Figure 4: Production of TFV was inhibited by DN and RNA interference of Tsg101.

(A) HepG2 cells were transfected with 0.1, 0.2, 0.4, or 0.8 μg of pTsg101-N/C/F-myc. Protein samples were collected and analyzed by Western blot, and GAPDH was included as control. (B) HepG2 cells were infected with TFV for 1 h after transfection with 0.8 μg of pTsg101-N/C/F-myc or pCMV-myc and then cultured for 66 h. Cells and supernatant were lysed to extract DNA. Samples were then subjected to qPCR using MCP primers to quantify TFV genomic concentration. Statistical analysis was performed using Student’s t-test (0.05 < *p ≤ 0.1, 0.01 < **p ≤ 0.05, 0.01 ≤ ***p). (C) 100 nM of siRNA specific for Tsg101 and control RNA (siGFP) were transfected into cells. The cells were collected from 24 h to 72 h and then subjected to Western blot. (D) 100 nM siTsg101 or 100 nM siGFP were transfected into the cells 6 h prior to infection with TFV for 1 h and then cultured for 66 h. Cells and supernatant were lysed to extract DNA. Samples were then subjected to qPCR using MCP primers to quantify TFV genomic concentration. Statistical analysis was performed using Student’s t-test (0.05 < *p ≤ 0.1, 0.01 < **p ≤ 0.05, 0.01 ≤ ***p). (E) In vivo confocal microscopy analysis was performed after HepG2 cells were infected with TFV for 1 h and cultured for 66 h. Cells were processed for immunostaining as described under “Materials and Methods”. Arrow indicates co-localization of TFV MCP and endogenous Tsg101.