STAT3 regulated the transcription of Mcl-1 promoter and is essential to cell survival of macrophages. (A-B) STAT3 regulates Mcl-1 promoter activation. Mcl-1 (0.5 μg) promoter-report construct plus 2.5 μg of STAT3 dominant-negative (DN) (A) or activated STAT3 (B) expression plasmids or control plasmid were transiently transfected into RAW264.7 cells. After 24 hours, cells were harvested for luciferase assay (A) or treated by NaSal (20 mM) for an additional 24 hours before luciferase assay (B). Luciferase activity was corrected for protein concentration as described above. (C-D) STAT3 controls survival in the RAW 264.7 macrophage cell line. Cell death assays were performed employing RAW 264.7 cells transfected with a GFP-expressing plasmid (0.5 μg) plus 2.5 μg STAT3 DN (C), or activated STAT3-expressing plasmids (D), or control plasmid. Cells were harvested 24 hours after transfection for detection of GFP-positive cells by FACS (C) as described7 or cells were treated with NaSal (20 mM) for another 24 hours before the detection of GFP-positive cells (D). (E) STAT3 regulates Mcl-1 expression and macrophage survival. Differentiated primary human macrophages were transfected with 12 μg/mL lipofectamine plus 400 nM AS STAT3 ODN or the control ODN (AS, 5 bp) in OptiMem medium as described in “Materials and methods.” Cells were harvested after 48 hours and apoptosis was determined by detection of subdiploid DNA (DNA fragmentation, % < 2N DNA). Cell lysates also were employed for Western blot analysis to detect the expression of STAT3 and Mcl-1 using monospecific antibodies. * indicates P < .05, ** indicates P < .01 determined by Student t test for paired samples compared with control. The results in each panel are presented as the mean ± 1 SE of experiments performed in triplicate, which are representative of 3 independent experiments.
