Extended Data Fig. 8: AC roles in CD82-facilitated vascular leakage and CD82-tuned RhoA-versus-Cdc42 balance.
(a) The monolayers were treated with indicated ceramide species (20 µM) for 24 h and then examined in FITC-dextran (250 kDa) diffusion assay (mean±SD; n=4 experiments). (b) The cells were treated with 25-HC (0.2 µM) for 3 h, followed by flow cytometry analysis on AC levels at the cell surfaces (mean±SD; n=4 experiments). (c)-(d) The monolayers were treated with 25-HC (0.2 µM) for 3 h and examined for endothelial permeability (mean±SD, n=4 experiments) with FITC-dextran (250 kDa) diffusion assay, under VEGF-A164 (50 ng/mL) stimulated (c) or basal (d) condition. (e) The cells were preincubated with intact ALOD4 peptide (3.0 µM) at 37 °C for 30 min and then examined by flow cytometry with Alexa488-conjugated ALOD4 for the AC level (mean±SD; n=3 experiments). (f)-(g) The monolayers were preincubated with intact ALOD4 peptide (3.0 µM) at 37 °C for 30 min, followed by FITC-dextran diffusion assay (mean±SD, n=4 experiments), under VEGF- or LPS-stimulated (f) or basal (g) condition. (h) The cells were treated with PBS, simvastatin (5.0 µM for 24 h), or intact ALOD4 peptide (3.0 µM for 30 min), followed by LPS stimulation for 24 h. TNF-α in the cell culture supernatants were measured by ELISA (mean±SD; n=4 experiments). (i) The cells were pretreated with simvastatin (5 µM) for 24 h and then examined for RhoA and Cdc42 activities with G-LISA assays. Their relative functionality is present as the ratio of Cdc42 activity to RhoA activity (mean±SD). (j) The cells were treated with PBS or simvastatin (5.0 µM) for 24h, stained for Cdc42-GTP and CD44, and imaged with confocal microscopy. Arrows: Cdc42-GTP-localized cell-cell junction. (k) The live cells were stained with fluorescent OlyA and ALOD4, fixed, and imaged with structured illumination microscopy. (l) The cells were treated with DMSO, simvastatin (5.0 µM), or cholesterol (0.1 mM) for 24 h, followed by the Cdc42-FARP1 PLA. (m) MDA-MB-231 cells were incubated with His-tagged ALOD4 at 37 °C for 30 min and lyzed by sonication for immunoprecipitations with the indicated Abs, shown in Fig. 6f, followed by immunoblot with the indicated mAbs.
