Fig. 6.

CDK6 is a targeted gene of FOXO3a/BRD4. a Schematic depiction of the CDK6 promoter and CDK6 reporter luciferase constructs used. Two consensus FOXO3a-binding motifs are indicated. b Effects of WT and KR mutant of FOXO3a, BRD4 on the activity of CDK6 promoter luciferase in the presence or absence of MK2206. c Effects of WT-FOXO3a, BRD4 on the activation of WT or mutant CDK6 promoter luciferase in the presence of MK2206. For b and c, ^*/#/**p < 0.01. d FOXO3a, BRD4, H4ac4, and RNA-Pol II association at the CDK6 promoter as assessed by ChIP. ^*/⁑p < 0.01 when FOXO3a-ChIP or H4ac-ChIP in either MK2206-treated group or MK2206 plus JQ1-treated group is compared with control group. ^#/‡p < 0.01 when BRD4-ChIP or RNA-Pol II-ChIP in MK2206-treated group is compared with control group. e Cells treated with MK2206 were analyzed by sequential ChIP. The first-round of immunoprecipitation used anti-IgG, anti-FOXO3a or anti-BRD4 antibodies, followed by a second-round of immunoprecipitation using anti-IgG, anti-FOXO3a, or BRD4 antibodies. Pulldown DNA was analyzed by RT-PCR using specific CDK6 promoter primers and nonspecific primers. ^*/#/‡/⁑p < 0.01 when individual group with specific antibodies (FOXO3a and BRD4) used in the first-round of ChIP is compared with the corresponding group using IgG in the first-round of ChIP. f Effects of FOXO3a-knockdown, MK2206 treatment, or both on the association of FOXO3a, BRD4, RNA-Pol II, and H4ac at the CDK6 promoter as assessed by ChIP. ^*/#/⁑p < 0.01 when specific ChIP in MK2206-treated group is compared with the corresponding ChIP in control, or FOXO3a-knockdown or combination groups. g WT or KR mutant of FOXO3a was expressed in BT474 cells with endogenous FOXO3a knockdown. Rescue CDK6 expression was analyzed by western blotting; growth inhibition was assessed by cell count. ^*p < 0.01 when WT-FOXO3a group is compared with either vector or KR-FOXO3a group. h WT or HN mutant of BRD4 was expressed in BT474 cells with endogenous BRD4 knockdown. Rescue CDK6 expression and growth inhibition were assessed as in g. ^*p < 0.01 when WT-BRD4 group is compared with either vector or HN-BRD4 group. From b to h, data are representative of three experiments performed in triplicate as the mean ± SD