EPO suppresses p53-dependent apoptosis.

(A) TUNEL assay of DP16.1/p53ts cells grown at 37°C or at 32°C for 24 hours, with or without EPO (1 U/mL). Apoptotic cells were identified by their fluorescence (TdT-FITC) after the fragmented DNA was labeled with biotin-dUTP and avidin-FITC. The numbers shown in each panel represent the percentage of total cells undergoing apoptosis. (B) Pulse labeling with BrdU to detect DNA synthesis. DP16.1/p53ts cells were cultured at 37°C or at 32°C for 12 hours, or 24 hours with or without EPO and then labeled with BrdU for 30 minutes to detect cells undergoing DNA synthesis. Cells were fixed and incorporated BrdU was detected with FITC-conjugated BrdU antibody. BrdU-FITC staining indicates active DNA synthesis and PI staining reveals the DNA content and. hence, the cell cycle position of a cell. (C) DP16.1/p53ts cells were cultured at 32°C for the time periods indicated in the presence or absence of EPO. Cell extracts were prepared and subjected to SDS-PAGE and Western blotting for pRB protein. ppRB indicates hyperphosphorylated RB; pRB, hypophosphorylated RB. (D) Northern blot for Waf-1 mRNA expression. Total RNA was extracted from DP16.1/p53ts cells grown at 37°C or at 32°C for 16 hours with or without EPO. Then 10 μg RNA from each sample was separated on a 1% agarose gel containing formaldehyde and transferred to a nylon membrane. The RNA blot was probed sequentially for Waf-1 andGapdh.