*2.5. The E*ff*ect of ABCC1 and ABCC4 Knockdown on Breast Cancer Proliferation and Migration*

Having established that the ABCC proteins could be knocked down, the effect of this on cell proliferation was investigated. In Figure 6a, it can be seen that one of the knockdowns of ABCC1 in MCF-7 cells caused a decrease in clonogenic capacity. The double knockdown of both ABCC1 and ABCC4 caused an even larger impact. For MDA-MB-231 cells, only one of the double knockdowns had a significant effect (Figure 6b). In contrast, when examining the bulk proliferation, it can be seen in Figure 6c,d that knockdown had no significant effect on the growth of either cell line.

≥ an ANOVA with a Dunnett's **Figure 6.** Combined knockdown of ABCC1 and ABCC4 affects the clonogenic capacity of breast cancer cells. (**a**,**b**) Clonogenic capacity of breast cancer cells following the siRNA-mediated knockdown of ABCC1 or ABCC4 was analyzed using a colony formation assay. (**c**,**d**) Proliferation of breast cancer cells following the siRNA-mediated knockdown of ABCC1 or ABCC4 was analyzed using an MTT assay. Data are mean ± sem, n ≥ 6. Data were analyzed by an ANOVA with a Dunnett's post hoc test. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 and \*\*\*\* *p* < 0.0001significantly lower than with the negative siRNA treatment.

Next, the effect of ABCC knockdown on cell migration was investigated. Figure 7a,b shows the average results from the scratch assays. With the MCF-7 cells (Figure 7a), a knockdown with ABCC4 siRNA #35 caused a significant decrease in migration, and with the MDA-MB-231 cells (Figure 7b), both ABCC4 knockdowns caused a significant decrease in migration. To examine this further, invasion rather than just migration was investigated (Figure 7c,d). With the MCF-7 cells, no significant effects were observed; however, with MDA-MB-231 cells, the knockdown with ABCC4 siRNA #35 and one of the double knockdowns did cause a significant decrease in invasion.

≥ ≥ Dunnett's post **Figure 7.** siRNA knockdown of ABCC4 affects the migration of breast cancer cells. (**a**,**b**) Migration of breast cancer cells following the siRNA-mediated knockdown of ABCC1 or ABCC4 was analyzed using a scratch assay. Data are mean ± sem, n ≥ 9. (**c**,**d**) Migration of breast cancer cells following the siRNA-mediated knockdown of ABCC1 or ABCC4 was also investigated using a cellular invasion assay. Data are mean ± sem, n ≥ 3. Data were analyzed by an ANOVA with a Dunnett's post hoc test. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 significantly lower than with the negative siRNA treatment.

These results with the ABCC knockdowns correlate well with the inhibitor studies, with ABCC1 and the double knockdown having an impact on cellular proliferation, whilst ABCC4 and the double knockdown affect migration.
