Treatment of CB cells with 5azaD/TSA modifies the expression of transcription levels of genes and their products implicated in HSC self-renewal. (A) Effects of 5azaD/TSA treatment on the relative transcript levels of genes (HOXB4, BMI 1, GATA 2, NOTCH 1, P21, P27, C-MYC, GATA 1, and MPO) were measured by real-time quantitative PCR. Total RNA was extracted from CB CD34⁺ cells (day 0) or cells obtained after 5 and 9 days of culture in the presence of cytokines with or without 5azaD/TSA treatment. Relative mRNA levels in cultured cells (day 5 and day 9) to primary CB cells (day 0) was determined by real-time quantitative PCR. GAPDH was used as internal calibrator (control gene), the standard curve method was used for relative mRNA quantitation. Measurements were obtained in triplicate and a negative control (lacking the cDNA template) was included for each assay. (B) Detection of HOXB4, Bmi-1, and P21 proteins in the cells cultured with or without 5azaD/TSA treatment in the presence of cytokines after 9 days using Western blotting analyses as described in “Materials and methods.” Equal loading of protein was verified with anti–β-actin antibody on the same membrane.