Fig. 6

Protection of barrier functionality by AC-CM is independent of HIF-1. a Immunostaining of confluent RBE4 labeled for ZO-1 (green) and cell nuclei (DAPI, blue). Cells were cultured in RBE4 or AC conditioned media under normoxia or hypoxia for 24 h. Hypoxia-induced disruption of ZO-1 (arrows) and inter-endothelial gap formation (asterisks) is seen at cell–cell borders. Scale bar = 50 μm. b Phalloidin (white) and DAPI stain (blue) of confluent RBE4 monolayers after 24 h normoxic or hypoxic exposure with AC-CM or RBE4 media. Arrows indicate hypoxic stress fiber formation, asterisks highlight inter-endothelial gap formation. Enhanced images, scale bar = 25 μm. c Permeability assays were performed on confluent RBE4 on Transwell inserts. Cells were treated with RBE4 media or AC-CM for 24 h. Results are compared to normoxic baseline (dotted line). 2way ANOVA, mean ± SD, n = 3–4, *p < 0.05, **p < 0.01 compared to RBE4 media