Upregulation of SHP1 by 5-AZA correlates with downregulation of the JAK3/STAT3 signaling in ALK+ ALCL cells. (a) Western blot analysis showed that 5-AZA induced time-dependent increase in SHP1 in Karpas 299 cells. In the same experiment, decreases in JAK3, pJAK3 and STAT3 were identified, although the decrease in pSTAT3 was relatively delayed. (b) Western blot analysis revealed that SU-DHL-1 cells treated with 5-AZA in 5 days showed restoration of SHP1 and downregulation of pJAK3, JAK3 and pSTAT3, similar to the changes seen for Karpas 299 cells. (c) Densitometric measurement of the band intensity for pJAK3, JAK3 and pSTAT3 shown in (a). For each day, the band intensity of SHP1, JAK3, pSTAT3 and pJAK3 in cells treated with 10 μ M of 5-AZA was normalized to that from cells treated with solvent only (negative controls). pJAK3 was also normalized to the level of JAK3 for each drug dosage and each day. Compared to the untreated samples, pJAK3 decreased more than JAK3, and substantial decrease (i.e. >50% reduction) of pSTAT3 was identified later than that of JAK3 and pJAK3. (d) Western blot analysis showed that 5-AZA induced decreases of some but not all of the STAT3 downstream targets in Karpas 299 cells. Mcl-1, Bcl-2 and SOCS-3 were downregulated, whereas cyclin D3 and survivin showed no detectable changes. A similar pattern of changes was identified in SU-DHL-1 cells, except that downregulation in cyclin D3 was also detected in this cell line.