Figure 6

Protein expression and functional analysis of Ct-OATP1B3 in colon cancer cell lines. (A) Quantification of Ct- and Lt-OATP1B3 mRNA expression levels in human pooled hepatocytes (Hep50) and colon cancer cells (LS180 and HCT116) was separately performed by qPCR. The black and white bars indicate Ct- and Lt-OATP1B3 mRNA levelsc ± S.D. of three independent experiments, each performed in duplicate. The dashed line indicates the QL value. (B) The membrane protein expression of Ct-OATP1B3 in colon cancer cells was examined by Western blotting using rabbit anti-OATP1B3 antibodies that recognize the C-terminal peptide, which was supposed to be the common region among both isoforms. The soluble membrane fraction of human liver tissue (HL) was used as a positive control. Na⁺/K⁺ ATPase was used for a plasma membrane marker. (C) The CCK-8 uptake was examined by transport analysis using LS180 and HCT116 cells. The CCK-8 uptake (1 μM) level was determined in the absence (black bar) or presence (gray bar) of BSP (100 μM). The CCK-8 uptake in cancer cells is expressed as the relative ratio against the uptake of CCK-8 without BSP at 37°C. The white bar indicates transporter-independent CCK-8 accumulation level, which was determined at 4°C. This experiment was performed three times in duplicate.