U2OS-SRA is a modified human osteosarcoma cell line that expresses the scavenger receptor A (SRA) [30 (link)]. U2OS-SRA cells were grown at 37° C in a 5% CO2 humidified incubator and in McCoy’s 5A medium supplemented with 10% fetal bovine serum, 1 mg/ml geneticin as a selection for SRA, 100 units/ml penicillin, and 100 ug/ml streptomycin. For stable STARD4 knockdown, U2OS-SRA cells were transfected with pGFP-V-RS shRNA encoding the target sequence 5’-GCAAGCACTTTAACCAACTTCTATGGTGA-3’ (Origene) using HiPerFect transfection reagent (Qiagen) according to manufacturer’s instructions. Selection of stable knockdown STARD4 U2OS-SRA cells was done with 5 μg/ml puromycin. Cells for wide-field microscopy were plated on 35-mm plastic dishes, the bottoms of which were replaced with poly-D-lysine-coated coverslips.
U2OS cells were transiently transfected with pCMV-Tag2b (Stratagene, Santa Clara, CA) plasmid containing shRNA resistant FLAG-tagged hSTARD4 or with mCherry-FKBP-hSTARD4 by electroporation using Amaxa Nucleofector II Device with the Amaxa Cell Line Nucleofector Kit V and Nucleofector Program X-001 or using FuGENE 6 transfection reagent (Promega, Madison, WI). STARD4 rescue experiments were performed 1 d after transfection.
U2OS cells were transiently transfected with pCMV-Tag2b (Stratagene, Santa Clara, CA) plasmid containing shRNA resistant FLAG-tagged hSTARD4 or with mCherry-FKBP-hSTARD4 by electroporation using Amaxa Nucleofector II Device with the Amaxa Cell Line Nucleofector Kit V and Nucleofector Program X-001 or using FuGENE 6 transfection reagent (Promega, Madison, WI). STARD4 rescue experiments were performed 1 d after transfection.