Small interfering RNA (siRNA) transfection

siRNAs specifically targeting human ABCG2 (siRNAs #1 and #2, IDs s18056 and s18057), CD44 (siRNA #1 and #2, IDs s2681 and s2682), and SOX9 (siRNAs #1 and #2, IDs s13306 and s532658) were obtained from Life Technologies (Carlsbad, CA, USA). Cells were plated in six-well plates (1x10⁵ per well) and transfected with siRNAs for 3 days in antibiotic-free media using siRNA Lipofectamine RNAiMAX and OPTI-MEM I reduced serum medium (Invitrogen, Carlsbad, CA, USA), in accordance with the Lipofectamine protocol. After 72 h of transfection, the cells were used for experiments. siRNA transfection was confirmed by immuno blotting (Fig. S1). The efficacy of knockdown by siRNAs was determined by calculating the inhibition of protein expression with the following results: siSOX9#1 90.1%, siSOX9#2 93%, siABCG2#1 12%, siABCG2#2 61%, siCD44#1 81%, and siCD44#2 83% (Fig. S1).