Figure 6

SEC62 silencing and TFP treatment additively affect cell growth and migration in PC3 cells. A, Cells were seeded in 6-cm dishes and transfected with SEC62 siRNA or control siRNA 24 h and 48 h after seeding. Twenty-four hours after the second transfection, 5 × 10³ PC3 cells were seeded in a 96-well ePlate and growth was examined using the xCELLigence RTCA system. After 300 min, the cells were treated with 10 nM thapsigargin or 0.1 nM thapsigargin in the presence of DMSO (0.1%, solvent control) or TFP (8 μM). All samples were measured in triplicate. B, The slopes of the growth curves shown in A between 8–72 h were calculated using the RTCA software. The error bars indicate standard deviations. C, Cells were treated with SEC62 siRNA or control siRNA as described in A. Twenty-four hours after the second transfection, cells were seeded in normal growth medium without FBS and supplemented with either 4 μM TFP or 0.1% DMSO (control) in the top chamber of a BD-Falcon Fluoroblok migration system (24-well format). The lower chamber contained the same medium with 10% FBS as an attractant. After 72 h, migrated PC3 cells were fixed with methanol and DAPI stained. Migration was analyzed by fluorescence microscopy. D, Migrated cells from C were automatically counted using the NIS-Elements AR Software (Nikon).