*2.3. RT-PCR Analysis*

To measure the gene expression levels at the RNA level of BCRP, reverse-transcription polymerase chain reaction (RT-PCR) was performed. Total RNA was isolated from Hela, Caco-2, MCF-7, and SW620 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA); complementary DNA (cDNA) was synthesized from 2 µg of the RNA extracted from cells, using the PrimeScript RT reagent Kit (TaKaRa, Shiga, Japan). cDNA was then amplified by PCR using human-specific primers: BCRP, 5′ -TTC TCC ATT CAT CAG CCT CG-3′ (forward) and 5′ -TGGTTGGTCGTCAGGAAGA-3′ (reverse); GAPDH 5 ′ -GAA GGT GAA GGT CGG AGT C-3′ and 5′ -GAAGATGGTGATGGGATTTC-3′ (reverse). Reverse transcription PCR (RT-PCR) was performed in a T-100TM thermal cycler (Bio-Rad, Hercules, CA, USA) using AccuPower PCR Premix (Bioneer, Daejeon, Korea), according to the manufacturer's protocol. The thermocycler conditions used for amplification were 95 ◦C for 5 min (hot start), 94 ◦C for 45 s, 55 ◦C for 30 s, and 72 ◦C for 30 s in 30 (BCRP) or 26 (GAPDH) cycles. Subsequently, the resultant products were analyzed by separation on a 1.5% agarose gel in tris-acetate/ethylenediaminetetraacetic acid (EDTA) buffer.

#### *2.4. FACS-Cellular Accumulation Study*

The cellular accumulation of quercetin was measured by FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA, USA). For FACScan analysis, 2 × 10<sup>5</sup> HeLa cells/well were seeded into 6-well cell culture plates on the day before the experiment. On the following day, cells were treated with vehicle or quercetin and 1 µM MX. A time course experiment was conducted on HeLa cells following treatment with quercetin (1 and 100 µM) for 2, 4, and 6 h. After treatment, the cells were harvested by trypsinization and transferred to a fluorescence-activated cell sorting (FACS) tube, pelleted by centrifugation (1500 rpm, 5 min), and then resuspended in 200 µL of PBS. Flow cytometry analysis was performed using red fluorescence. A minimum of 10,000 cells were acquired per sample.

#### *2.5. Cytotoxicity Assay*

To determine the cytotoxic efficacy (i.e., the anticancer activity) of mitoxantrone associated with its intracellular accumulation, we performed the Cell Counting Kit-8 assay (CCK-8 assay kit; Dojindo Molecular Technologies, Kumamoto, Japan) following the manufacturer's instructions. HeLa cells (at a density of 1 × 10<sup>4</sup> cells per well) were seeded and cultured overnight in 96-well plates. Then, the medium was replaced with fresh medium containing the test drugs (mitoxantrone alone, mitoxantrone with 1 µM or 100 µM quercetin); the antiproliferation potential was examined at different drug concentrations after 24 h of incubation [28]. Additionally, 1 µM Ko143 was used as a positive control for BCRP inhibition. The absorbance was measured at a wavelength of 450 nm using a microplate reader (BioTeK, Highland Park, WI, USA).
