MDA-MB-231, NRK, and HeLa S3 cell lines were grown according to American Type Culture Collection (Manassas, VA) guidelines. The stable MDA-MB-231 cell lines that overexpress GOLPH3-IRES-GFP, GOLPH3-R90L-IRES-GFP, or IRES-GFP were constructed with the pBABE-Puro retroviral system (Morgenstern and Land, 1990 (link)). Empty retroviral expression vector pBABE-Puro and packaging vectors pUMVC and pVSV-G were kind gifts from Jing Yang (University of California, San Diego). The expression vector of wild-type GOLPH3 was generated by serial cloning to combine GOLPH3 (Dippold et al., 2009 (link)) and IRES-hrGFP (Stratagene/Agilent, Santa Clara, CA) into the BamHI and EcoRI sites of pBABE-Puro vector. Expression and packaging vectors were transfected into HEK293T packaging cells using TransIT-LT1 (Mirus, Madison, WI). Viral supernatants harvested at 48 and 72 h after transfection were filtered through a 0.45 μm filter (Thermo Fisher Scientific, Waltham, MA) and incubated with target MDA-MB-231 cells in the presence of 6 μg/ml protamine (Sigma, St. Louis, MO). 24 h after the second viral infection, 0.5 μg/ml puromycin (InvivoGen, San Diego, CA) was used to select and maintain stable cell pools. The cell lines overexpressing IRES-GFP and R90L-IRES-GFP were generated in similar manner.