Involvement of extracellular signal-regulated kinase/mitogen-activated protein kinase pathway in multidrug resistance induced by HBx in hepatoma cell line

Figure 1 Restriction enzyme digestions of the plasmids. Lane M: markers (the brightest band of the marker presents the 500 bp); lane E: one of the split bands emerged at the exact position of 500 bp, and the other was the empty pcDNA3.0 vector as expected.

Figure 2 Confirmation of the successful transfection of HBx into HepG2 cells by RT-PCR. Lane M: markers (the brightest band of the marker presents the 500 bp); lane A: transfected cells; lane B: transfected cells treated with U0126; lane C: the control.

Figure 3 Western blot analysis of HBx protein in samples isolated from HepG2 cells with or without HBx transfection. Lane 1: the sample transfected with HBx; lane 2: the sample transfected with vector only.

Figure 4 Apoptosis index of three different groups of cells after treatment with 5-Fu. A: HepG2/pcDNA3 cells; B: HepG2/ pcDNA3-HBx cells; C: U0126 pretreated transfected cells. D: Apoptotic index quantitated by FACS.

Figure 5 Protein levels of MDR associated genes in different groups of cells. Lane1: HepG2/pcDNA3 cells; lane2: HepG2/pcDNA3-HBx cells; lane3: transfected cells treated with U0126. The primary antibody for A, B, C, D was β -actin, MDR-1, MRP-1 and LRP respectively.

Figure 6 RT-PCR analysis of the expression of MDR associated genes in transfected and control cells. Lane 1: HBx-expressing cells; lane 2: control cells; lane M: markers. A: Specific primers to amplify MDR-1 gene; B: Specific primers to amplify MRP-1 gene; C: Specific primers to amplify LRP gene.

Figure 7 Increased phosphorylation of ERK induced by HBx in hepatoma cells analyzed by Western blot. Lane 1: trans-fected cells after treated with U0126; lane 2: HBx-expressing cells ; lane 3: control cells. A: Specific antibody for phosphory-lated-ERK; B: Specific antibody for total ERK.