Fig. 3

Reciprocal crosstalk between NSCLC cells and HUVECs induces activation of GSK-3β in multicellular tumor spheroid models. a NCI-H460 or A549 cells were cultured under 2D and 3D conditions for 2 days. The levels of TGF-β1 in culture supernatants were measured using ELISA. Results are presented as mean ± standard error of the means. *P < 0.05 versus 2D condition. b NCI-H460 or A549 cells were cultured in 2D and 3D culture systems for 2 days. Lysates were analyzed by immunoblotting using anti-TGF-β1 and anti-β-actin antibodies. c Gene expression heat map representing fold changes greater that 1.5 in samples from co-cultures of NCI-H460 cells with HUVECs under 2D and 3D conditions for 1 day. Categorization of Akt pathway-related genes. d The expression levels of GSK-3β and phosphorylated GSK-3β were determined by immunoblotting of samples from co-cultures of NCI-H460 cells or A549 cells with HUVECs under 2D and 3D conditions. e and f The ratio of p-AKT/AKT (e) and p-GSK-3β/ GSK-3β (f) was calculated