Journal: EMBO Reports

Article Title: PIWI proximity proteome reveals Set1-mediated piRNA biogenesis for transposon silencing at telomeres

doi: 10.1038/s44319-026-00901-6

Figure Lengend Snippet: ( A ) Immunoprecipitation of GFP or GFP-Set1 proteins from ovaries using anti-GFP antibody (GFP-IP) after crosslinking treatment. Tubulin serves as a loading control. ( B ) Proportion (%) of miRNA and piRNA in ovarian small RNAs. miRNA: reads mapping to miRNA precursors. piRNA: 23–29-nt reads (excluding rRNA, tRNA, snoRNA, snRNA, and miRNA mappers), or 23–29-nt reads mapping to TE consensus sequences. Control: single replicate of gfp -GLKD and GFP-Set1 WT -rescue conditions. set1 -GLKD: biological duplicate of set1 -GLKD condition. Bars: mean values, dots: individual values from biological replicates ( n = 2). ( C ) Fold changes (FC) of piRNA levels in set1 -GLKD compared to the control condition (RPM/RPM). Dots: individual TE families. TEs having few mappers (RPM < 5) were excluded. ( D ) Track view of piRNAs mapping to HTT family members. ( E ) Bar graph showing mean FC of TE-mapping piRNA levels in the mutants compared to the controls (RPM/RPM). Public data: panx KO from GSE71374 , rhi KO from GSE126578 . Dots: individual FC values from biological replicates ( n = 2). .

Article Snippet: Rabbit anti-GFP Living Colors , Clontech , 632592.

Techniques: Immunoprecipitation, Control

Journal: EMBO Reports

Article Title: PIWI proximity proteome reveals Set1-mediated piRNA biogenesis for transposon silencing at telomeres

doi: 10.1038/s44319-026-00901-6

Figure Lengend Snippet: ( A ) Immunoprecipitation of GFP-Set1 proteins from ovaries using anti-GFP antibody (GFP-IP) without crosslinking treatment. W/o: sibling control without GFP-Set1 expression. Tubulin serves as a loading control. ( B ) Ping-pong signature analysis on piRNAs mapping to telomeric TE families. Z10 scores (reflecting 10-nt overlap frequency within piRNA groups) were highlighted. Values are means of biological replicates ( n = 2). ( C ) Bar graph showing mean fold change (FC) of piRNA abundance in mutants relative to their respective controls. Raw data from GSE126578 and GSE55824 were re-analyzed using the same pipeline. GSE126578 includes biological replicates ( n = 2), and individual values are shown as dots.

Article Snippet: Rabbit anti-GFP Living Colors , Clontech , 632592.

Techniques: Immunoprecipitation, Control, Expressing, Analysis

Journal: EMBO Reports

Article Title: PIWI proximity proteome reveals Set1-mediated piRNA biogenesis for transposon silencing at telomeres

doi: 10.1038/s44319-026-00901-6

Figure Lengend Snippet: ( A ) Venn diagram showing overlap of Set1-binding peak-associated genes and TEs between biological replicates ( n = 2). Peak called by MACS2 comparing GFP-Set1 WT and GFP, or GFP-Set1 E1613K and GFP ( p < 0.01, Poisson test). ( B ) Track view for CUT&Tag signals of GFP proteins (green) and H3K4me3 (black) on TE consensus sequences. ORFs were predicted by ORF Finder. ( C ) Alignment of HeT-A{}6278 with GFP-Set1 E1613K binding signals to HeT-A consensus sequence. ( D ) Track view for anti-GFP CUT&Tag signals at 42AB locus (partial). Blue and red: piRNAs mapped to the plus and minus strands, respectively.

Article Snippet: Rabbit anti-GFP Living Colors , Clontech , 632592.

Techniques: Binding Assay, Sequencing

Journal: EMBO Reports

Article Title: PIWI proximity proteome reveals Set1-mediated piRNA biogenesis for transposon silencing at telomeres

doi: 10.1038/s44319-026-00901-6

Figure Lengend Snippet: ( A ) Venn diagram for genes and TEs having GFP-Set1 WT - or GFP-Set1 E1613K -binding peaks. Peaks called by MACS2 ( p < 0.01, estimated by Poisson distribution model). ( B ) Scatter plot comparing fold differences of CUT&Tag signals (GFP-Set1 - GFP) on genes and TEs identified by peak calling. Mean values of biological replicates ( n = 2). Signals on exons of genes and TEs were counted. r : Pearson correlation coefficient. ( C ) Heatmap shows GFP-Set1 enrichment within genes expressed in ovaries ( n = 8196, genes analyzed in Fig. ). Log2 FC of mean CPM + 1 pseudocount (GFP-Set1/GFP). Gene bodies were scaled to 5 kb. ( D ) Track view on aub gene showing signals from anti-GFP (green) or anti-H3K4me3 (black) CUT&Tag. Mean CPM of biological replicates ( n = 2). ( E ) Heatmap shows GFP-Set1 localization on TE consensus sequences. >3 kb TE families were selected. Log2 FC of mean CPM + 1 pseudocount (GFP-Set1/GFP). ( F ) Track view on HeT-A consensus sequence showing signals from anti-GFP (green) or anti-H3K4me3 (black) CUT&Tag. ( G ) Track view at the subtelomeric cluster 3 locus. Blue and red: piRNAs mapping to genomic plus and minus strands, respectively. .

Article Snippet: Rabbit anti-GFP Living Colors , Clontech , 632592.

Techniques: Binding Assay, Sequencing

Journal: EMBO Reports

Article Title: PIWI proximity proteome reveals Set1-mediated piRNA biogenesis for transposon silencing at telomeres

doi: 10.1038/s44319-026-00901-6

Figure Lengend Snippet: ( A ) Track view at subtelomeric cluster 3 or 38C locus. Light green: signals from anti-Rhi CUT&Tag. Dark green: GFP-Set1-EK signals in set1 -GLKD (anti-GFP CUT&Tag). Blue and red: piRNAs mapped to genomic plus and minus strands, respectively. Mean CPM from biological replicates ( n = 2). ( B ) Quantification of anti-Rhi CUT&Tag signals. Bars: mean FPKM (fragments per kilobase per million mapped reads), dots: individual FPKM, from biological replicates ( n = 2). ( C ) Working model. Set1 interacting with HTT sequences cooperates with Piwi for piRNA cluster formation in subtelomeric regions. Set1 provides target templates for Piwi-piRNA complexes and/or precursor transcripts for piRNA production. Fragmented HeT-A 3’UTR retaining antisense TSS plays a crucial role in the latter process. Cooperation between Set1 and Piwi promotes H3K9me3 deposition and Rhi accumulation. Once this piRNA cluster status is established, Set1 and Piwi become dispensable. Similar mechanisms operate in the silencing of stand-alone TEs. Germline cells lacking the noncanonical functions of Set1 not only fail to establish piRNA clusters at subtelomeric loci but also exhibit aberrant Rhi distribution and piRNA overproduction at certain loci. .

Article Snippet: Rabbit anti-GFP Living Colors , Clontech , 632592.

Techniques:

Journal: EMBO Reports

Article Title: PIWI proximity proteome reveals Set1-mediated piRNA biogenesis for transposon silencing at telomeres

doi: 10.1038/s44319-026-00901-6

Figure Lengend Snippet: ( A ) Track view at 42AB or 20A locus. Light green: signals from anti-Rhi CUT&Tag. Dark green: GFP-Set1-EK signals (anti-GFP CUT&Tag). Blue and red: piRNAs mapped to genomic plus and minus strands, respectively. ( B ) Quantification of anti-Rhi CUT&Tag signals. Bars: mean FPKM, dots: individual FPKM, from biological replicates ( n = 2). Analyzed regions within 42AB are indicated in ( A ). ( C ) Track view in the subtelomeric region on chromosome 2R. ( D ) Track view at subtelomeric cluster 3 showing H3K9me3 signal (mean CPM from biological replicates ( n = 2)). embKD: knockdown at embryonic stages. Raw data: GSE83238 .

Article Snippet: Rabbit anti-GFP Living Colors , Clontech , 632592.

Techniques: Knockdown

Journal: EMBO Reports

Article Title: PIWI proximity proteome reveals Set1-mediated piRNA biogenesis for transposon silencing at telomeres

doi: 10.1038/s44319-026-00901-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-GFP Living Colors , Clontech , 632592.

Techniques: Drosophila, shRNA, Recombinant, Sequencing, Plasmid Preparation, Protease Inhibitor, Mass Spectrometry, Membrane, Reverse Transcription, SYBR Green Assay, Multiplex Assay, Purification, Staining, Gel Extraction, Cloning, Cross-linking Immunoprecipitation, Qubit, Software, Imaging, Confocal, Microscopy, Real-time Polymerase Chain Reaction

Journal: Nature Microbiology

Article Title: Specialized RNA decay fine-tunes monogenic antigen expression in Trypanosoma brucei

doi: 10.1038/s41564-026-02289-4

Figure Lengend Snippet: a , b , Fluorescence microscopy analysis of ESB2 12myc localization following ESB3 and ESAP1 RNAi ( a , b ), 6myc ESB3 localization following ESB2 and ESAP1 RNAi ( b ) and 6myc ESAP1 localization following ESB2 and ESB3 RNAi ( b ). c , ESB2, ESB3 and ESAP1 protein levels following VEX2, ESB2, ESB3 and ESAP1 RNAi. The values were derived from protein blotting analysis of three biological replicates per cell line and are represented in a floating bar graph in which the box spans between minimum and maximum values, the centre line is the median, and all datapoints are depicted. Two-tailed unpaired t -tests were applied; NS, non-significant; ** P < 0.01. d – i , Fluorescence microscopy analysis of Pol-I localization following VEX2, ESB1, ESB2, ESB3 and ESAP1 knockdown ( d , e ); ESB1 12myc , ESB2 12myc , 6myc ESB3 and 6myc ESAP1 localization following VEX2 knockdown ( f , g ); 6myc VEX2 localization following ESB2, ESB3 and ESAP1 knockdown ( h , i ). The graphs in b , e , g and i depict mean values of two biological replicates; >100 G1 cells per condition were analysed; all analyses were performed at 24 h post-induction. j , Heatmap summarizing the microscopy analyses; C1, clone 1; C2, clone 2. k , Diagram depicting the complex network of co-dependencies at the ESB and interface with the SLAB. The direction of the arrows indicates that the protein upstream is required for the localization of the protein downstream to its corresponding nuclear compartment. l , Fluorescence microscopy colocalization analysis of GFP-myc ESB2 and 6HA ESB3. The images in a , d (left), h and l were acquired using a Zeiss AxioObserver, whereas the images in d (right) and f were acquired using a Zeiss LSM980 Airyscan 2. All correspond to 3D projections by brightest intensity of 0.1-μm stacks. DNA was stained with DAPI (cyan or grey). Scale bars, 2 or 5 μm. m , Immunoprecipitation of GFP-myc ESB2 using GFP nanobody-coated beads followed by protein-blot analysis using an anti-HA antibody to detect the prey ( 6HA ESB3) and an anti-GFP antibody to detect the bait ( GFP-myc ESB2). IP, immunoprecipitation. The protein blots are representative of four independent experiments; a 6HA ESB3 single tagged cell line was used as a negative control. n , Violin plots depicting a comparative transcriptomic analysis between VEX2 ( n = 3), ESB2 ( n = 5), ESB3 ( n = 3) and ESAP1 ( n = 3) RNAi cell lines for ‘active’ ESAGs ; n values correspond to biological replicates. log 2 (FC), fold change in transcript abundance between RNAi (24 h post-induction) and the parental cell line. The violins span between minimum and maximum values, centre lines correspond to the mean and all datapoints are shown. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple-comparison test; **** P < 0.0001. Diagram in m created in BioRender; Faria, J. https://BioRender.com/oia6hdu (2026).

Article Snippet: The following primary antibodies were used: mouse α-myc (Millipore, clone 4A6, 1:10,000), mouse anti-Ty (BB2, Invitrogen, 1:5,000), mouse monoclonal anti-HA (Sigma, clone HA-7, 1:2,000), rabbit polyclonal anti-GFP (LifeTech, 1:2,000) and mouse α-EF1α (Millipore, clone CBP-KK1, 1:30,000).

Techniques: Fluorescence, Microscopy, Derivative Assay, Two Tailed Test, Knockdown, Staining, Immunoprecipitation, Negative Control, Comparison

Journal: Cells

Article Title: Host SNARE Proteins Mediate Lysosome and PVM Fusion to Support Plasmodium Liver Infection

doi: 10.3390/cells15070584

Figure Lengend Snippet: Localization of host SNARE proteins at the Plasmodium PVM during liver-stage infection. ( A – C ) Confocal live cell imaging of GFP-tagged SNARE proteins VAMP7-GFP ( A ), VAMP8-GFP ( B ), and Stx7-GFP ( C ) in P. berghei -infected HeLa cells expressing mCherry (parasite cytoplasm, red) at 6, 24, and 48 hpi. SNARE localization is shown in green. ( D – G ) Immunofluorescence analysis of Vti1B ( D ), VAMP7 ( E ), VAMP8 ( F ), and Stx7 ( G ) in HeLa cells fixed at 0.5, 1, and 1.5 hpi. SNAREs were detected with anti-GFP (green) or anti-Vti1B (green); the PVM was labeled with anti-UIS4 (red); and DAPI (blue) stained nuclei. ( H ) Localization of SNARE proteins (anti-Vti1B/anti-GFP in green) with the PVM marker UIS4 (red) at 24 hpi, visualized by expansion microscopy (5-fold expanded). Merged channels (yellow) highlight PVM-SNARE protein association. Nuclei were counterstained with DAPI (blue). Scale bars: 5 μm ( A – G ); 10 μm ( H ).

Article Snippet: The cells were then incubated in 10% FCS/PBS for 1 h, at room temperature with primary antibodies: Vti1B (mouse mAb 1:1000 (1:500 PS-ExM), BD Transduction Laboratories #611404, Allschwil, Switzerland), UIS4 (rabbit 1:1000, P. sinnis ; chicken 1:10000, Proteogenix, Schiltigheim, France), hLAMP1 (mouse mAb 1:1000 (1:500 PS-ExM), DSHB H4A3 (Iowa City, USA); rabbit pAb 1:1000, Cell Signaling #9091, Allschwill, Switzerland), GFP (rabbit pAb 1:1000, Origene TA100030, Herford, Germany; mouse mAb 1:1000, Roche AQ160, Basel, Switzerland), and α-tubulin (guinea pig pAb 1:500, ABCD AA345, Geneva, Switzerland).

Techniques: Infection, Live Cell Imaging, Expressing, Immunofluorescence, Labeling, Staining, Marker, Microscopy