Cho s sfm 2

EtDO-P4 (D-threo-1-(3′,4′-ethylenedioxy)-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol), a nanomolar inhibitor of GSL synthesis, was provided by Dr. James A. Shayman (Department of Internal Medicine, University of Michigan, Michigan, USA). For the EtDO-P4 treatment experiments, 5 × 10⁶ cells were cultured on 100 mm plates in MEM-α (Minimum Essential Medium Eagle-α; Gibco, Grand Island NY) supplemented with 10% dFBS (dialyzed fetal bovine serum; Gibco) for 24 hours^{31 (link)}. Cells were washed twice with serum-free medium (CHO-S-SFM II; Gibco) before culturing them for an additional 48 hours in serum-free medium containing 1 µM EtDO-P4.

5.0 × 10⁶ of EC2-1H9 cells or the engineered cells were seeded in T175 culture flask containing MEM-α supplemented with 10% (v/v) dFBS, 3.5 g/L glucose, 1% (v/v) Ab-Am solution, and 20 nM MTX. The culture medium was replaced with serum-free medium (CHO-S-SFM II; Gibco) in 3 days. After 2 days, the culture medium was collected, filtered using 0.45 μm filter (Sartorius, Göttingen, Germany), and dialyzed against phosphate buffer saline (PBS, pH 7.4) at 4 °C, overnight. To purify rhEPO, cultured medium was subjected to EPO Purification Gel (MAIIA Diagnostics, Uppsala, Sweden) according to the manufacturer’s instructions. Purified rhEPO was dialyzed against distilled water at 4 °C, overnight. The concentration was measured using a Quant-iT™ protein assay kit (Invitrogen) and stored at −80 °C until use.
The sialic acid level of the purified rhEPO was examined using the OPD-labeling method as previously described^{26 (link)}. Briefly, sialic acid moieties from the purified rhEPO were released using 0.5 M NaHSO₄ at 80 °C for 20 min. Released sialic acid was labeled with OPD (o-phenylenediamine-2HCl; Sigma) at 80 °C for 40 min. The level of OPD-labeled sialic acid was determined using C18 reversed-phase column (Shim-pack CLC-ODS; Shimadzu, Kyoto, Japan) with 474 scanning fluorescence detector (excitation at 230 nm and emission at 420 nm, Waters).

The TransIT-X2 Dynamic Delivery System (Mirus) was used for transient transfection of miRNA mimics into medium and low Qp CDCLs. Transient transfections were carried out on static cell culture. Cells in mid-exponential phase were seeded in T25 flasks at a concentration of 3 × 10⁶ cells/flask in 5 mL of CHO S SFM II (Gibco) media. Cells were allowed to grow for 24 h. Cells were fed prior to transfection in accordance with the manufacturer’s specifications. IDT miRNA mimic was allowed to complex with CHO S SFM II media and TransIT-X2 (Mirus) reagent for 20–25 min before being added in a dropwise fashion to the appropriate flask. After 20 h cells were passaged, resuspended in 15 mL Lilly propriety media and split into technical triplicates. Cells were grown in 4 mL of Lilly proprietary media in 50 ml spin tubes (Helena-BioSciences) in an ISF1-XC climo-shaker, at 37 °C, 80% humidity and 5% CO₂. VCD/mL was monitored using the ViaCount™ assay on a Guava® EasyCyte benchtop cytometer for 5 days. On day 5, cell pellets and cell free supernatant samples were taken for further analysis.