Targeting Apoptosis Regulators in TNBC

Retrovirus-encoded short-hairpin RNAs (shRNAs), shNOXA, shBIM, and shPUMA, were cloned into the pSuper puro vector (Oligoengine; Seattle, WA, USA). The target sequences were as follows: 5′-GGAAACGGAAGATGGAATA-3′ (shNOXA#1), 5′-GCTACTCAACTCAGGAGAT-3′ (shNOXA#2), 5′-CTACCTCCCTACAGACAGA-3′ (shBIM), 5′-GGGTCCTGTACAATCTCAT-3′ (shPUMA). Lentiviral shRNA-expressing constructs were cloned into the plko.1 vector (Addgene; Cambridge, MA, USA). The target sequences were as follows: 5′-CCAGCCAGAAAGCACTACAAT-3′ (sh-KLF4), 5′-GAACTGCACTTCAGCAATAAT-3′(sh-BNIP3), and 5′-CCTAAGGTTAAGTCGCCCTCG-3′ (sh-control). The constructs were transfected into 293T packaging cells along with the packaging plasmids (Addgene) and the lentivirus-containing supernatants were used to transduce TNBC cells. Retroviral or lentiviral infection was performed as previously described [40 (link)]. Infected cells were selected using 1 μg/mL puromycin (Sigma-Aldrich) for 3 days.
siRNA transfections were performed using Opti-MEM and Lipofectamine RNAi Max (Invitrogen, Waltham, MA, USA) with a final siRNA concentration of 10 μM siRNA. Cells were transfected at a concentration of 10 nM for 24 h. The siRNAs used in this study were silencer negative control (4390843) and si-ATF4 (s1704) (Silencer Select siRNAs from Ambion, Austin, TX, USA).