*3.4. Culture of Cell Lines*

Human stably P-gp-expressing cells (ABCB1/Flp-InTM-293) were established and cultured in DMEM as in a previous study [27]. The human cervical epithelioid carcinoma cell line HeLaS3 was purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan), and the multi-drug resistant human cervical cancer cell line KBvin was kindly provided by Dr. Kuo-Hsiung Lee (University of North Carolina, Chapel Hill, NC, USA). All cancer cell lines were cultured in RPMI-1640 containing 10% FBS, at 37 ◦C in a humidified atmosphere of 5% CO2.

#### *3.5. Calcein AM Uptake Assay*

The calcein AM uptake assay was performed to evaluate the inhibitory effect of the test compounds on human P-gp efflux function. To be brief, 1 <sup>×</sup> 105 cells/well were seeded in 96-well black plates overnight. Before starting the assay, the cells were washed and pre-incubated with warm Hanks- balanced salt solution (HBSS) for 30 min. Then, the test compounds were added, and incubation was carried out for 30 min. Calcein-AM was added after washing with warm PBS. The BioTek Synergy HT Multi-Mode Microplate Reader was utilized to detect calcein fluorescence (excitation/emission wavelength = 485 nm/528 nm) at 37 ◦C every 3 min for 30 min. Each experiment was performed at least three times, each in triplicate on different days.
