## 3 Results and Discussion
### Improvement of EPO Production  
We then investigated the effect of the 30 Kc19\(\alpha\) protein on EPO production in CHO cells. EPO-CHO cells were treated with 30 Kc19\(\alpha\) under the following three conditions based on the date of medium replacement with serum-free DMEM/F12: pretreatment, cotreatment, and dual treatment. For pretreatment, 30 Kc19\(\alpha\) was added to the complete medium at day 2 after inoculation and cells were incubated for 24 h. The medium was then replaced with serum-free DMEM/F12. For cotreatment, the medium was replaced with a fresh complete medium on day 2 after inoculation followed by serum-free DMEM/F12 containing 30 Kc19\(\alpha\) on day 3. Cells were further incubated for 3 days (Figure 4a). As a result, although supplementation with 16 uM 30 Kc19\(\alpha\) resulted in a certain enhancement of EPO production in both cases, the extent of such an enhancement was not statistically significant (Figure 4b). Next, we combined to pretreatment and cotreatment protocol for dual treatment. In this condition, cells were treated with 30 Kc19\(\alpha\) once in complete medium for 24 h and then again in serum-free DMEM/F12 for 3 days. The results show that the cumulative EPO production in serum-free DMEM/F12 for 3 days was increased nearly three times in cells treated dually with 16 uM 30 Kc19\(\alpha\) compared to that in the nontreated control (Figure 4c). Figure 2: Intracellular delivery of recombinant 30 Kc19\(\alpha\) protein into EPO–CHO cells. EPO–CHO cells were treated with 16 uM 30 Kc19\(\alpha\) for 4 h in a complete medium. Intracellular delivered 30 Kc19\(\alpha\) was then visualized by immunofluorescence analysis using anti-T7 tag primary antibody and Alexa Fluor 488-conjugated secondary antibody (green). The cell-penetrating property of 30 Kc19\(\alpha\) was compared to that of full-length 30 Kc19. Cell nucleus was stained by DAPI (blue). Scale bar, 100 μm. Figure 4: Effects of 30 Kc19\(\alpha\) treatments on rhEPO production in CHO cells. (**a**) Schedule for the treatment with 30 Kc19\(\alpha\) of EPO–CHO cells. Conditioned media of cell cultures treated (**b**) once (pretreated and cotreated) and (**c**) twice (dually treated) with 30 Kc19\(\alpha\) were collected on day 3 of culture in serum-free medium. Titers of rhEPO were determined with an enzyme-linked immunosorbent assay (ELISA). Error bars indicate standard deviations (\(n=3\) per group). Statistical significance was determined using Student’s \(t\)-test. *, \(p<0.05\); n.s., not significant compared to the nontreated control. Figure 3: Effect of 30 Kc19\(\alpha\) on viability and viable cell growth of CHO cells. After 48 h of incubation in a complete medium, EPO–CHO cells were treated with the indicated concentration of 30 Kc19\(\alpha\) or 30 Kc19 for a further 24 h in fresh complete medium. The medium was replaced with serum-free DMEM/F12 containing each protein. (**a**) Cell viability and (**b**) viable cell concentration of EPO–CHO cells before and after the medium change. Error bars indicate standard deviations (\(n=3\) per group). Based on the previous study showing that EPO production in CHO cells can be enhanced by treatment with the recombinant 30 Kc19 protein [24], the effects of 30 Kc19\(\alpha\) and the 30 Kc19 protein on EPO production were compared. As a result of the dual treatment, while the EPO production was increased by 30 Kc19\(\alpha\) treatment in a dose-dependent manner, it showed no significant differences between cells treated with 30 Kc19\(\alpha\) at 16 \(\upmu\)M and cells treated with 30 Kc19 at the same concentration (16 \(\upmu\)M) (Figure 5). These results demonstrate that 30 Kc19\(\alpha\), which is a truncated form of 30 Kc19, has the same function as the full-length 30 Kc19. Both can be intracellularly delivered to improve EPO production in CHO cells. Nevertheless, 30 Kc19\(\alpha\) is considered advantageous as a medium supplement over the full-length 30 Kc19 due to its physicochemical characteristics.