Journal: Molecular Therapy. Nucleic Acids

Article Title: CAD increases the long noncoding RNA PUNISHER in small extracellular vesicles and regulates endothelial cell function via vesicular shuttling

doi: 10.1016/j.omtn.2021.05.023

Figure Lengend Snippet: sEV-incorporated PUNISHER is transferred into recipient ECs (A) PKH67-labeled sEVs (green) were cultured with recipient ECs for 0, 0.5, 6, and 24 h. The nuclei of the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue), and images were taken by using immunofluorescence microscopy. Scale bar, 20 μm. (B) PUNISHER expression was assessed in target ECs that were treated with sEV PUNISHER downregulated or sEV mock transfected using qRT-PCR. CT values were normalized to GAPDH and were expressed as fold change (∗p < 0.05, n = 3, by Student’s t test). (C) PUNISHER expression was analyzed in PUNISHER -siRNA- or control-siRNA-transfected ECs and the corresponding sEVs by qRT-PCR. GAPDH was used as an endogenous control (∗p < 0.05, ∗∗∗p < 0.001, n = 3−4, by Student’s t test). (D−F) PUNISHER expression was assessed in target ECs that were treated with PBS, sEVs, sEV PUNISHER downregulated , or sEV mock transfected using copy number analysis (∗p < 0.05, ∗∗∗p < 0.001, n = 4, by 1-way ANOVA with Bonferroni multiple comparisons test). (E) Transwell experiments with normal, siScr, si PUNISHER , and sihnRNPK in donor cells and recipient cells. PUNISHER expression was quantified in donor and target ECs that were treated with sEVs from cells transfected with siRNAs. GAPDH was used as a control. (∗∗p < 0.01, ∗∗∗p < 0.001, Student’s t test). HCAECs, human coronary artery ECs.

Article Snippet: HCAECs (PromoCell) were cultured in EC growth media with endothelial growth media supplement mix (PromoCell; #C-39225) under standard cell culture conditions (37°C, 5% CO 2 ) in a standard humidified incubator.

Techniques: Labeling, Cell Culture, Staining, Immunofluorescence, Microscopy, Expressing, Transfection, Quantitative RT-PCR, Control

Journal: eLife

Article Title: Human immunocompetent Organ-on-Chip platforms allow safety profiling of tumor-targeted T-cell bispecific antibodies

doi: 10.7554/eLife.67106

Figure Lengend Snippet: ( A ) Diagram of Colon and Duodenum-Intestine chip seeding, beginning with fragmented primary human organoids seeded into the epithelial channel of the chip. Primary intestinal endothelial cells, either colon or small-intestinal depending on corresponding epithelial tissue, are seeded into the vascular channel and the chip is cultured to maturity under flow and mechanical deformations. ( B ) Immunofluorescence staining of CEA and nuclei in colon or dudodenum chips versus matched colon or duodenum organoids. ( C ) Immunohistochemistry analysis of CEA (brown coloration) in a conventional, static model of the intestinal barrier: organoid-derived intestinal cell seeded on ECM-coated transwell membranes. ( D ) Flow cytometry-based quantification of CEA binding sites expressed by intestinal barriers cultured in transwells. High CEA-expressing cancer cell lines MKN5 and A549 serve as positive controls. ( E ) Treatment of PBMC with CEA-targeted TCBs in the absence of target does not induce activation, confirming target-dependent mode of toxicity observed in the Intestine-Chips.

Article Snippet: Human Large Intestine Microvascular Endothelial Cells (cHIMEC) (Cell Systems) were thawed at passage five and cultured in Endothelial Cell Growth Medium (EGM-2MV) (PromoCell) supplemented with Endothelial Cell Growth Medium MV2 Supplement Pack (PromoCell) and 1% v/v primocin (InvivoGen).

Techniques: Cell Culture, Immunofluorescence, Staining, Immunohistochemistry, Derivative Assay, Flow Cytometry, Binding Assay, Expressing, Activation Assay