Polybrene

Murine C3 shRNA, human TWIST1 cDNA, or human C3 cDNA carrying lentiviri were produced by the core facility for Molecular cloning and Lentivirus production system in the Department of Cancer Biology at UT MDACC. Briefly, murine C3 shRNA (or scrambled sequence), human TWIST1 cDNA, or human C3 cDNA was cloned into pGreen Puro-Lentiviral vectors (pGreenPuro^™, System Biosciences, Mountain View, CA) and tagged with green fluorescent protein (GFP). Twenty micrograms of these cloned lentiviral vectors were transfected into HEK293T cells along with 15 μg of packaging plasmid (2^nd generation psPAX2, Addgene, Cambridge MA) and 15 μg of envelope plasmid (2^nd generation pMD2G) using FuGENE transfection reagent (Promega, Madison, WI) in accordance with the manufacturer’s protocol. Supernatants containing the lentivirus were collected, filtered, and added to the cancer cells in the presence of 6 μg/ml Polybrene (Promega). Twenty-four hours later, 4 μg/ml of puromycin was added to the media for 7 days. Transduction efficiency of the cells was calculated by dividing the number of GFP-expressing cells by the total number of cells, and was found to be 100% in all of the experiments. Transduced cells were analyzed by qRT-PCR assay to determine the level of C3 or TWIST1 mRNAs.

Murine C3 shRNA, C3aR shRNA, C5aR shRNA, and C5L2 shRNA carrying lentiviri were produced by the core facility for molecular cloning and lentivirus production system in the Department of Cancer Biology at UT MDACC. Briefly, murine C3 shRNA, C3aR shRNA, C5aR shRNA, C5L2 shRNA, or scrambled sequence was cloned into pGreenPuro lentiviral vectors (pGreenPuro; System Biosciences) and tagged with GFP. A total of 20 μg of these cloned lentiviral vectors was transfected into HEK293T cells along with 15 μg of packaging plasmid (2^nd generation psPAX2; Addgene) and 15 μg of envelope plasmid (2^nd generation pMD2G) using FuGENE transfection reagent (Promega) in accordance with the manufacturer’s protocol. Supernatants containing the lentivirus were collected, filtered, and added to the cancer cells in the presence of 8 μg/ml Polybrene (Promega). Twenty-four hours later, 4 μg/ml of puromycin was added to the media for 7 days. Transduction efficiency of the cells was calculated by dividing the number of GFP-expressing cells by the total number of cells and was found to be 100% in all of the experiments. Transduced cells were analyzed by quantitative real-time PCR assay to determine the level of C3 mRNA silencing.