Methylation status. PCR on NotI-digested and undigested DNA from (A) MM JAG2-positive and -negative (MUTZ5) cell lines with primers located on either side of the NotI site. The JAG2-positive line shows the absence of a band when digested (hypomethylated promoter region), whereas the negative line shows no difference (hypermethylated promoter region). (B) A similar PCR pattern was obtained on NotI-digested and undigested DNA from 2 MM JAG2-positive samples (4438, 2477) and (JAG2-negative) tonsil plasma cells using the same primers. The JAG2-positive samples show a reduction of band when digested (hypomethylated promoter region), whereas the JAG2-negative cells show no difference (hypermethylated promoter region). The profile with MM patient DNA presents a reduction of band intensity, certainly due to the nonpurity of the plasma cells after purification. ND indicates not digested; D, digested. (C) JAG2 expression profile in the MUTZ5 cell line before (left panel) or after incubation with 5-azacytidine (right panel). Blocking of methylation with 5-AZA induced JAG2 expression in the negative cell line, comparable to the levels observed in MM. (D) Representation of the JAG2 expression levels differential between controls, MGUS/smoldering MM, and MM samples per cell lines. (E) RT-PCR with HS-1 and GAPDH-specific primers on RNA extracted from MRC5 cells after coculture with 0.5 million and 1 million MM cells. The induction of HES-1 expression is proportional to the activation signal per number of MM cells in the coculture (right panel). (F) IL-6 detection assay by ELISA for the assessment of IL-6 secretion with MRC5 cells alone, MRC5 cells cocultured with K620 cells (MM) in an insert (no contact between the two cell types), and MRC5 cells cocultured with K620 cells. (G) VEGF secretion measured by ELISA in the MM cells alone (RPMI 8226 and U266) or when cocultured with the MRC5 cells. Error bars (panels D and F) represent standard deviation.