Fig. 4

rhEpo induces Yamanaka factor expression in breast cancer cells in vitro and in vivo. SUM159PT-ZsGreen-cODC cells were depleted of intrinsic stem cells by sorting for the ZsGreen-negative population. a These cells were plated and, the following day, were treated with 1 IU/ml rhEpo or vehicle followed 1 h later by irradiation with 0 or 8 Gy. Five days later, the cells were sorted for ZsGreen-negative and ZsGreen-positive cells. Expression of the Yamanaka factors were measured by qRT-PCR and normalized to GAPDH and the ZsGreen-negative population from the control sample. Bars represent the average fold increase in mRNA expression obtained from three independent repeats ± SEM. Statistical significance was determined by multiple t tests with a false discovery rate of 1%. * p < 0.05, ** p < 0.01. b BCIC-depleted SUM159PT-ZsGreen-cODC cells were implanted subcutaneously into the flanks of female 6–8-week-old NSG mice. Injection sites were monitored weekly for tumor formation. When the tumors reached ~ 3–5 mm, the mouse was treated intraperitonally with 500 IU/kg rhEpo or vehicle. Three hours after rhEpo treatment, the mice were treated with a 4-Gy dose to the tumor using a single beam. The mice were administered daily injections of rhEpo or vehicle (i.p.) for a total of 5 days. After the final treatment, the tumors were harvested, fixed, embedded, and sectioned. The sections were stained for Klf4 and Sox2, representative images shown. The % stained cells were quantified using a non-biased automated nuclei counting software. The graphs represent the % Klf4- or Sox2-stained cells normalized to the 0 Gy, vehicle-treated group from four individual animals per group ± SEM. Statistical significance was determined by Student’s t tests. * p < 0.05