Immortalized HMEC and MCF-10A cells; human breast cancer cells MDA-MB-231, MDA-MB-468, Hs578t, and BT549; and human embryonic kidney (HEK) 293T cells were obtained from the American Type Culture Collection and cultured under standard conditions. The standard Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) protocol was followed for all transient plasmid transfections.

Lentiviruses were generated using the pLKO.1 vector and packaging plasmids (psPAX2 and pMD2.G) in the HEK293T cell line. Annealing oligonucleotides targeting TROJAN were synthesized (Sangon Biotech) and cloned into the pLKO.1-Puro vector. Supernatants were collected and filtered through a 0.45-μm syringe filter. Target cells were infected with lentivirus in the presence of polybrene (6 mg/ml; Sigma-Aldrich) at a multiplicity of infection of 0.7. Stably transduced cells were generated by puromycin selection for 3 days beginning 24 hours after infection. The knockdown efficiency and specificity of all the shRNAs were validated with qPCR or immunoblotting. The two shRNAs producing the best knockdown efficiency were used in subsequent studies (shTROJAN-1, 5′-GCAGTCTCTTAAGCAGATTGA-3′; shTROJAN-2, 5′-GCAACTGCTGTTAATGAAAGT-3′; shZMYND8, 5′-CGGAGTAATAAATCCAGTT-3′).

The siRNA transfections were conducted with the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The target sequence for siZNF592 was 5′-GCCAGATCCTGATGATCCA-3′.

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