GRASP depletion–mediated Golgi destruction decreases cell adhesion and migration via the reduction of a5ß1 integrin

Ahat, Erpan; Xiang, Yi; Zhang, Xiaoyan; Bekier, Michael E.; Wang, Yanzhuang

For cell attachment, a total of 3 × 10 5 control or GRASP siRNA-treated cells were seeded on fibronectin-coated plates and incubated in serum-free medium for 30 min. Twelvewell plates were coated with fibronectin from human plasma (Sigma, F0895) at a concentration of 1 µg/ml in Tris-buffered saline (pH 7.5) at 4°C for 16 h and blocked with 2% bovine serum albumin (BSA) for 30 min. HeLa cells transfected with the indicated siRNAs, or GRASP-KO HeLa cells with or without GRASP or integrin rescue, were detached using 20 mM EDTA in phosphate-buffered saline (PBS), pelleted, and resuspended in serum-free DMEM. The cells were seeded on the plate (3 × 10 5 cell per well, three wells for each condition) and cultured in serum-free DMEM at 37°C for 30 min. Cells were then gently washed four times with PBS, and the attached cells were treated with 20 mM EDTA, collected by centrifugation, resuspended in 200 µl of growth medium, and counted using a hemocytometer. Attachment was presented as the percentage of attached cells out of the total number of cells.