Fig 3.

ACD is detected preferentially in the Side Population of liver cancer cells. (A-B) FLOW CYTOMETRY sorting images of side population (SP) and non-side population cells (NSP). The side population is based on the ability of the ABCG2 transporter to efflux Hoechst 33342 (Ho). (B) In order to identify the population of cells that efflux Ho specifically by the ABCG2 transporter, we used Verapamil to block the activity of the ABCG2 transporter. (A) Here we show that the SP comprises 0.28% of the total cell population of Huh-7 liver cancer cells. (C) Side-population (SP) and non-SP hepatocellular carcinoma cells were plated initially at low concentrations, then allowed to proliferate, followed for 5 weeks, and tested for the presence of ACD-NRCC. At one week, SP cells did not exhibit ACD-NRCC, but when left to proliferate and differentiate while generating non-SP cells, demonstrated increasing levels of ACD-NRCC (p=0.0034); the non-SP cells neither generated SP cells nor demonstrated ACD-NRCC (p=0.024). While ACD-NRCC was never detected in NSP or CD133-negative cells (Vide Infra, figure 4), the maximal detection rate in total cells, in SP cells or in CD133+ cells seem to be constant suggesting that per a given condition the rate of ACD-NRCC is constant (steady state rate).