SMAD1 is silenced by DNA methylation of its promoter region in the majority of DLBCL cell lines. In silico analysis of ∼1000 cell lines of the various indicated origins that are publicly available through the CCLE database with respect to SMAD1 expression (A) and SMAD1 gene methylation (B). DLBCL cell lines are highlighted in red. (C) Correlation between SMAD1 expression and gene methylation in DLBCL cell lines in the CCLE database. (D) SMAD1 expression, as determined by western blotting of a panel of DLBCL cell lines. ABC DLBCL cell lines are in blue, and GCB DLBCL cell lines are in green. The classification of RC-K8 is controversial. Tubulin serves as loading control. (E) Methylation analysis by bisulfite sequencing of 5 regions of the SMAD1 promoter in SMAD1-expressing (SU-DHL-6) vs SMAD1⁻ (RC-K8, SU-DHL-4, RIVA) cell lines, as determined by bisulfite sequencing. Each circle represents 1 CG dinucleotide. Each line represents 1 clone. Two to 4 clones were sequenced per sample. ●, methylated cytosine; ○, unmethylated cytosine; ×, aligned mismatches between genomic and bisulfite sequences.