Puromycin

Human ER-positive breast cancer cell lines MCF7 and T47D (ATCC), and HEK293T (ATCC) cells were cultured in DMEM (Hyclone) with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Hyclone) at 37 °C in a humidified 5% CO₂ atmosphere. Tamoxifen-resistant (TamR) MCF7 and T47D cells were generated as described previously [63 (link)]. In brief, cells were cultured in the presence of increasing concentrations of 4-hydroxytamoxifen (Sigma-Aldrich) starting at 0.5 μM, and finally gradually increased up to 5 μM 4-hydroxytamoxifen when the growth of the cells could not be inhibited in this concentration. In parallel, parental MCF7 and T47D cells were cultured under identical conditions without tamoxifen. SP600125, etomoxir and puromycin was obtained from MedChem Express.

NOZ, GBC-SD, and human embryonic kidney 293 T (HEK293T) cells were purchased from the Health Science Research Resources Bank (Osaka, Japan), the Cell Bank of Type Culture Collection of Chinese Academy of Science (China), and the American Type Culture Collection (American), respectively. NOZ cells were cultured in William’s E medium (Hyclone), GBC-SD and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone). All cell lines were supplemented with 10% fetal bovine serum (Gibco) and penicillin-streptomycin (Hyclone), incubated in a humidified chamber with 5% CO₂ at 37 °C, and ensured to be mycoplasma-negative cultures by monthly mycoplasma testes. Gemcitabine (GEMZAR) was purchased from Eli Lilly (American), demcitabine and puromycin were purchased from MedChem Express (American).

Lentiviral vectors expressing human short hairpin RNA (shRNA) targeting PSMD9 (shPSMD9, GenePharma Inc, Shanghai, China) or scrambled control (shNC) were used to generate stable cell clones expressing shPSMD9 or a nonspecific shRNA as the control. The sequence of the shRNA used was as follows: GCAAGUGGAUGAU GAGAUUTT. Lentiviral vectors expressing human mRNA targeting PSMD9 (GenePharma Inc., Shanghai, China) or scrambled control (negative control) were used to generate stable cell clones overexpressing PSMD9 or a nonspecific RNA as the control. Clones infected with lentivirus were selected using 1 mg/mL puromycin (#HY‐K1057, MedChemExpress; USA).

Before the 24-h transfection, we plated 4 × 10⁶ HEK293T cells in 10-cm dishes. The cells were cotransfected with pLVX-miR-193a, pLVX-NC (negative control), MSCV-WT1, and MSCV-NC with packaging and envelope vectors. After 48 h, the viruses were harvested from the supernatant, and filtered through 0.45-μm low–protein binding polysulfonic filters (Merck-Millipore, Billerica, MA, USA). We inoculated 2 × 10⁵ MDA-MB-231 or BT549 cells in 6-well plates. Cultures that were approximately 30–60% confluent were transfected with lentivirus pLVX-miR-193a or pLVX-NC, followed by 8 μg/mL polybrene (Sigma-Aldrich) to increase infection efficiency. Subsequently, the cells were transfected with MSCV-WT1 or MSCV-NC. Positive clones were selected via 1-week puromycin (2 μg/mL, MedchemExpress, Princeton, NJ, USA) selection.

HEK293T cells (4 × 10⁶) were plated in 10 cm dish. After 24 h, LVX-puro-miR-193a and MSCV-puro-WT1 vectors were cotransfected with packaging and envelope vectors into HEK293T cells. Virus was harvested from the supernatant at 48 h posttransfection, and was mixed with 8 μg/ml polybrene (Sigma-Aldrich, St. Louis, USA) to increase the infection efficiency. To select the cells stably expressing miR-193a, 2 μg/ml puromycin (Medchemexpress, Princeton, USA) was added into the supernatant for 1 week.

Cell lines (LOVO, HUVEC, CT26 and SW48) were obtained from the American Type Culture Collection. The SW48, LOVO, and HUVEC were incubated in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose content (Invitrogen, CA, USA) containing 10% fetal bovine serum (FBS; gibco, USA) and 1% penicillin/streptomycin, CT26 was maintained in RPMI1640 (Invitrogen, CA, USA) supplemented with 10% FBS and 1% penicillin/streptomycin. All cells were cultured in humidified atmosphere of 5% CO2 in air at 37 °C. SU11274 (Medchem express, USA), a specific chemical inhibitor of Met; Anti-HGF antibody (Sinobiological, China), a neutralizing antibody for HGF; recombinant human HGF (Peprotech, USA). GFP lentiviral particles were purchased from Genechem Co (china, shanghai). The transfection of lentiviral was conducted following instruction of manufacturer. For lentiviral transduction, 5000 cells/well were seeded on 96 well tissue culture plates and infected the following day with lentiviral particles (Santa-Cruz Biotechnology, Dallas, TX) at a MOI of 10 in the presence of 10 mg/ml polybrene, purchased from). GFP-expressing cells were selected with 2 μg/ml puromycin (Medchem express, USA) and enriched by three cycles of fluorescence-activated cell sorting (FACS).

HEK-293T cells were co-transfected with the lentiviral constructs, packaging plasmid (psPAX2) and envelope plasmid (pMD2.G) at a ratio of 4:3:1 using jetPRIME® (Polyplus, Illkirch, France). The viruses were collected, concentrated, and added to the cells using polybrene (Cat. HY-112,735; MedChemExpress, Monmouth Junction, NJ, USA) for 24 h. The infected cells were further selected with 2 µg/mL puromycin (Cat. S7417; Selleck) or 400 µg/mL hygromycin B (Cat. ST1389; Beyotime, Shanghai, China), to obtain stably transfected cells.