MDA-MB-231 and MDA-MB-468 human breast carcinoma cell lines were acquired from ATCC and cultured in RPMI 1640 medium. MVT1 mouse mammary tumor cells have been described previously [23 (link)] and were grown in DMEM medium. Media contained 10% fetal bovine serum (FBS), 2% pen strep, 1% l -glutamine. Cells were kept at 37°C with 5% CO2.
Small interfering RNA (siRNA)-mediated knockdown of RALA and RALB was achieved through the transfection of MDA-MB-231 or MDA-MB-468 cells with 50 pMol of siRNA targeting human RALA (GCAGACAGCUAUCGGAAGA; Dharmacon, Lafayette, CO, USA), RALB (GAAAGAUGUUGCUUACUAU, Dharmacon) or simultaneously targeting both isoforms (GAGCUAAUGUUGACAAGGU; Dharmacon) or non-targeting control siRNA pool (D-001810-10-05, Dharmacon) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 72 h.
Human RALA (TL309957V) and RALB (TL309956V) targeting shRNA lentiviral particles and a non-targeting control (TR30021V) were purchased from OriGene (Rockville, MD, USA). MDA-MB-231 cells were transduced with lentiviral particles and selected using 5 μg/mL puromycin for > 7 days. Similarly, mouse RALA targeting shRNA lentiviral particles and a non-targeting control (sc-41843; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used to achieve knockdown of RalA in MVT1 cells. Again, cells underwent selection in puromycin-containing medium for > 7 days.
Small interfering RNA (siRNA)-mediated knockdown of RALA and RALB was achieved through the transfection of MDA-MB-231 or MDA-MB-468 cells with 50 pMol of siRNA targeting human RALA (GCAGACAGCUAUCGGAAGA; Dharmacon, Lafayette, CO, USA), RALB (GAAAGAUGUUGCUUACUAU, Dharmacon) or simultaneously targeting both isoforms (GAGCUAAUGUUGACAAGGU; Dharmacon) or non-targeting control siRNA pool (D-001810-10-05, Dharmacon) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 72 h.
Human RALA (TL309957V) and RALB (TL309956V) targeting shRNA lentiviral particles and a non-targeting control (TR30021V) were purchased from OriGene (Rockville, MD, USA). MDA-MB-231 cells were transduced with lentiviral particles and selected using 5 μg/mL puromycin for > 7 days. Similarly, mouse RALA targeting shRNA lentiviral particles and a non-targeting control (sc-41843; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used to achieve knockdown of RalA in MVT1 cells. Again, cells underwent selection in puromycin-containing medium for > 7 days.