Journal: Cell Reports Medicine
Article Title: RSAD2 : A pathogenic interferon-stimulated gene at the maternal-fetal interface of patients with systemic lupus erythematosus
doi: 10.1016/j.xcrm.2025.101974
Figure Lengend Snippet: RSAD2 depletion alleviates placental vascular injury caused by lipid accumulation (A) Schematic diagram showing the decidual and placental architecture and the localization of different cell types within these structures. (B and C) OCT-embedded frozen placental sections from poly(I:C)- or saline-treated pregnant WT mice were stained for BODIPY 493/503 (used to stain LDs, green). In (B), the whole placental images were obtained by a Pannoramic MIDI scanner (3DHISTECH; Hungary) and analyzed using Case Viewer software. Scale bar, 500 μm. (C) Local enlargement of the placental labyrinth, obtained by a confocal LSM980 microscope and analyzed using ZEN 2.6 software. CK17/19 is used to stain trophoblast cells. Scale bar, 20 μm. Representative images from at least three placentas per genotype from at least two litters are shown. (D) GSEA of genes expressed in the placentas of poly(I:C)-treated mice compared to those expressed in the placentas of WT mice (saline-treated), showing inhibition of vasculogenesis pathway. (E) Placentas from poly(I:C)- or saline-treated pregnant WT or Rsad2 −/− mice were fixed in PFA. Paraffin-embedded sections were then stained for CD31 (used to stain blood vessel) with DAB. Representative blood vessel images in labyrinth zone from three placentas of three pregnant mice per genotype and per treatment are shown. Scale bar, 50 μm. (F) Quantification of the labyrinth zone area occupied by blood vessels, visualized using CD31 staining as described in (E) and analyzed using Case Viewer software, n = 240. Error bars indicate SEM, and statistical analyses were performed using the unpaired t test. For measuring vascular space, a researcher was blinded to the samples and asked to quantify the area within ∼10 blood vessels from each image irrespective of the orientation of the vessel cross-section. Blood vessels were defined using CD31 staining and were quantified from at least eight different fields of view from three different placentas for each group. (G and H) GSEA of genes expressed in the placentas of Rsad2 −/− mice compared to those expressed in the placentas of WT mice (poly[I:C]-treated), showing the enrichment of genes associated with the vasculogenesis (G) and erythrocyte development (H) pathways. (I) Comparison of FPKM values of Aplnr (Apelin receptor), Kdr (VEGF receptor 2), Tie1 (Tie1, angiopoietin receptor 1), Tek (Tie2, angiopoietin receptor 2), Apln (Aplin), and Apela (Elabela) in mouse placentas, quantified using RNA-seq; the unpaired t test was used for statistical analyses. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns , not significant.
Article Snippet: BODIPY FL C12 , Thermo Fisher , Cat# D3822.
Techniques: Saline, Staining, Software, Microscopy, Inhibition, Comparison, RNA Sequencing
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![Transformed, metastasized P53 null FTE harbor mutational hallmarks of HGSOC. Whole-genome sequencing was used to characterize mutations present in transformed P53 null FTE originating from P53 null FTE/hrMSC organoids. A, Oncoprint including mutations in genes commonly mutated in HGSOC. This includes correlations to COSMIC single- and double-base signatures that are shown [single-base substitution (SBS) and double-base substitution (DBS), respectively]. B, Summary statistics of mutational analysis including somatic synonymous and nonsynonymous mutations. C, Representative graph of single-base mutations for one sample exhibiting the pan-cancer mutational signature (signature 5). D, Gene set enrichment analysis on STIC stroma relative to normal stroma from DSP. E, Enrichment score of stromal genes associated with oxidative stress–induced senescence derived from our DSP dataset. F, Gene set enrichments shared between STIC stroma and STIC epithelium. G, IF and ( H ) flow cytometry of hrMSCs stained with the general oxidation probe CellROX Green and CellROX DR, respectively. Representative cells are shown at 20× magnification with a 100 μm scale bar. In G and K , individual data points correspond to individual nuclei. More than 3 fields per group were taken to analyze 150–200 individual cells. In H – J , individual data points correspond to separate wells. I, Flow cytometry analysis of MSC CellROX DR after treatment with the antioxidant Trolox. J, Flow cytometry analysis of carboxyfluorescein succinimidyl ester (CSFEL)-labeled FTE cells cocultured with <t>MSCs</t> ( n = 3). MFI data were normalized for cell number. FTE CellROX values are displayed. K, 53BP1 foci per FTE nuclei after 24-hour coculture with hrMSCs <t>±10</t> <t>μmol/L</t> Trolox. L, Simple linear regression correlating WT1 relative fluorescence intensity (RFI) with CellROX DR determined by flow cytometry. Individual data points represent single cells. M, CellROX DR MFI following lentiviral overexpression of WT1 in nMSCs. N, CellROX DR MFI following lentiviral shRNA KD of WT1 in hrMSCs. O, FTE or ovarian cancer cells were cocultured with either CellTrace-labeled ( O ) nMSCs or ( P ) hrMSCs. MSCs were assessed for changes in CellROX DR fluorescence. For G – P , P values were determined by the Student‘s t test. Q, WT1 overexpression induces MSC and FTE oxidative stress, resulting in increased FTE DNA DSBs.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0807/pmc12130807/pmc12130807__cd-24-0805_f5.jpg)
![RSAD2 depletion alleviates placental vascular injury caused by lipid accumulation (A) Schematic diagram showing the decidual and placental architecture and the localization of different cell types within these structures. (B and C) OCT-embedded frozen placental sections from poly(I:C)- or saline-treated pregnant WT mice were stained for <t>BODIPY</t> 493/503 (used to stain LDs, green). In (B), the whole placental images were obtained by a Pannoramic MIDI scanner (3DHISTECH; Hungary) and analyzed using Case Viewer software. Scale bar, 500 μm. (C) Local enlargement of the placental labyrinth, obtained by a confocal LSM980 microscope and analyzed using ZEN 2.6 software. CK17/19 is used to stain trophoblast cells. Scale bar, 20 μm. Representative images from at least three placentas per genotype from at least two litters are shown. (D) GSEA of genes expressed in the placentas of poly(I:C)-treated mice compared to those expressed in the placentas of WT mice (saline-treated), showing inhibition of vasculogenesis pathway. (E) Placentas from poly(I:C)- or saline-treated pregnant WT or Rsad2 −/− mice were fixed in PFA. Paraffin-embedded sections were then stained for CD31 (used to stain blood vessel) with DAB. Representative blood vessel images in labyrinth zone from three placentas of three pregnant mice per genotype and per treatment are shown. Scale bar, 50 μm. (F) Quantification of the labyrinth zone area occupied by blood vessels, visualized using CD31 staining as described in (E) and analyzed using Case Viewer software, n = 240. Error bars indicate SEM, and statistical analyses were performed using the unpaired t test. For measuring vascular space, a researcher was blinded to the samples and asked to quantify the area within ∼10 blood vessels from each image irrespective of the orientation of the vessel cross-section. Blood vessels were defined using CD31 staining and were quantified from at least eight different fields of view from three different placentas for each group. (G and H) GSEA of genes expressed in the placentas of Rsad2 −/− mice compared to those expressed in the placentas of WT mice (poly[I:C]-treated), showing the enrichment of genes associated with the vasculogenesis (G) and erythrocyte development (H) pathways. (I) Comparison of FPKM values of Aplnr (Apelin receptor), Kdr (VEGF receptor 2), Tie1 (Tie1, angiopoietin receptor 1), Tek (Tie2, angiopoietin receptor 2), Apln (Aplin), and Apela (Elabela) in mouse placentas, quantified using RNA-seq; the unpaired t test was used for statistical analyses. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns , not significant.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0327/pmc11970327/pmc11970327__gr5.jpg)
