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 cultures, HeLa and MDA-MB-231 cells were grown in DMEM (Invitrogen) containing 10% super calf serum (Gemini, and glutamine at 37°C in a 5% CO 2 incubator. For knockdown of GRASP55 and/or GRASP65, HeLa cells were plated at 40% confluency in six-well plates and 3 µl of a 50 µM siRNA stock was added to 250 µl of Opti-MEM. In a separate tube, 5 µl of transfectamine RNAiMAX (Invitrogen) was mixed with 250 µl of Opti-MEM and incubated for 5 min at room temperature. The two mixtures were combined and incubated at room temperature for 20 min and then added to the cells in 2 ml DMEM containing 10% super calf serum. siRNAs for human GRASP55 (AACTGTCGAGAAGTGATTATT) and GRASP65 (CCTGAAGGCACTACTGAAAGCCAAT) (Xiang and Wang, 2010; Xiang et al., 2013) were purchased from Ambion and Invitrogen, respectively. Control nonspecific siRNAs were purchased from Ambion. GRASP-KO cell lines were described previously (Bekier et al., 2017) .