*2.4. Knockdown of ABCC1 and ABCC4 Using siRNA*

Knockdown of ABCC1 and ABCC4 was carried out using siRNA. Two different siRNA sequences for each protein were tested, alongside a negative siRNA. The effectiveness of the knockdown was measured by both RT-qPCR to monitor the mRNA levels and Western blotting to monitor the protein levels. As can be seen in Figure 5, the knockdown of either ABCC1 or ABCC4 was effective in both cell lines. In addition, since the two proteins have overlapping substrate specificity, the dual knockdown of both proteins was also undertaken (Figure 5e).

**Figure 5.** ABCC1 and ABCC4 can be successfully knocked down in breast cancer cells using siRNA. Gene knockdown in breast cancer cells was performed using the INTERFERin-siRNA transfection protocol, with two different ABCC1 siRNAs (#30 or #31), two different ABCC4 siRNAs (#34 or #35),

or in combination (#30/#34 or #31/#35). A negative control siRNA was also used. The effectiveness of the knockdown was measured by both RT-qPCR and Western blotting. (**a**) Knockdown of ABCC1 in MCF-7 cells, (**b**) knockdown of ABCC4 in MCF-7 cells, (**c**) knockdown of ABCC1 in MDA-MB-231 cells, (**d**) knockdown of ABCC4 in MDA-MB231 cells and (**e**) double knockdown of both transporters in each cell line. Uncropped images can be found in Figure S4. RT-qPCR data are mean ± sem, n = 3. Data were analyzed using a one-way ANOVA with a Dunnett's post hoc test. \* *p* < 0.05 and \*\* *p* < 0.01 significantly lower than the negative siRNA sample.
