Psuper puro vector

A BDNF shRNA construct was designed using an online design program from MIT (http://sirna.wi.mit.edu/home.php). The 19-nucleotide hairpin-type shRNAs with a 9-nucleotide loop were cloned into the pSUPER-puro vector (OligoEngine, USA) according to manufacturers’ protocol. shRNA sequences are included in the Supplementary File. SVOG cells were transfected using Lipofectamine 2000 with the pSuper-puro shRNA constructs and selected for 4 days in media containing 2 μg/ml puromycin. The cells were collected and screened by western blot for target expression knockdown.

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).

Gene-specific 19nt shRNAs were designed based on a previously described algorithm using an in-house Perl script [49] (link). All shRNA sequences were BLASTed to ensure specificity. Synthesized oligomers were annealed and ligated into the pSuper.puro vector (Oligoengine). To make the Oct4/Nanog/Esrrb combinatorial shRNA constructs, ClaI-XhoI sites were used to insert H1-shRNA cassettes digested with BstBI-XhoI. The shRNA encoding sequences are: Oct4– GAAGGATGTGGTTCGAGTA (shRNA_#1) and GCGAACTAGCATTGAGAAC (shRNA_#2), Nanog– GAACTATTCTTGCTTACAA, Esrrb– GATTCGATGTACATTGAGA and Jarid2 – TCACTGTCCTCCCAAATAA.