SMAD1 expression is restored by DAC treatment. SMAD1 expression at the transcript (A) and protein (B) levels after DAC treatment (1 μM for 96 hours) of 4 DLBCL cell lines, as determined by qRT-PCR and western blotting. (A) Results from 4 (RIVA, SU-DHL-4, and RC-K8) and 2 (SU-DHL-6) independent experiments are pooled. SU-DHL-6 cells express SMAD1 prior to DAC treatment; all other cell lines are initially (pre-DAC) SMAD1⁻. (C) S1PR2 expression at the messenger RNA level after 1 μM DAC treatment (1 μM for 96 hours) of the cell lines in panel A. Pooled results from 4 (RIVA, SU-DHL-4 and RC-K8) and 2 (SU-DHL-6) independent experiments are shown. (D) SMAD1 expression of SMAD1^WT and SMAD1^KO SU-DHL-4 cells after exposure to DAC or vehicle control. The red asterisks (*) in panels B and D mark the correct band corresponding to SMAD1. Note that in SU-DHL-4 cells, and to a lesser extent in RIVA and RC-K8 cells (B), our SMAD1 antibody recognizes a second faster migrating band that is not SMAD1, because it is not lost in SMAD1^KO SU-DHL-4 cells, and that goes away upon DAC treatment. (E) Relative S1PR2 expression, as determined by qRT-PCR, of SMAD1^WT and SMAD1^KO SU-DHL-4 cells after DAC (1 μM for 96 hours) and TGF-β (2 ng/mL for 24 hours) treatment. Pooled results from 3 independent experiments are shown. Bar graphs show mean + SEM. *P < .05, **P < .01, Student t test.