Figure 3

A mutation in the putative EF hand motif of Sec62 affects cell migration, growth and ER calcium efflux in a dominant-negative manner. A, Sequence of the human Sec62 protein. Transmembrane domains 1 and 2 are indicated with a solid underline. The predicted EF hand motif is indicated with a dotted underline. In the plasmid-encoded SEC62 _D308A , amino acid D308 (red) was replaced with an alanine. B, Sec62 protein levels in stably transfected HEK293 cells were analyzed by western blot analysis. C, HEK293 cells stably transfected with pIRES-GFP-SEC62-WT, pIRES-GFP-SEC62 _D308A or pIRES-GFP (control plasmid) were seeded in normal growth medium without FBS 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 cells were fixed with methanol and DAPI stained. Migration was analyzed by fluorescence microscopy using a 10-fold objective magnification. Migrated cells in at least five individual images were automatically counted using NIS-Elements AR Software (Nikon, Düsseldorf, Germany). The mean values and standard deviation are shown in the diagram. D, Stably transfected HEK293 cells (5 × 10³) were seeded in a 96-well ePlate and growth was examined in the xCELLigence RTCA system. After 300 min, 6 nM thapsigargin (left panel) or 0.1% DMSO (solvent control, right panel) was added to each well. All samples were measured in triplicate. E, Stably transfected HEK293 cells were seeded on glass slides in 6-cm dishes and loaded with FURA2-AM. Forty-five minutes later the cells were used for Ca²⁺ imaging as described in the legend for Figure 2. After 60 s incubation with EGTA buffer, the cells were treated with 1 μM thapsigargin. The curves shown in the diagram represent the mean cytosolic Ca²⁺ concentration of 158 cells (pSEC62-WT), 159 cells (pSEC62 _D308A ) and 160 cells (control plasmid).