Fig. 3.

Annexin A1 (ANXA1) signalling mediates effects of TMAO on hCMEC/D3 cells. A Total cellular expression of ANXA1 in hCMEC/D3 cells treated for 24 h with 0.4 μM TMA or 40 μM TMAO. Data are expressed as mean ± s.e.m., n = 5–7 independent experiments. B Medium ANXA1 content of hCMEC/D3 monolayers treated for 24 h with 0.4 μM TMA or 40 μM TMAO. Data are expressed as mean ± s.e.m., n = 7 independent experiments. C Assessment of paracellular permeability of monolayers of wild-type hCMEC/D3 cells, or hCMEC/D3 cells stably transfected with either a scramble shRNA sequence, or one of three shRNA sequences targeting ANXA1 (clone 57/61—20.6 ± 5.6% reduction, clone 60A—47.3 ± 1.5% reduction, clone 60B—67.5 ± 1.1% reduction) to a 70-kDa FITC-dextran tracer following treatment for 24 h with 40 μM TMAO. Data are expressed as mean ± s.e.m., n = 4 independent experiments. D Assessment of TEER of monolayers of wild-type hCMEC/D3 cells, or hCMEC/D3 cells stably transfected with either a scramble shRNA sequence, or one of three shRNA sequences targeting ANXA1 (clone 57/61—20.6 ± 5.6% reduction, clone 60A—47.3 ± 1.5% reduction, clone 60B—67.5 ± 1.1% reduction) following treatment for 24 h with 40 μM TMAO. Data are expressed as mean ± s.e.m., n = 4 independent experiments. E Assessment of paracellular permeability of hCMEC/D3 cells to a 70-kDa FITC-dextran tracer following treatment for 24 h with 40 μM TMAO, with or without 10 min pre-treatment with the FPR2 antagonist WRW₄ (10 μM). Data are expressed as mean ± s.e.m., n = 3 independent experiments. F Assessment of TEER of hCMEC/D3 cells following treatment for 24 h with 40 μM TMAO, with or without 10 min pre-treatment with the FPR2 antagonist WRW₄ (10 μM). Data are expressed as mean ± s.e.m., n = 3 independent experiments