The sequence of primers used for amplification is listed in Table 1. mRNA or miRNA levels were normalized using GAPDH or U6 RNA as a internal reference gene and compared with non-SP cells. The relative amount of each miRNA to U6 RNA was described using the 2-∆∆Ct method [15]. Table 1 Reverse transcription and stem-loop primers for real-time RT-PCR Gene name Reverse transcription primer (5′-3′) PCR primers (5′-3′)

F: forward primer R: reverse primer miR-21 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACA F: CGCGCTAGCTTATCAGACTGA     R: GTGCAGGGTCCGAGGT miR-10b GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCACAAA F: CGTCGTACCCTGTAGAACCGA R: GTGCAGGGTCCGAGGT miR-470* GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCTTCT Selleck CB-5083 F: GTGCGAACCAGTACCTTTCTG R: GTGCAGGGTCCGAGGT miR-34c-3p GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCTGGC F:GGTGGAATCACTAACCACACG find more R: GTGCAGGGTCCGAGGT let-7i* GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGCAAG F: TAGTACTGCGCAAGCTACTGC R: GTGCAGGGTCCGAGGT miR-200a* GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCCAGC

F: GAGTGCATCTTACCGGACAGT R: GTGCAGGGTCCGAGGT miR-148b* GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCCTGA F: GGCGCAAGTTCTGTTATACAC R: GTGCAGGGTCCGAGGT U6 CGCTTCACGAATTTGCGTGTCAT F: GCTTCGGCAGCACATATACTAAAAT R: CGCTTCACGAATTTGCGTGTCAT Western blotting analysis Cells sorted by FACS were washed twice with ice-cold PBS and then incubated with ice-cold cell lysis buffer (1% Nonidet P-40, 50 mmol/L HEPES, pH7.4, 150 mmol/L NaCl, 2 mmol/L ethylenediaminetetraacetic acid, 2 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium

vanadate, 1 mmol/L sodium fluoride, and 1× protease inhibitor mixture) to extract protein. The Terminal deoxynucleotidyl transferase protein concentrations of the lysates were measured using a Bradford protein assay kit (Bio-Rad). All samples were separated in 12% SDS polyacrylamide gels. Signal were revealed by primary antibodies and IRDye700-labeled secondary antibody. The signal intensity was determined by Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, NE). Results SP cells are present in rat HCC cancer cell and fetal liver cells The existence of the SP fraction in primary fetal liver cells and in HCC cells was confirmed by staining with Hoechst 33342 dye to generate a Hoechst learn more blue-red profile. A small fraction of low-fluorescing cells in the lower-left region of each profile was gated as SP. The appearance of this fraction was blocked by verapamil, an inhibitor of transport via multidrug resistance proteins (Figure 1A-D). Both fetal liver cells and HCC cells contained a distinct fraction of SP cells. The SP of fetal liver cells was calculated to be 0.15% ± 0.02% (mean ± SEM), and that of HCC cells was calculated to be 0.20% ± 0.08%. Once identified, the cells in the SP gate were sorted into a centrifuge pipe by FACS.