Geneticin g418 sulfate

The Eker rat-derived uterine leiomyoma (ELT-3) cell lines were kindly provided by Lin-Hung Wei (Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan). ELT-3 cells transfected with luciferase reporter genes (ELT-3-LUC) were previously established in our laboratory. In addition, the primary cultures of human leiomyoma cells were isolated from uterine leiomyoma tumor tissue specimens, which were collected from women (30–40 years of age, n = 6) undergoing myomectomy at the Department of Oncology, National Taiwan University Hospital (Taipei, Taiwan). According to a previous study all the human tissue specimens were approved by the Institutional Review Board and Ethics Committee of the National Taiwan University Hospital (permit number: 201210072RIC). The process of purification of the leiomyoma cells was as described previously [29 (link)], and leiomyoma cells from passages 2–7 were used in this study. Both ELT-3-LUC and leiomyoma cells were cultured in DMEM/F12 containing 10% FBS, 1% antibiotics [10,000 units·mL⁻¹ penicillin, 10,000 μg·mL⁻¹ streptomycin, and 25 μg·mL⁻¹ amphotericin with 8.5 g·L⁻¹ NaCl], and 0.6 mg·mL⁻¹ Geneticin^® G418 Sulfate (Thermo Fisher Scientific, Waltham, MA, USA; ELT-3-LUC only); both cell lines were incubated at 37 ℃ with 5% CO₂.

Two isoforms of PIP5K1C (PIP5K1C-1 [NM_001195733.2] and PIP5K1C-2 [NM_012398.3]) were detected in HFF1 cells using isoform-specific primer pairs from the cDNA made from HFF1 RNA. Isoform-3 (NM_001300849.2) was not detected. PCR-amplified PIP5K1C-1 and PIP5K1C-2 DNA were then cloned into the pcDNA™3.4 TOPO™ TA vector (ThermoFisher, A14697) following the manufacturers’ protocol. Melanoma A375 cells were transfected, and clones with the correct orientation of amplified PCR were selected that expressed the PIP5K1C isoform protein.
To make overexpressing PIP5K1C cell lines, A375 cells were plated in 24-well plates (10⁵ cells/well) and then transfected independently with PIP5K1C-1, PIP5K1C-2, or pCMV-GFP expression vectors. At 24 h post-transfection, cells were selected for resistance in 1000 µg/mL Geneticin/G418 Sulfate (Thermo Fisher, 10,131,035), which killed all the untransfected cells. After 14 days of G418 selection, cells expressing the PIP5K1C isoform were obtained based on the green fluorescent protein signal from cells in pCMV-GFP transfected wells. These cells were then tested for their sensitivity to increasing concentrations of WX8.

Lentiviral pseudoparticles were produced by transfecting pcz-VSV-G, pCMV-dR8.74 and pWPI with the respective gene of interest into HEK 293T cells with Lipofectamine 2000 (Thermo Fisher, 11668019). Supernatants were harvested after 24 and 48 h, pooled and filtered through 0.45 µM filters (Sarstedt, Nümbrecht, Germany; Filtropur 0.45, 83.1826) und used to infect target cells. Target cells were selected and maintained in DMEM complete, containing Geneticin (G418-Sulfate, ThermoFisher Scientific, 11811031, 750 µg/mL final concentration).

SARS-CoV-2 Pseudovirus (COV-PS02), expressing S-glycoprotein on the surface of the Lentivirus, and HEK293/ACE2 cells, genetically engineered to overexpress angiotensin-converting enzyme 2, were purchased from Creative Diagnostics (Shirley, NY, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and geneticin (G-418 sulfate) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Cell Titer-Glo Luminescent cell viability assay kit was purchased from Promega (Madison, WI, USA). HEK293/ACE2 was maintained at 37 °C, 5% CO₂ in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 0.5 mg/mL of G418. The cells were sub-cultured within 48 h intervals.

Briefly, the pTEJ-8 expression vectors encoding wild-type CXCR7, kindly provided by Thue W. Schwartz (University of Copenhagen, Denmark), were cotransfected with the pPUR selection vector encoding puromycin resistance (Clontech Laboratories, Palo Alto, CA, USA) into U87.CD4 or U87-MG cells by the use of FuGENE HD transfection reagent (Promega, USA). After puromycin (2 μg ml⁻¹) selection, CXCR7-expressing cells were isolated from the puromycin-resistant cell cultures by incubation of the cells with mouse anti-human CXCR7 mAb clone 8F11-M16 (BioLegend, San Diego, CA, USA) and subsequent magnetic separation of chemokine receptor-positive cells with sheep anti-mouse immunoglobulin G (IgG)-conjugated M450 Dynabeads (ThermoFisher Scientific, Waltham, MA, USA). The transfected cells were cultured under selection in Dulbecco's modified Eagle's medium (DMEM; ThermoFisher Scientific) containing 10% fetal bovine serum (FBS; ThermoFisher Scientific), 0.01 M HEPES buffer (ThermoFisher Scientific), 0.2 mg ml⁻¹ Geneticin (G-418 sulfate; ThermoFisher Scientific), and 1 μg ml⁻¹ puromycin (Sigma-Aldrich, St. Louis, MO, USA).