Journal: bioRxiv

Article Title: Single-cell RNA Profiling Identifies Diverse Cellular Responses to EWSR1-FLI1 Down-regulation in Ewing Sarcoma

doi: 10.1101/750539

Figure Lengend Snippet: A , C2 in Dormant 2 populations clusters are characterized by increased transcripts for autophagic genes ( GABARAP, MAP1LC3B, MAP1LC3B2 ) and TGFβ receptors and cyclin-dependent kinase inhibitory genes ( CDKN1A, CDKN2A ). B , The relationship between CD63 marker and dormancy associated gene markers ( TGFBR2 and TGFBR3 ), proliferation markers (MKI67, CDKN2A) and EWSR1 and FLI1 in each subpopulation (Pearson correlation). (C0: Stem-like (blue), C1:Committed to the cell-cycle (Red), C2;Dormant-like (Green), C3;Transitional dormant state (Orange).

Article Snippet: The primary antibodies used were human Cyclin B1-153Eu (Fluidigm; 3153009A), Cyclin D-141Pr (Cell Signaling;2978BF), CD271-149Sm (Fluidigm;3149017B), CD40-142Nd (Fluidigm; 3142010B), CD79a-158Gd (Cell Signaling;13333BF), CD49b-161Dy (Fluidigm; 3161012B), CD49e-160Gd (Fluidigm; 3160015B), CD63-150Nd (Fluidigm; 3150021B), CDKN2A-155Gd (Abcam;Ab186932), CELSR2-165Ho (Abcam; Ab189045), FGFR1-159Tb (Cell Signaling;9740BF), MKI67-147Sm (Cell Signaling; 9449BF), MYC-176Yb (Fluidigm;3176012B), TGFBR2-170Er (Abcam;Ab 78419),TGFB1-163Dy (Fluidigm;3163010B), SOX2-169Tm (Cell Signaling;3579BF).

Techniques: Marker

Journal: bioRxiv

Article Title: Single-cell RNA Profiling Identifies Diverse Cellular Responses to EWSR1-FLI1 Down-regulation in Ewing Sarcoma

doi: 10.1101/750539

Figure Lengend Snippet: A , Enrichment of a fraction of EW8 cells with positive dormancy gene features ( CDKN2A, TGFBR2 ) determined by CyTOF in the Dormant 1 and Dormant 2 populations, showing bimodal kinetics over time. B, (VISNE PLOTS) overview of the shift in the frequency of the positive dormant cells over time (120h). C , Distribution of cells with dormant features (TGFBR2+, CDKN2A+, MK167−) in siControl, Dormant 1 and Dormant 2 populations combined. D , Left , Illustration of the gating strategy for detecting cells with high levels of the membrane marker CD63 that are also expressing dormant features (high TGFRB2 and high CDKN2A). CD63-positive, TGFRB2 High , CDKN2A High represents <1% of the population of exponentially growing EW8 cells. Center, and right panels , visualization of the rare fraction of cells isolated from dissociated CDX-ES1 and CDX-EW8 xenografts with dormant characteristics with ViSNE plots. E, Cells dissociated from ES4, ES1 and EW8 xenografts were FACs sorted for high or low CD63 expression, and their ability to migrate was determined with the chemotaxis assay. (P-values are from unpaired t-test).

Article Snippet: The primary antibodies used were human Cyclin B1-153Eu (Fluidigm; 3153009A), Cyclin D-141Pr (Cell Signaling;2978BF), CD271-149Sm (Fluidigm;3149017B), CD40-142Nd (Fluidigm; 3142010B), CD79a-158Gd (Cell Signaling;13333BF), CD49b-161Dy (Fluidigm; 3161012B), CD49e-160Gd (Fluidigm; 3160015B), CD63-150Nd (Fluidigm; 3150021B), CDKN2A-155Gd (Abcam;Ab186932), CELSR2-165Ho (Abcam; Ab189045), FGFR1-159Tb (Cell Signaling;9740BF), MKI67-147Sm (Cell Signaling; 9449BF), MYC-176Yb (Fluidigm;3176012B), TGFBR2-170Er (Abcam;Ab 78419),TGFB1-163Dy (Fluidigm;3163010B), SOX2-169Tm (Cell Signaling;3579BF).

Techniques: Marker, Expressing, Isolation, Chemotaxis Assay

Journal: bioRxiv

Article Title: Single-cell RNA Profiling Identifies Diverse Cellular Responses to EWSR1-FLI1 Down-regulation in Ewing Sarcoma

doi: 10.1101/750539

Figure Lengend Snippet: A , Fluorescence images of EW8 cells transfected with siEWSR1 or siControl; EW8 Cells were fixed after 48h immunostained and subsequently cells were imaged via fluorescent microscopy to study the induction of the autophagy marker LC3B and lysosomal marker CD63 when EWSR1-FLI1 is suppressed. B , Quantification of FAM134B co-localization presented as Pearson’s correlation coefficient (r); ****P < 0.0001, t-test, n=15 fields (∼150 cells) in the dormant cells shown in A using the colocalization tool in imageJ. C , EW8 cells were treated with siEWSR1 or siControl, before being stained for ER resident protein SEC61B. The expansion of ER is shown following EWSR1-FLI1 down regulation (boxed areas). D , Quantification of cells with expanded ER after masking nuclei. **** P, 0.0001, t-test, error bars indicate s.d., n=150 cells.

Article Snippet: The primary antibodies used were human Cyclin B1-153Eu (Fluidigm; 3153009A), Cyclin D-141Pr (Cell Signaling;2978BF), CD271-149Sm (Fluidigm;3149017B), CD40-142Nd (Fluidigm; 3142010B), CD79a-158Gd (Cell Signaling;13333BF), CD49b-161Dy (Fluidigm; 3161012B), CD49e-160Gd (Fluidigm; 3160015B), CD63-150Nd (Fluidigm; 3150021B), CDKN2A-155Gd (Abcam;Ab186932), CELSR2-165Ho (Abcam; Ab189045), FGFR1-159Tb (Cell Signaling;9740BF), MKI67-147Sm (Cell Signaling; 9449BF), MYC-176Yb (Fluidigm;3176012B), TGFBR2-170Er (Abcam;Ab 78419),TGFB1-163Dy (Fluidigm;3163010B), SOX2-169Tm (Cell Signaling;3579BF).

Techniques: Fluorescence, Transfection, Microscopy, Marker, Staining

Journal: Cell reports

Article Title: Transplants foster B cell alloimmunity by relaying extracellular vesicles to follicular dendritic cells

doi: 10.1016/j.celrep.2025.115832

Figure Lengend Snippet: (A) Donor H2 Ag in splenic FDCs (inset, arrows) after transplantation of B6 (H2 b ) hearts in BALB/c mice. Original magnification ×200. Scale bars, 20 μm. (B) Quantification with ImageJ of donor H2 Ag spots per FDC network on spleens of BALB/c mice transplanted with B6 hearts. (C) Donor H2 Ag spots per FDC network on BALB/c LNs draining B6 skin allografts assessed using ImageJ. (D) Deep SIM of donor H2 Ag spots in FDCs of a BALB/c LN draining a B6 skin graft. Scale bar, 2 μm. ROI, region of interest. (E and F) 2P-microscopy of FDCs in the spleen of a BALB/c mouse transplanted with a B6 heart releasing RFP-sEVs (E) and a BALB/c LN draining a B6 skin graft releasing RFP-sEVs (F). Arrows: graft-RFP-sEVs on the surface (red) or inside (yellow) of FDCs (green). CellTrace Violet-B cells indicate B cell follicles. FDCs were labeled with AF488-CD21/CD35 Ab. Original magnification ×25. Scale bars, 20 μm. (G) Colocalization by confocal microscopy of donor (B6, IA b + H2K b ) allo-Ags in FDCs of LNs draining CMV Cre/+ LSL -RFP-CD63 B6 skin grafted in BALB/c mice. Scale bar, 10 μm. Mander’s colocalization coefficient of graft-sEVs and donor IA b + H2K b Ab analyzed using Imaris. Each triangle represents one FDC network. In (E) and (F), RFP spots per FDC network z stacks were quantified within 90-μm-thick z stacks using Imaris. Experiments were done on 4 spleens per POD (A and B), 6 graft-dLNs per POD (C), 3 graft-dLNs (D), 4 spleens (E), 4 graft-dLNs (F), and 12 graft-dLNs (G). In (B), (C), (E), and (F), comparisons were made by multiple unpaired two-tailed Student’s test. Error bars, means ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; NS, not significant.

Article Snippet: Anti-human / mouse CD63 (Clone LAMP3/ 2788) , NeoBiotechnologies , Cat # 967-MSM8-P1.

Techniques: Transplantation Assay, Microscopy, Labeling, Confocal Microscopy, Two Tailed Test

Journal: Cell reports

Article Title: Transplants foster B cell alloimmunity by relaying extracellular vesicles to follicular dendritic cells

doi: 10.1016/j.celrep.2025.115832

Figure Lengend Snippet: (A) FACS analysis of B6 LN FDC-enriched suspensions incubated with CM-DiI-allo (BALB/c)-sEVs. FDCs with remnant B cells attached were excluded by gating CD19 Neg FDCs. One of two experiments. (B) Three-dimensional reconstruction by STED microscopy of a B6 FDC following incubation with CM-DiI-allo (BALB/c)-sEVs from the experiment in (A). Top: sEVs bound to the FDC. Bottom: 180° rotation to visualize internalized sEVs at the FDC midsection. Scale bars, 5 μm. (C) IEM of an FDC (pseudo-colored) in the dLN of a BALB/c mouse injected in the footpad with 10-nm-gold CD21/35 Ab to label FDCs and allo (B6)-sEVs coated with 5-nm-gold H2K b -IA b Abs. Insets: allo-sEVs next to an FDC. N, nucleus; ROI, region of interest. Scale bars, 200 nm (D) IEM of allo (B6)-sEVs in endocytic vesicles of a BALB/c LN FDC, 3 h after footpad injection of the sEVs. Scale bars, 100 nm (E) IEM of footpad-injected allo (B6)-sEVs (5 nm gold) internalized into the labyrinthine infoldings of FDCs heavily labeled with 10-nm-gold CD21/CD35 Ab. N, nucleus. Scale bar, 300 nm. (F) Diagram of pHluorin-CD63-mScarlet-tagged sEVs internalized by FDCs in dLNs and then recycled to the extracellular space, where the sEVs emit green flashes when exposed to neutral pH. Illustration created with BioRender. (G) IEM of pHluorin labeled with 6 nm gold (arrows) on the B6 sEV surface. Scale bar, 50 nm. (H) Time-lapse by 2P-microscopy on an explanted BALB/c LN of recycling of pHluorin-CD63-mScarlet-tagged B6 sEVs from intracellular compartments of FDCs (arrows at 0 and 45 seconds) to the surface of the FDC and extracellular milieu (arrow at 90 seconds), where the sEVs emitted green flashes. Scale bar, 50 μm. For (C)–(E) and (G), original magnifications were ×20,000 and ×80,000.

Article Snippet: Anti-human / mouse CD63 (Clone LAMP3/ 2788) , NeoBiotechnologies , Cat # 967-MSM8-P1.

Techniques: Incubation, Microscopy, Injection, Labeling

Journal: Cell reports

Article Title: Transplants foster B cell alloimmunity by relaying extracellular vesicles to follicular dendritic cells

doi: 10.1016/j.celrep.2025.115832

Figure Lengend Snippet: (A) Isolation of sEVs from culture supernatants of WT or Rab27a KO B6 mouse skin explants under pro-inflammatory conditions. Diagram created with BioRender. (B) Microscopy of cryosections of BALB/c LNs draining WT or Rab27a KO B6 skin allografts. Donor H2 Ag was detected using a cocktail of biotin-IA b and -H2K b Abs. Original magnification ×200. Representative of eight LNs per variable. Scale bars, 30 μm. (C) Quantification with ImageJ of donor (B6) H2 Ag Pos spots on FDCs in skin graft-dLNs pooled from eight BALB/c recipients per POD (left). DSAs (FACS) in sera of BALB/c mice transplanted with WT or Rab27a KO B6 skin (right). Each symbol represents one recipient. (D) EM of CM-DiI-labeled sEVs from supernatants of human DCs. Original magnification ×80,000. Scale bar, 200 nm. (E) Western blot with sEV markers Tsg101 and CD63, and lack of the endoplasmic reticulum marker GRP94, on the human sEVs used in the experiments in (F)–(I). (F) Quantification (Imaris) of CM-Dil Pos spots in FDCs on cryosections of human spleens incubated with CM-DiI-human allo-sEVs untreated or opsonized with normal human serum, unprocessed or heat de-complemented. (G) Representative cryosections of human spleen incubated (or not, control) with CM-DiI-human allo-sEVs (red) untreated or opsonized with normal or heat-decomplemented human serum. FDCs are labeled with CD35 Ab (green). Arrows indicate CM-DiI-human allo-sEVs retained on the tissue cryosections. Original magnification ×400. Scale bar, 10 μm. (H) Overlapping of CM-DiI-human allo-sEVs and human splenic FDCs. (I) STED microscopy revealed that the CM-Dil Pos spots (circles) by fluorescence microscopy in human FDCs are clusters of sEVs. Scale bars, 0.5 μm. In (C) and (F), comparisons were by two-tailed Student’s test. Error bars, means ± SD; ** p < 0.01, *** p < 0.001, and **** p < 0.0001; NS, not significant.

Article Snippet: Anti-human / mouse CD63 (Clone LAMP3/ 2788) , NeoBiotechnologies , Cat # 967-MSM8-P1.

Techniques: Isolation, Microscopy, Labeling, Western Blot, Marker, Incubation, Control, Fluorescence, Two Tailed Test