Hypermethylation of an alternative promoter in breast tumors downregulates SEPT9_v3. (A) Quantification of DNA methylation at CpG sites upstream of the SEPT9_v3 transcription start site in cell lines targeted by MassARRAY. The unsupervised clustering distributes cell lines into two groups: hypomethylated and hypermethylated (left panel). Differences in the overall methylation percentages between these two groups are shown (right panel). White boxes represent CpG sites that were not analyzable. (B) Quantification of SEPT9_v1 and SEPT9_v3 isoform mRNA levels in human cell lines. (C) Expression of SEPT9 (top panel), SEPT9_v3 isoform (middle panel) and α-tubulin (bottom panel) detected by Western blot in the panel of breast cancer cell lines. Note that with the labeling of recombinant SEPT9_v1 and _v3 by an antibody recognizing both of these isoforms , the anti-SEPT9_v3 antibody recognizes the bottom band of the high-molecular-weight isoform doublet.