Journal: Advanced Science

Article Title: Large Oncosomes Induce Pro‐Metastatic Stromal‐Myeloid Reprogramming

doi: 10.1002/advs.78406

Figure Lengend Snippet: Large oncosomes (LOs) engage convergent cytosolic DNA‐ and RNA‐sensing pathways in recipient stromal cells. (A) Heatmap of transcripts uniquely upregulated, Log2 fold change of differentially expressed, and incongruent genes between LO‐treated and small extracellular vesicles (sEV)‐treated human bone marrow‐derived mesenchymal stem cells (BM‐MSCs) relative to vehicle (adjusted p < 0.05). (B) Reactome pathway enrichment analysis of the gene set shown in (A). (C) Gene set enrichment analysis (GSEA) of the Kyoto Encyclopedia of Genes and Genomes (KEGG) cytosolic nucleic acid–sensing pathway in LO‐treated versus sEV‐treated human BM‐MSCs. (D) Direct stochastic optical reconstruction super resolution microscopy (dSTORM) imaging of PC3‐derived LOs labeled with a pan‐EV membrane dye (cyan) and anti‐double stranded DNA (dsDNA) antibody (red). LO‐associated DNA was assessed under four conditions: no permeabilization (External DNA), permeabilization only (internal + external DNA), DNase + permeabilization (DNA digested), and sequential DNase digestion followed by permeabilization (internal DNA). DNase treatment for Internal DNA was performed prior to permeabilization to selectively remove exposed (external) DNA while preserving membrane‐protected internal DNA. Scale bars, 1 and 2 µm. (E) Competitive enzyme‐linked immunosorbent assay (ELISA) quantification of 2'3'‐cyclic GMP‐AMP (2′,3′‐cGAMP) in PC3 cell lysates, PC3‐derived sEV, and LO. (F) Secretable alkaline phosphatase (SEAP) reporter activity in Interferon Stimulated Genes (ISG) reporter cells treated with LO in the presence or absence of stimulator of interferon genes (STING) inhibitor. Poly(I:C) and poly(dA:dT) were included as positive controls. (G–I) Reverse transcription quantitative PCR (RT–qPCR) analysis of interferon regulatory factor 3 ( Irf3 ) and 7 ( Irf7 )(G‐H),, and interferon alpha ( Ifna1)interferon (I) expression in mouse bone marrow cells isolated from wild‐type or Sting Gt mice following LO treatment for 24 h. Expression was normalized to beta actin ( Actb) . (J) Fluorescent western blot confirming STING knockout in human primary BM‐MSCs generated using CRISPR–Cas9. (K) RT–qPCR analysis of viperin ( RSAD2) expression in STING‐knockout and scramble‐control human BM‐MSCs following LO treatment for 24 h. Expression was normalized to Actb. (L) Heatmap showing expression of nucleic acid‐sensing pathway genes in human BM‐MSCs treated with LO or sEV relative to vehicle, based on RNA‐seq data. (M) Immunofluorescence imaging of PC3 cells stained with anti‐double stranded RNA (dsRNA) antibody (red) or isotype control and counterstained with DAPI. Representative images and higher‐magnification views are shown. (N) dSTORM super‐resolution imaging of PC3‐derived LO labeled with a pan‐EV membrane dye (cyan) and anti‐dsRNA antibody (magenta). LO‐associated dsRNA was assessed under four conditions: no permeabilization (External dsRNA), permeabilization only (internal + external dsRNA), RNase III + permeabilization (dsRNA digested), and sequential RNase III digestion followed by permeabilization (internal dsRNA). RNase III treatment for Internal dsRNA was performed prior to permeabilization to selectively remove exposed (external) dsRNA while preserving membrane‐protected internal dsRNA. Scale bars, 1 and 2 µm. (O) Flow cytometry analysis of PC3‐derived LO (> 1 µm) stained with anti‐dsRNA antibody or isotype control. (P–R) RT–qPCR analysis of Ccl5 (P), Irf3 (Q), and Irf7 (R) expression in bone marrow cells isolated from B6;129SF2/J and B6;129‐Mavs tm1Zjc /J (Mavs‐deficient) mice and treated in vitro with PC3‐derived LO. (S) RT–qPCR analysis of RSAD2 expression in human BM‐MSCs treated with LO pre‐incubated with DNase I or RNases. (T) Comparative analysis of gene expression changes in recipient human BM‐MSCs and corresponding EV preparations. Data are shown as LO/sEV ratios. Green bars indicate expression changes in recipient cells, whereas red bars indicate expression levels in EVs. Left, genes altered only in recipient cells and absent or negligible in EV cargo; right, genes showing opposite‐direction changes in recipient cells versus EVs, where response to sEV < response to LO in recipient cells but expression in sEV > expression in LO in EVs. For all in vitro experiments, data represent at least three independent experiments with at least three biological and three technical replicates. Statistical analyses are described in Section .

Article Snippet: Cells seeded in round cover class, after 24 h cells were fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit, and stained with dsRNA J2 antibody (Jena Bioscience, rabbit‐raised, 1:100 for cell imaging), rhodamine‐phalloidin (Cytoskeleton Inc., #PHDR1, 100 nM), WGA‐AF488 or WGA‐AF555 (Invitrogen, # W11261 /# W32464 , 1 μg/mL), and DAPI (Invitrogen, #62248, 1 μg/mL) across different experiments.

Techniques: Derivative Assay, Analysis, Gene Set Enrichment Analysis, Super-Resolution Microscopy, Stochastic Optical Reconstruction Microscopy, Imaging, Labeling, Membrane, Preserving, Competitive ELISA, Enzyme-linked Immunosorbent Assay, Activity Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Isolation, Western Blot, Knock-Out, Generated, CRISPR, Control, RNA Sequencing, Immunofluorescence, Staining, Super Resolution, Flow Cytometry, In Vitro, Incubation, Gene Expression

Journal: Science Advances

Article Title: Lineage-specific and gestational age–dependent DNA methylation programming orchestrates human placental development

doi: 10.1126/sciadv.aee0560

Figure Lengend Snippet: ( A ) Pie chart showing the proportion and number of methylated1 and escapee1 5-kb bins from TE to the first-trimester CTB. ( B ) Pie chart showing the proportion and number of methylated2 and escapee2 5-kb bins from the first- to third-trimester CTB. ( C ) Box plots showing the DNA methylation, H3K9me3, H3K9ac, and expression levels of genes and repeats undergoing methylation (genes, 25,044; repeats, 3,799,552) or escapee (genes, 10,225; repeats, 232,372) in the first-trimester CTB from TE to the first trimester. Significance between the groups was evaluated by Student’s t test. ( D ) GO enrichment analysis for methylated1 and escapee1 genes from TE to the first-trimester CTB. ( E ) qRT-PCR analysis of representative methylated1 or escapee1 genes in human embryonic stem cell (hESC), hTSC, and the first-trimester CTB. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) is set as an endogenous control. Data are presented as means ± SD (three biological replicates). ( F ) Box plots showing the DNA methylation, H3K9me3, H3K9ac, and expression levels of genes and repeats undergoing methylation (genes, 22,098; repeats, 2,847,005) or escapee (genes, 15,384; repeats, 1,217,495) in the third-trimester CTB from the first to third trimesters. Significance between the groups was evaluated by Student’s t test.

Article Snippet: The membranes were blocked with protein-free rapid blocking buffer and next incubated with EED (85322S, Cell Signaling Technology), EZH2 (39901, Active motif), SUZ12 (3737S, Cell Signaling Technology), H3K9me3 (39161, Active motif), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 60004-1-Ig, Proteintech), H3K27me3 (C15410195, Diagenode), H3 (4499, Cell Signaling Technology), CDH1 (3195S, Cell Signaling Technology), CDH2 (ab18203, Abcam), EpCAM (MA5-12436, Thermo Fisher Scientific), MMP2 (40994S, Cell Signaling Technology), and DNMT1 (24206-1-AP, Proteintech) overnight at 4°C on a shaker.

Techniques: DNA Methylation Assay, Expressing, Methylation, Analysis, Quantitative RT-PCR, Stem Cell, Control, Significance Assay

Journal: Molecular Neurobiology

Article Title: Neural Expression of IL-16 Mediates Neuropathic Pain after Sciatic Injury in Mice

doi: 10.1007/s12035-026-06280-2

Figure Lengend Snippet: Effects of systemic treatment with an anti-IL-16 or an anti-CD4 antibody on the hypernociception measured 2 days after sciatic ligation in male and female mice. a General schema of the experimental behavioural procedure. b , c The s.c. administration of an anti-IL-16 antibody 1 h before testing dose-dependently inhibited neuropathic thermal hyperalgesia ( b ; 1–10 µg/kg; n = 5–6) and mechanical allodynia ( c ; 0.01–1 µg/kg; n = 5–7) in male mice. d , e Inhibitory effect evoked in female mice after the s.c. administration of the anti-IL-16 antibody of neuropathic thermal hyperalgesia ( d ; 10 µ/kg; n = 5) and mechanical allodynia ( e ; 1 µg/kg; n = 6) 1 h before testing. f , g The s.c. administration of an anti-CD4 antibody 1 h before testing dose-dependently inhibited neuropathic thermal hyperalgesia ( f ; 1–10 µg/kg; n = 5–7) and mechanical allodynia ( g ; 0.01–0.1 µg/kg; n = 5–6) in male mice. h , i Inhibitory effect evoked in female mice after the s.c. administration of the anti-CD4 antibody of neuropathic thermal hyperalgesia ( h ; 10 µ/kg; n = 5) and mechanical allodynia ( i ; 0.1 µg/kg; n = 6–8) 1 h before testing

Article Snippet: Subsequently, samples were incubated overnight at 4 °C in a humidified chamber with either a rabbit polyclonal anti-IL-16 antibody (1:100; Invitrogen PA5-82,627), a rabbit recombinant monoclonal anti-CD4 antibody (1:200; Proteintech 86,300–3-RR).

Techniques: Ligation

Journal: Molecular Neurobiology

Article Title: Neural Expression of IL-16 Mediates Neuropathic Pain after Sciatic Injury in Mice

doi: 10.1007/s12035-026-06280-2

Figure Lengend Snippet: Time course of anti-IL-16 and anti-CD4 antibodies on thermal hyperalgesia measured 2 days after sciatic ligation in mice. Effects of systemic administration of an anti-IL-16 or an anti-CD4 antibody on hypernociception measured 9 days after sciatic ligation. a Schema of time course of the study of the antihyperalgesic effects induced by the anti-IL-16 or anti-CD4 antibodies in mice submitted to nerve ligation 2 days before. b , c Withdrawal latencies measured in mice 2 days after nerve ligation by the unilateral hot plate test 0, 1, 2, 4 and 6 h after the s.c. administration of anti-IL16 antibody ( b ; 10 µg/kg; n = 6), an anti-CD4 antibody ( c ; 10 µg/kg; n = 6) or the corresponding IgG2bk isotype ( b , c ; IgG2bk 10 µg/kg; n = 6 in each case). Each point represents the mean and corresponding standard error. • p < 0.05, •• p < 0.01 compared with the measure done at time 0, before drug administration; ** p < 0.01 compared with right paws of mice treated with IgG2bк isotype at the corresponding time, two-way ANOVA followed by Tukey’s test

Article Snippet: Subsequently, samples were incubated overnight at 4 °C in a humidified chamber with either a rabbit polyclonal anti-IL-16 antibody (1:100; Invitrogen PA5-82,627), a rabbit recombinant monoclonal anti-CD4 antibody (1:200; Proteintech 86,300–3-RR).

Techniques: Ligation, Hot Plate Test

Journal: Molecular Neurobiology

Article Title: Neural Expression of IL-16 Mediates Neuropathic Pain after Sciatic Injury in Mice

doi: 10.1007/s12035-026-06280-2

Figure Lengend Snippet: Inhibition of neuropathic hypernociception by i.sc. injection of an anti-CD4 antibody. Expression of CD4 receptors in sciatic nerves and up-regulation after nerve injury. Absence of CD3 labelling in the sciatic injured nerve and lack of effect of lymphocyte depletion on neuropathic thermal hyperalgesia. ( a – c ) Behavioural testing of the effects induced after the intra-sciatic administration of the anti-CD4 antibody in mice submitted to nerve ligation 2 days before. A timeline schema of the procedure is represented in ( a ). The i.sc. administration of an anti-CD4 antibody (50 ng; 1 h before testing) prevents thermal hyperalgesia ( b ; n = 5) and mechanical allodynia ( c ; n = 5) in mice submitted to nerve ligation 2 days before. In ( b and c ), means and their corresponding standard errors are represented, • p < 0.05, •• p < 0.01 compared with mice submitted to sham surgery; ** p < 0.01 compared with values obtained in injured mice treated with IgG2bκ, two-way ANOVA followed by Tukey’s test. ( d ) Representative example of CD4 labelling by immunofluorescence in a nerve coming from a mouse submitted to sham surgery ( d1 ) or nerve ligation ( d2 ) 2 days before. The image in d2 ) corresponds to the proximal side of the ligature closest to DRG. The white arrow indicates the ligation site. ( e ) Percentage of CD4 + immunoreactive surface measured in nerve sections coming from mice submitted to sham surgery or sciatic ligation ( n = 3). Means and their corresponding standard errors are represented, ** p < 0.01 compared with mice submitted to sham surgery, Student’s t test. ( f ) Immunofluorescence image showing a CD4 + cell located in the sciatic nerve compatible with a Schwann cell with its nucleus stained with DAPI, ( g1 – g4 ) CD4 expression in a cross section performed at the first ligation of a sciatic nerve proximal to DRG showing a myelinated axon. Bright field image showing the histological structure compatible with the axon profile and its myelin sheath ( g1 ), DAPI staining (blue, g2 ) and CD4 labelling (green, g3 ) are shown. The merged image ( g4 ) shows a more precise localization of CD4 immunofluorescence. ( h1 – h4 ) CD4 expression in a cross section performed at the first ligation of a sciatic nerve proximal to DRG showing CD4 labelling in a Schwann cell surrounding a myelinated axon. Bright field image without staining shows the histological structure compatible with a Schwann cell surrounding an axon ( h1 ), DAPI staining (blue, h2 ) and CD4 labelling (green, h3 ) are shown. The merged image ( h4 ) shows a more precise localization of CD4 immunofluorescence. ( i ) Representative examples of confocal images of cross sections of injured sciatic nerves labelled with DAPI (blue, i1 ), CD4 (green, i2 ) and the neural marker βIII-tubulin (red, i3 ). The merged image shows the colocalization of CD4 and βIII-tubulin in an axon ( i4 , yellow). ( j ) Representative examples of confocal images of injured sciatic nerves labelled with DAPI (blue, j1 ), CD4 (green, j2 ) and the neural marker βIII-tubulin (red, j3 ). The merged image ( j4 ) shows the labelling of an axon (red) surrounded by a CD4 + cell compatible with the shape of a Schwann cell (green). ( k ) Representative examples of confocal images of injured sciatic nerves labelled with DAPI (blue, k1 ), CD4 (green, k2 ), the Schwann cell marker S100β (red, k3 ). The merged image shows the colocalization of CD4 and S100β + Schwann cell ( k4 , yellow). ( l1 - l4 ) Immunofluorescence images of DAPI (blue) and CD3 (red) labelling in thymus cells ( l1 ) and longitudinal nerve sections coming from mice submitted to nerve ligation 2 days before ( l2 - l4 ). Contrasting with the clear-cut lymphocyte morphology observed in the thymus ( l1 ), no CD3 labelling is observed in nerve ( l2 - l4 ) and the sporadic red labelling signal that appeared in some nerve sections was not associated to DAPI-stained nuclei (Fig. l4 ). ( m - n ) Lack of effect of lymphocyte depletion on the hyperalgesic effect evoked by nerve ligation and on the antihyperalgesic effect evoked by the administration of an anti-IL-16 antibody. m ) Schematic representation of the experimental procedure used to evaluate the functional effect of an anti-IL-16 antibody in lymphocyte-depleted mice submitted to sciatic nerve ligation. n ) Decreased withdrawal latencies are shown in lymphocyte-depleted mice 2 days after nerve ligation. The s.c. administration of the anti-IL-16 antibody (10 µg/kg) reversed this thermal hyperalgesia measured by the unilateral hot plate test 1 h later. Means and their corresponding standard errors are represented, •• p <0.01 compared with the contralateral paw; ** p <0.01 compared with values obtained in injured mice treated with IgG2bκ, two-way ANOVA followed by Tukey’s test.

Article Snippet: Subsequently, samples were incubated overnight at 4 °C in a humidified chamber with either a rabbit polyclonal anti-IL-16 antibody (1:100; Invitrogen PA5-82,627), a rabbit recombinant monoclonal anti-CD4 antibody (1:200; Proteintech 86,300–3-RR).

Techniques: Inhibition, Injection, Expressing, Ligation, Immunofluorescence, Staining, Confocal, Marker, Morphology, Functional Assay, Hot Plate Test, Behavioral Assay