Molecular analysis of the U266 MM cell line. (A) Northern blot analysis of human MM cell lines showing cyclin D1 overexpression in U266. The length of the cyclin D1 transcripts is shown in kb. β-actin hybridization is shown for loading quantification. (B) Southern blot analysis of the IGH locus in the U266 cell line. The DNA was digested with BamHI restriction enzyme, and the nylon filter was subsequently hybridized with the probes specified below. The germ-line bands are indicated by dashes. The arrow indicates the cloned “illegitimate” IGHrearranged fragment. (C) Molecular cloning of the chromosomal breakpoint in the U266 cell line. The breakpoint and the respective 11q13 germ-line region are shown; the probes used for Southern and Northern blot analyses are shown in the diagram. The vertical arrow indicates the breakpoint position. Chromosome 14 is represented by open boxes with black or stippled boxes showing different IGHregions and chromosome 11 is represented as a solid line. Restriction enzyme symbols: B, BamHI; R, EcoRI; H,HindIII; X, XhoI. (D) The nucleotide sequence of the breakpoint region and its alignment with 14q32 and 11q13 germ-line sequences are shown.