Figure 2: Effect of midkine overexpression on wild type cell survival. A midkine overexpressing SK-N-SH cell line (SK-N-SH HMK) was created as previously described [6]. (a) Midkine expression in the medium of wild type (WT), doxorubicin resistant (DoxR), and HMK cells were analyzed using western blot. SK-N-SH WT, DoxR, and HMK were cultured in 25 cm flasks to 80% confluence. Medium was then exchanged for 2 mL of low fetal bovine serum culture medium and cultured for an additional 48 hours. Cultured medium was then collected and frozen at −80°C prior to being subjected to western blot. The volume of medium loaded on the gel was normalized based on cell count in the flask prior to medium collection. Cell membranes were isolated through a well-established protocol. We have previously demonstrated that midkine transfected cells (HMK) have acquired some doxorubicin resistance [6]. The MTT cell survival assay was performed to determine if SK-H-SH HMK cells have acquired resistance to other chemotherapeutic drugs including (b) etoposide and (c) cisplatin. (d) SK-N-SH WT, DoxR, and HMK cells were treated with or without doxorubicin at 10−7 M for 24 hours prior to collection of cell lysates. Proteins were extracted and probed using western blot for Pgp and β-actin.