Fig. 6
From: Reactive astrocytic S1P3 signaling modulates the blood–tumor barrier in brain metastases
Knockdown of astrocytic S1PR3 strengthened the BBB and BTB in vitro. Immortalized human astrocytes were transduced with either control shRNA (shControl-1, shControl-2) or shS1PR3 (shS1PR3-1, shS1PR3-2), and used in TEER and permeability assays of the BBB and BTB (see legend to Fig. 5). a RT-PCR for S1PR3 mRNA (top) and immunofluorescent staining (bottom) for S1P3 protein (green). Nuclei are DAPI stained (blue) (scale bar = 50 μm). b TEER assays using either shControl-1, -2 or shS1PR3-1, -2 astrocytes in the cultures. S1PR3 knockdown elevated TEER at 24 h, indicative of a tighter BBB and BTB, (n = 6). c Permeability of 100 µg mL−1 doxorubicin from luminal to abluminal side of the inserts in the BBB and BTB, (n = 6). Astrocytic S1P3 knockdown (using shS1PR3-1 and shS1PR3-2) reduced permeability. d Staining of endothelial cell monolayers in TEER assays for VE-cadherin (red) demonstrating increased membrane staining (white arrows) when shS1PR3-1, -2 astrocytes were included. Nuclei were DAPI stained (blue) (scale bars = 10 μm for set-1 and 20 μm for set-2). For (b) and (c), Mann–Whitney statistical analysis was performed. Graphs represent the median and the error bars the 95% confidence intervals, from independent transwell BBB/BTB cultures (n) from multiple experiments. **P < 0.01, ***P < 0.001
