Fig. 7. (Top panel) Western blot analysis of N-cadherin expression of CHO-B2 and 5 integrin transfected cell lines grown as 2D or 3D cultures. Cell lysates were prepared from cells grown on tissue culture plastic and cells grown as 3D spheroids. N-cadherin was detected by immunoblot analysis. Note the presence of a 130 kDa band, corresponding to the published molecular weight of N-cadherin. Note also that transfection of CHO cells with 5 integrin did not result in increased N-cadherin expression irrespective of whether cells were grown in conventional tissue culture or as spheroids. (Bottom panel) Assessment of cadherin function by fast aggregation assay. Cells from near-confluent plates of CHO-B2 (A,B), CHO-P3 (C,D), CHO-A5 (E,F) and CHO-Ncad (G,H) were detached by trypsin/calcium (0.05% trypsin/2 mM CaCl₂) treatment. Cells were stained with the membrane intercalating dye PKH-2 and resuspended at a concentration of 1x10⁶ cells/ml in 3 ml of either calcium/magnesium-free HBSS (A,C,E,G) or HBSS with 2 mM Ca²⁺ (B,D,F,H), transferred to shaking flasks and placed on a gyratory shaker at 37°C and 120 rpm. Aggregation was monitored 1 hour later and imaged by fluorescence microscopy. Note that only the CHO-Ncad cell line aggregated in a calcium-dependent manner (G,H).