Fig. 4.

CHIR-99021, a GSK-3β inhibitor, suppresses the EndMT process. a NCI-H460 cells were co-cultured with HUVECs in 2D and 3D-culture systems at a ratio of 5:5, and then treated with the indicated drugs for 24 h. Cells were harvested and immunoblotted with anti-CD31, anti-VE-cadherin, anti-α-SMA, anti-vimentin, and anti-β-actin antibodies. b A549 were co-cultured with HUVEC under 2D and 3D condition at ratio of 5:5, and then treated with 2 μM CHIR-99021, 5 μM MSAB, 20 μM SB-216763, and 20 μM IWR-1. After 24 h, lysates were analyzed by immunoblotting using indicated antibodies. c NCI-H460 cells were co-cultured with HUVECs under 2D and 3D conditions and then treated with 2 μM CHIR-99021, 5 μM MSAB, and co-treated with both 2 μM CHIR-99021 and 5 μM MSAB. After 24 h, lysates were analyzed by immunoblotting using anti-CD31, anti-VE-cadherin, and anti-β-actin antibodies. d NCI-H460 cells were co-cultured with HUVECs under 3D conditions, and then treated with 2 μM CHIR-99021 for 24, 48, and 72 h. e NCI-H460 cells were co-cultured with HUVECs under 3D conditions, and then treated with 0.5, 1, and 2 μM CHIR-99021 for 24 h. Lysates were analyzed by immunoblotting. f Representative images of MCTSs containing NCI-H460 cells and HUVECs treated with CHIR-99021 for 1 day. Shown are spheroids stained with H&E, and immunohistochemically stained for CD31. Scale bars = 50 μm. g NCI-H460 cells were co-cultured with HUVECs in a 3D culture system, and then treated with 1 μM CHIR-99021 for 24 h. Lysates were analyzed by immunoblotting using anti-phospho-GSK-3β (Tyr216), anti-phospho-GSK-3β (Ser9), anti-GSK-3β, anti-CD31, and anti-β-actin antibodies