NaSal treatment down-regulates the expression of Mcl-1 and suppresses the activation of the Mcl-1 promoter. (A) Effect of NaSal on Mcl-1 and Bcl-x_L expression. Monocyte-differentiated macrophages were treated with NaSal (20 mM) for 12 to 48 hours, then cell lysates were collected and Western blot analysis performed using the antibodies indicated. (B) Effect of NaSal on Mcl-1 mRNA expression. Macrophages were treated with NaSal for the times indicated, then total RNA was extracted and used for RT-PCR using primers specific to Mcl-1. No indicates normal macrophages without NaSal treatment. (C) Mcl-1 expression protects RAW264.7 cells from NaSal-induced death; 0.5 μg of GFP-expressing plasmid and a total of 3 μg plasmid were included in each arm of the experiment. Control or Mcl-1—expressing plasmid (2.5 μg) were transfected where indicated. After 24 hours, the RAW264.7 cells were treated with NaSal (20 mM) for another 24 hours. GFP-positive cells were detected by fluorescence-activated cell-sorter, as previously described.⁷  (D) Effect of NaSal on Mcl-1 promoter activation. 1 μg Mcl-1 promoter-reporter construct was transiently transfected into RAW264.7 cells, which were then treated with NaSal (20 mM), LY294002 (50 μM), and control (dimethyl sulfoxide [DMSO]) as indicated for 24 hours. Cells were harvested, and luciferase activity was detected, and the values corrected for protein concentration. * indicates P < .05 and ** indicates P < .01 as determined by Student t test for paired samples compared with control (No indicating no NaSal [medium alone] or DMSO), as indicated. The results in panels C and D are presented as the mean ± 1 SE of experiments performed in triplicate, which are representative of 3 independent experiments.