5-Azacytidine (5-AC) treatment of the myeloma line U266. The U266 cell line was totally methylated at SHP1, as shown by positive amplification only in M-MSP and not U-MSP at day 0 (D0) before treatment with 5-AC. On treatment with 5-AC, positive amplification appeared in U-MSP on day 2 (D2), indicating SHP1 demethylation. The U-MSP amplification became progressively stronger until day 5 (D5), indicating increasing demethylation of SHP1. The progressive demethylation of SHP1 was associated with increasing reexpression of SHP1, as shown by increasing amplification intensity of SHP1 mRNA by reverse transcription PCR (RT-PCR). Western blot analysis of STAT3 showed that before treatment (D0), phosphorylated STAT3 was constitutively expressed. With progressive demethylation and reexpression of SHP1, there was progressive down-regulation of phosphorylated STAT3, without significant changes in the amount of nonphosphorylated STAT3. By day 5 (D5), phosphorylated STAT3 was almost undetectable. Comparable protein loading was shown by β-actin. M indicates molecular weight marker; B, blank; D0 to D5, days 1 to 5 after 5-AC treatment; NC, normal control; PC, positive control with methylated DNA.