Aza treatment induced the expression of C/EBPδ. (A) Relative levels of mRNA expression of C/EBP family members' transcripts in Aza-cytidine–exposed and nonexposed U937 cells. Indicated are means and standard deviations. (B) Relative levels of C/EBPδ mRNA in Aza-treated or untreated U937 and NB4 cells by real-time RT-PCR. Expression levels were normalized to GAPDH levels. Results shown are mean ± SD of 3 independent experiments. (C) Western blot analysis of C/EBPδ expression in lysates from Aza-treated U937 cells. (D) Densitometry of the Western blot shown in panel C normalized to actin verified induction of C/EBPδ protein after Aza treatment of U937 cells. (E) Western blot showing expression of C/EBPδ in different leukemic cell lines. Total protein lysate was subjected to Western blotting and probed with anti-C/EBPδ and antiactin antibodies. (F) Bisulfite sequencing for C/EBPδ promoter methylation analysis in leukemic cell lines. Filled squares indicate methylated and open squares nonmethylated CpG dinucleotides. The transcriptional start site and the location of the analyzed CpGs (n = 32) are indicated. Individual clones were sequenced.