Rhuepo

HepG2 cells were labeled with Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE/CFSE, final concentration: 1 μM, R&D Systems, Minneapolis, USA) for 7 min at 37 °C. Then, cells were washed and re-suspended in culture medium for additional 15 min to stabilize the CFSE staining. After the final wash step, cells were cultured in a 48-well microplate (5 × 10⁴ cells/well) overnight prior to rHuEPO (R&D Systems, Minneapolis, USA) or soluble-EPOR (R&D Systems, Minneapolis, USA) treatments. After being treated for the indicated times, cells were analyzed by flow cytometry.

Human CD34+ progenitor cells (AllCells Inc., Emeryville, CA) were isolated from bone marrow using CD34 immunomagnetic purification (Miltenyi Biotec, Auburn, CA), per the manufacturer’s protocol. Differentiation of EPCs was induced with rHuEpo (0.1 U/mL), IL-3, IL-6, and stem cell factor (SCF) (R&D Systems, Minneapolis, MN). The use of CD36+/CD34- expression as markers for erythroid lineage development has been previously described [19 (link), 30 (link)]. EpoR function was analyzed by exposing the culture at various time points to a range of rHuEpo concentrations from 0 U/mL (rHuEpo formulation buffer “vehicle” control: 100 mM NaCl, 20 mM NaCitrate, 0.25% human serum albumin [HAS], pH 6.9) to 300 U/mL for 5 or 30 minutes, which includes the physiological range of endogenous Epo (5–30 mU/mL) and exceeds the pharmacological levels observed in patients treated with ESAs (reported to be a mean of 1 U/mL in plasma) [31 ]. Thus, the upper range of the titration was at least 300-fold higher than the theoretical exposure of tumor cells in patients administered ESAs. EpoR cell-surface expression was determined by flow cytometry using the EpoR specific antibody MAb307 (R&D Systems) incorporating 7-amino-actinomycin-D (7-AAD; Invitrogen) staining to select live cells with intact plasma membranes.

Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT, Sigma-Aldrich). Cells were seeded in a 96-well microplate (4500 cells/well) and incubated at 37 °C overnight before rHuEPO (R&D Systems, Minneapolis, USA) or soluble-EPOR (R&D Systems, Minneapolis, USA) treatments. After culture finished, the cells were incubated with a medium containing 5 mg/mL MTT for 4 h at 37 °C. After precipitated formazan was dissolved in 150 ul DMSO, then the absorbance was detected on a BioTek Elx 800 ELISA reader (Winooski, VT, USA) at a wavelength of 570 nm.