Overexpression of a SUMO-mutant form of FOXK2 partially impairs FOXK2-mediated FOXO3 regulation. MCF-7 cells were transfected with the empty vector (pCMV5), wild-type FOXK2 and SUMO-mutant FOXK2 vector (K527/633 R) and collected for analysis of FOXO3 expression by western blot ( a ) and qRT-PCR ( b ). Following transfection with the empty vector (pCMV5) wild-type FOXK2 and SUMO-mutant FOXK2 vector (K527/633 R), MDA-MB-231 cells were treated with 10 nM paclitaxel for the indicated times and subjected to western blot analysis, where FOXO3 levels were determined by Western blot ( c ) and qRT-PCR ( d ). For 4a and 4c, the relative expression levels of FOXK2 and FOXO3 were determined based on the expression levels of the target gene product versus the reference, Tubulin, and the values shown under the respective western blot bands. The intensities of the unsaturated western blot bands were determined using the ImageJ software. Bars represent average ± s.d. of three independent experiments. Statistical significance was determined by Student's t -test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, significant; ns = not significant). e MCF-7 transfected with the empty vector (pCMV5), wild-type FOXK2 and SUMO-mutant FOXK2 vector (K527/633 R) were harvested for chromatin immunoprecipitation assays using the IgG as negative control and anti-FOXK2 antibody. After reversal of cross-linking, the coimmunoprecipitated DNA was amplified by qRT-PCR, using primers amplifying the FOXK2 binding-site containing region in the FOXO3 promoter