 300 U/ml IFNα from day 9 to 15 p.i. and HBV cccDNA was determined thereafter by qPCR relative to the prion protein (Prnp) gene (n=4 biological replicates).

B-D dHepaRG-TR-X cells were infected with an HBx-deficient HBV (HBV∆x) at an MOI of 200 virions/cell. Cells were treated with tetracycline to induce expression of HBx (+HBx) and/or 1000 U/ml IFNα from day 10 to 17 p.i. HBeAg (B) was measured by ELISA (OD: optical density) and HBV cccDNA (C) by qPCR relative to Prnp (n=3 biological replicates). cccDNA amplicons were analyzed by differential DNA denaturation PCR (3D-PCR) (D) to visualize deamination.

E-G dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell. Cells were treated with tetracycline (Tet (A3A)) or/and 300 U/ml IFNα from day 9 to 18 p.i. cccDNA amplicons were analyzed by 3D‑PCR (E). Marked amplicons were sequenced and analyzed for nucleotide composition (F) in comparison to the wildtype HBV sequence (n=3-5 biological replicates). HBV infection level was determined by measuring cccDNA, intracellular HBV DNA by qPCR relative to Prnp or HBeAg by ELISA (G) (n=6 biological replicates from two independent experiments).

Data information: Data are represented as mean +/- standard deviation (SD), nd: not detected. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant by Student's unpaired t test with Welch's correction.
Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20 (2021)
Figure 2

Figure 2 - Differential gene expression analysis indicates ISG20 as candidate nuclease.

A A3A expressing dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell and treated 7 days after infection with 300 U/ml IFNα (+ IFNα) or without (- IFNα). Microarray data was analyzed for upregulated nucleases after 6 or 72 hours of treatment, respectively.

B,C dHepaRG cells were infected with HBV at an MOI of 100 virions/cell and treated 9 days later with 300 U/ml IFNα (B) or 200 U/ml IFNγ (C) for indicated times (h: hours). Expression of indicated genes was analyzed by qRT-PCR relative to TATA-box binding protein (TBP) mRNA (n=6 biological replicates of two independent experiments).

D dHepaRG cells were infected with HBV at an MOI of 100 virions/cell or not infected and treated 7 days later with 300 U/ml IFNα for 6 hours or 200 U/ml IFNγ for 24 hours. ISG20 mRNA levels were measured by qRT-PCR relative to TBP mRNA (n=6 biological replicates of two independent experiments).

E dHepaRG cells were infected with HBV at an MOI of 100 virions/cell and treated with 300 U/ml IFNα from day 7 to 9 p.i. or 200 U/ml IFNγ from day 7 to 10 p.i. Primary human hepatocytes were infected with HBV at an MOI of 100 virions/cell and treated with 500 U/ml IFNα or 200 U/ml IFNγ from day 4 to 6 p.i. Expression of ISG20 was detected by western blot.

Data information: Data are represented as mean +/-SD. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant by Student's unpaired t test with Welch's correction.
Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20 (2021)
Figure 3

Figure 3 - ISG20 is expressed in acute, self-limiting HBV infection but not in chronic hepatitis B.

A Liver tissue samples obtained from HBV-negative patients undergoing metastasis resection (Control), or patients with acute, self-limiting HBV infection or chronic hepatitis B, or chronic coinfection with HBV and hepatitis D virus (HDV) were stained for ISG20 by immunohistochemistry. For each clinical entity, tissue sections from three different patients are shown; scale bar: 50 µm.

B ISG20 positive area of each sample (n=3 biological replicates) was determined by Tissue IA image analysis software and is given in % of total tissue area scanned.

Data information: Data are represented as mean +/-SD. **p < 0.01, ns: not significant by one‑way ANOVA.
Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20 (2021)
Figure 4

Figure 4 - ISG20 localizes to nucleoli after interferon treatment.

A dHepaRG cells were infected with HBV at an MOI of 100 virions/cell and treated with 600 U/ml IFNα or 400 U/ml IFNγ from day 10 to 14 p.i. and ISG20 was stained by immunofluorescence.

B Primary human hepatocytes were infected with HBV at an MOI of 100 virions/cell and treated with 500 U/ml IFNα or 200 U/ml IFNγ from day 4 to 6 p.i. and ISG20 was stained by immunofluorescence.

C, D dHepaRG cells were infected with HBV at an MOI of 500 virions/cell, treated with 600 U/ml IFNα or 400 U/ml IFNγ from day 7 to 9 p.i. and stained by immunofluorescence for ISG20 and PML (C) or ISG20 and nucleophosmin (D).

E Primary human hepatocytes were infected with HBV at an MOI of 100 virions/cell, treated with 500 U/ml IFNα or 200 U/ml IFNγ from day 4 to 6 p.i. and stained for ISG20 and nucleophosmin. White arrowheads indicate double-positive nuclei.

Data information: Scale bars: 10 µm.
Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20 (2021)
Figure 5

Figure 5 - ISG20 knockdown mitigates interferon-induced control of cccDNA.

A-D dHepaRG cells were infected at an MOI of 100 virions/cell and transduced at day 7 p.i. with an adenoviral vector for expression of shRNA targeting ISG20 (AdV-shISG20). After 2 days, cells were treated with 300 U/ml IFNα or 200 U/ml IFNγ. ISG20 expression (A) was analyzed by western blot after 7 days of IFNα or 12 days of IFNγ treatment, respectively. The amount of ISG20/GAPDH was determined by signal density measurement. cccDNA (B) relative to Prnp (n=4 biological replicates), total intracellular HBV DNA (C) relative to Prnp and HBeAg (D) were measured 12 days after treatment (n=8 biological replicates of two independent experiments). cccDNA was measured after T5 digestion of DNA.

Data information: Data are represented as mean +/- SD. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant by Student's unpaired t test with Welch's correction.
Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20 (2021)
Figure 6

Figure 6 - ISG20 degrades dsDNA and ssDNA and is enriched on deoxyuridine(dU)-containing ssDNA.

A A 3D-PCR amplicon was digested in vitro with recombinant ISG20 and increasing concentration of manganese chloride.

B A subsequence of the 3D-PCR amplicon was used as (-) ssDNA oligomer, complementary (+) ssDNA and a dU-containing (‑) ssDNA, where all cytosines were replaced by uracils. These oligomers were digested in vitro with recombinant ISG20 with or without EDTA as indicated.

C Recombinant ISG20 and mA3A (mutant E72A, C72A) were mixed with indicated oligomers and analyzed in a single point binding mobility shift assay.

D dHepG2H1.3 cells were treated for one day with 1000 U/ml IFNγ and harvested proteins were subjected to an affinity purification-mass spectrometry assay (n=4 technical replicates). Oligomers used for pulldown were single-stranded or double-stranded DNA with and without dU as indicated.

Significantly enriched protein groups in the volcano plot were identified via two-sided Welch's t-tests (S0 = 1) corrected for multiple hypothesis testing applying a permutation-based FDR (FDR < 0.01, 250 randomizations). Dashed line and blue colored points indicate a significant enrichment according to the Welch's t-test cut-offs. Statistical significance of log2 iBAQ intensities of ISG20 enrichment was determined using a two-sided Welch's t-test.

Data information: Data are represented as mean +/- SD. ***p ≤ 0.001, n.s.: not significant (p > 0.05) by two-sided Welch's t-test.