Fig 2: ADM efflux effect of the Eca109/ABCG2 cells assayed by flow cytometry. The Eca109, Eca109/PCDNA3.1 and Eca109/ABCG2 cells were incubated at 37°C with 0.02 µg/ml ADM for 2 h and then without ADM for 1 h. The ADM fluorescence intensity, which represents the ADM content in the cells, was detected by flow cytometry. The ADM fluorescence intensity in the Eca109/ABCG2 cells was lower than in the Eca109 and Eca109/PCDNA3.1 cells. The ADM efflux effect of the Eca109/ABCG2 cells was more efficient than that of the Eca109 and Eca109/PCDNA3.1 cells. (A) Eca109, (B) Eca109/PCDNA3.1 and (C) Eca109/ABCG2 cells. ABC, ATP-binding cassette; ADM. adriamycin.

Fig 3: Analysis of cell cycle and apoptosis of GSCs with low or high ABCG2 expression upon DMC treatment(A) GSCs were transfected with either lenti-GFP-ABCG2 shRNA or lenti-GFP-ABCG2 to downregulate or overexpress ABCG2 in GSCs and treated with either 10µM or 30µM DMC for 24h followed by PI staining and flow cytometry analysis of the cell cycle. (B) TUNEL staining to determine apoptosis of GSCs transfected with or without lenti-GFP-ABCG2 shRNA and treated with 30µM DMC. (C) Histone-DNA ELISA analysis of apoptosis in GSCs tranfected with or without lenti-GFP-ABCG2 shRNA and treated with 30µM DMC.

Fig 4: Expression of the ABCB1, ABCC1, ABCG2 genes and P-gp, MRP1, BCRP proteins in BJAB/ADR cells. (A) The expression of the ABCB1 gene in the ADR-resistant BJAB/ADR cells. The mRNA level of MDR1 analyzed by qRT-PCR and normalized to the mRNA level of the housekeeping gene GAPDH. Compared with the sensitive cells BJAB, BJAB/ADR drug-resistant cells showed increased expression of ABCB1 mRNA. *P < 0.05, compared with control group. (B) The protein expression of P-gp on BJAB/ADR cells. The expression of P-gp was evaluated via flow cytometry analysis. BJAB/ADR cells showed higher expression of the membrane protein P-gp compared with the BJAB sensitive cells. (C,E) The qRT-PCR on the gene expression of ABCC1 and ABCG2, respectively. These results showed higher expression levels of both genes, but the increased expression was not as high as that of ABCB1. *P < 0.05, compared with control group. (D,F) The protein expression level of MRP1 and BCRP, separately. The results were obtained from flow cytometry analysis. These results indicated that higher expression levels of both protein, but the increased expression was not as high as that of P-gp.

Fig 5: In vivo effects of DMC on GSCs with low or high ABCG2 expressionBalb/c nude mice were xenografted with GSCs that were transfected with either lenti-GFP-ABCG2 shRNA or lenti-GFP-ABCG2 to downregulate or overexpress ABCG2 in GSCs, respectively. After 30 days, the mice were administered 10mg/kg or 30mg/kg DMC for 30 days. Then, relative tumor proliferation rates T/C % (A) and the tumor growth inhibition rates (TGI %) (B) were calculated.