Fig. 6

KLF9 knockdown confers resistance to doxorubicin-induced apoptosis and induces migration in BCa cells. Apoptotic response of A MCF7 and B MDA-MB-231 cells was evaluated using an assay based on the activity of caspase-3 and caspase-7 to cleave a peptide conjugated to a DNA-binding dye. Scrambled shRNA- and shKLF9-transduced cells were treated with CORT (100 nM), doxorubicin (DOX; MCF7: 1 μM, MDA-MB-231: 10 μM) or a combination of CORT and DOX for 24 h. CORT treatment did not influence DOX-induced apoptosis in (A) MCF7 (one-way ANOVA within shRNA type, P < 0.0001) and B MDA-MB-231 cells (one-way ANOVA; P < 0.0001). However, KFL9 knockdown reduced the apoptotic signal in DOX-treated MDA-MB-231 cells (one-way ANOVA; P < 0.0001). Cell migration was assessed through a wound healing assay where cells were treated with vehicle (100% ethanol) or CORT (500 nM) and representative images of the scratch at each timepoint are shown to the right of each plot. C In MCF7, CORT treatment did not influence migration while KLF9 knockdown slightly increased migration in vehicle-treated cells (two-way ANOVA; Treatment: P = 0.3935, Knockdown: P = 0.0767). D For MDA-MB-231, CORT treatment of scrambled shRNA-transduced cells promoted migration. KLF9 knockdown likewise enhanced cell migration in vehicle-treated cells (2-way ANOVA; Treatment: P = 0.0015, Knockdown). Bars represent mean ± SEM with statistically significant differences determined through two-way ANOVA for main effects of treatment or overexpression and one-way ANOVA (effects of hormone treatment within an shRNA type followed by Tukey’s post-hoc test: P < 0.05; means with the same letter are not significantly different) or Student’s t-test for individual effects of hormone treatment (^*) and knockdown (^#). All experiments were performed with N ≥ 4 replicates