Transdux max

The miRZip anti-miR-126-3p-GFP co-expressing plasmid (CS940MZ-1, a custom order from System Biosciences, with EEF1A1 promoter for anti-miR-126-3p) and control plasmid (MZIP000-PA-1, miRZip negative control) were purchased from System Biosciences. Virus were produced and transduced as previously described^{30 (link)}. Briefly, primary AML or enriched CD34+ cells were cultured in Gibco™ IMDM (Thermo Fisher Scientific, 12440061) supplemented with 20% FBS and growth factors including interleukin 3 (IL-3, 25 ng/mL), interleukin 6 (IL-6, 10 ng/mL), Fms-like tyrosine kinase 3 ligand (Flt-3 ligand, 100 ng/mL), stem cell factor (SCF, 50 ng/mL), and thrombopoietin (100 ng/mL) overnight before transduction. Transduction was performed with a multiplicity of infection (MOI) of 25 by spinoculation in the presence of 1× TransDux MAX™ (System Biosciences, LV860A-1). 32D cells were cultured in RPMI-1640 supplemented with 10% FBS, 20% WEHI-3 conditioned media, and antibiotics at 37 °C with 5% CO₂ and were transduced with MSCV-IRES-GFP (MIG)-based retroviruses or lentiviruses (CD530, pLKO.1 or HIV-7) (MOI = 10) by spinoculation in the presence of 8 ug/mL polybrene (American Bioanalytical, AB01643-00001). Two days after infection, GFP⁺ or RFP⁺ cells was sorted on a 5-Laser, BD FACSAria Fusion Cell Sorter. The target sequences for shRNAs are shown in Supplementary Table 7.

UCHL1 knockdown was performed by transducing human leiomyoma and myometrial cells with control or UCHL1 short-hairpin RNA (shRNA) lentiviral virus particles (sc-108080 and sc-42304-V, Santa Cruz Biotechnology, Dallas, TX, USA) using TransDux MAX (LV860A-1, System Biosciences, Palo Alto, CA, USA), according to the manufacturer’s protocol. Briefly, leiomyoma and myometrial cells (50,000 cells per well) were seeded in a 24-well plate in DMEM/F12 with 10% FBS the day before transduction until 50–70% confluent. After aspirating the medium from the cells, the transduction solution and lentiviral virus particles were added to each well and incubated for 72 h at 37 °C in 5% CO₂. Leiomyoma and myometrial cells with control shRNA or UCHL1 shRNA were subjected to RNA extraction, followed by quantitative real-time PCR analysis.

For the noninvasive assay of ectopic lesion growth in mice with endometriosis, the firefly luciferase gene was cloned into the pSMPUW-Hygro construct (Cell Biolabs, catalog number: VPK-214). Lentivirus carrying luciferase gene were generated 293 T cells by transfection with pSMPUW-Hygro containing luciferase gene and the Lenti-X high-titer lentiviral packaging system (ClonTech, catalog number: 631278). The recombinant lentivirus titer was measured using Lenti-X™ GoStix™ Plus (ClonTech, catalog number: 631280). IHEECs and IHESCs were transduced with lentiviral vectors carrying the luciferase expression cassette with TransDux MAX™ (System Bioscience, catalog number: LV860A-1). Luciferase-labeled IHEECs and IHESCs were then selected in the presence of 300 µg/ml hygromycin. The luciferase gene expression in these recombinant cells was validated using a luciferase activity assay kit (Promega, catalog number: E1980). All these recombinant cells were maintained with DMEM/F12 supplemented with 10% FBS and penicillin/streptomycin under drug selection.