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. 2012 Jul 2;7(7):e39448. doi: 10.1371/journal.pone.0039448

Figure 6. ATF3 is a negative regulator of cyclin D1 and cell proliferation.

Figure 6

(A) Enforced AFT3 expression reduces cell proliferation. LNCaP cells were transiently transfected with an ATF3 expression construct, a vector alone control, or with the AFT3 expression construct + ATF3 siRNAs. Proliferation rate was measured at the indicated times. ATF3 expression levels were verified by immunoblot (upper panels). Data are plotted as cell proliferation (fold) vs. condition (Vec, ATF3, ATF3+siATF3) ± SD (n = 3). (B–C) Knockdown of ATF3 increases cell proliferation. LNCaP cells were transiently transfected with siATF3 (or siCon) and the number of cells at day 0 (grey bars) & day 3 (black bars) were measured. Four independent ATF3 siRNAs (siATF3-1, -2, -3, and -4) were transiently transfected, and cell numbers were determined at day 3. Data are plotted as cell proliferation (fold) vs. condition ± SD (n = 3). (D) Effect of cholesterol depletion on ATF3 and cyclin D1 expression. LNCaP cell were treated in serum free media (SF; grey bars) or with CDM (black bars) for 16 h and the level of ATF3 and cyclin D1 were determined. Data were normalized to β-actin from the same blots. Immunoblot data are representative of the immunoblot result used in densitometry. Data are plotted as expression level (fold) vs. condition ± SD (n = 3). (E) ATF3 regulates cholesterol depletion-induced cyclin D1 expression (immunoblot analysis). LNCaP cells were transiently transfected with siATF3 (or siCon). After serum starvation for 16 h, cells were stimulated with 10% serum for the indicated times. Immunoblot analysis was performed to determine cyclin D1 expression in ATF3 deficient cells. (F) ATF3 regulates cyclin D1 expression (promoter reporter analysis). LNCaP cells were transfected with promoter construct of cyclin D1 containing a luciferase reporter and followed by additional incubation with ± serum for 6 h. Data are plotted as promoter activation (fold) vs. condition ± SD (n = 3). (G) Promoter activation of cyclin D1 upon cholesterol alteration requires ATF3 binding on promoter region (promoter reporter analysis). LNCaP cells were transfected with a luciferase construct of a wild type (WT) or an ATF3 binding site mutated cyclin D1 (MUT) promoter. Promoter activity was measured 6 h after treatment with various conditions (±FBS or CDM). Data are plotted as promoter activation (fold) vs. condition ± SD (n = 4). All experiments were performed a minimum of 3 times. *p<0.05, **p<0.01 (Student’s t-test).