Ctso plasmid

Dulbecco's minimum essential medium (DMEM), glutamine and penicillin/streptomycin/glutamine stock mix were purchased from Life Technologies, Inc. (Carlsbad, CA, USA). Fetal bovine serum (FBS) and charcoal-stripped FBS were from Invitrogen (Carlsbad, CA, USA). L-trans-Epoxysuccinyl-leucylamido (4-guanidino) butane (E-64) was from Sigma-Aldrich (St. Louis, MO USA). CTSO, MTDH, PABPC4L, LMNA, EEFiA1and control small interfering RNAs (siRNA) were purchased from Dharmacon (Thermo Scientific Dharmacon, Inc.). CTSO plasmid was purchased from OriGene (Rockville, MD, USA). Affinity purified rabbit and mouse antibodies against human BRCA1 and CTSO were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). ZNF423 antibody was purchased from Abcam (Cambridge, MA, USA). Actin, MTDH, PABPC4L, LMNA, and EEFiA1 antibodies were from cell signaling (Danvers, MA, USA). For standard PCR, HotStart Taq Plus DNA Polymerase was used (Qiagen, Germantown, MD, USA). Reagents and primers for real time PCR were purchased from Qiagen (Valencia, CA, USA). The protease inhibitor cocktail kit was obtained from Pierce Biotechnology (Rockford, IL, USA). 17β-estradiol (E2) and 4-hydroxytamoxifen (OH-TAM) were purchased from Sigma Aldrich (Saint Louis, MO USA). Olaparib was from Selleckchem (Houston, TX, USA).

Cells were plated at 70% confluence in culture medium supplemented with 10% FBS, and were transfected with empty vector or CTSO plasmid (OriGene) using lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the vendor's protocol. Cells were collected for protein analysis 48 hours after transfection. In some experiments, 24 hours after transfection, cells were treated with 10 μM E-64, a cysteine proteases inhibitor, for additional 24 hours. Cells were then collected for protein analysis.
Specific siGENOME siRNA SMARTpool reagents against a given gene as well as a negative control, siGENOME Non-Targeting siRNA, were purchased from Dharmacon Inc. (Lafayette, CO, USA). Cells were transfected with control siRNA, and specific siRNAs (10nM) in 96-well plates or 12-well plates using lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the vendor's protocol. For the purpose of cell growth assay, cells were plated in base medium supplemented with 5% charcoal stripped FBS for 24 hours, and then cultured in FBS-free RPMI 1640 media for another 24 hours before transfection. Different treatments were started 24 hours after transfection. For the purpose of testing gene expression level, cells were transfected with control siRNA and specific siRNAs (10nM) in 12-well plates using lipofectamine RNAiMAX for 48 hours.