Figure 5

Calmodulin antagonist treatment affects cell migration, which can be overcome by SEC62 overexpression. A, Human lung (BC01, H1299 and A549) and thyroid cancer cells (BHT101 and ML1) were seeded in normal growth medium without FBS and treated with either ophiobolin A or TFP, and seeded in the top chamber of a BD-Falcon Fluoroblock migration system (24-well format). The upper chambers were set in the lower chambers, which contained the same medium with 10% FBS as an attractant. After 72 h, migrated cells were fixed with methanol and DAPI stained. Migration was analyzed by fluorescence microscopy. To exclude the possibility that the effects seen in the migration assay were caused by effects on cell proliferation, the cells were also analyzed in the xCELLigence system. To this end, 1 × 10⁴ cells were seeded in a 96-well ePlate and growth was examined using the RTCA software. Three hundred minutes after seeding, the cells were treated with either ophiobolin A (100 nM), TFP (4 μM) or DMSO (0.1%, solvent control). All samples were measured in triplicate. B, HEK293 cells stably transfected with a plasmid encoding wild-type SEC62 (pSEC62-WT) or the respective control plasmid were seeded in normal growth medium without FBS and supplemented with TPA (10 nM) and ophiobolin A at the indicated concentrations in the top chamber. Migration was analyzed as described in A. C, The experiments described in B were performed in the presence of TFP instead of ophiobolin A at the indicated concentrations. D, Sec62 protein content was analyzed in stably transfected HEK293 cells by western blot analysis.