Figure 1

PTEN protein phosphatase activity is required for decreasing HCV RNA level after HCV infection. (a) Huh-7.5 cells were transfected with plasmids expressing wild-type Flag-PTEN (PTEN WT), truncated mutants, phosphatase deficient mutants, or empty vector. Forty eight hours after transfection, cells were infected with HCV-2a J6/JFH-1(p7-rLuc-2A) virus at a multiplicity of infection (MOI) of 1. Luciferase assay was performed using the cell lysates 72 hours post-infection (hpi). This experiment was performed three times. (b) The protein levels of PTEN proteins and β-actin after transfection were determined by Western blotting using anti-Flag and anti-β-actin antibodies, respectively. (c) Cell viability was determined by MTT assay 72 hpi. This experiment was performed three times. (d) Huh-7.5 cells expressing an inducible PTEN shRNA or non-silencing control shRNA were treated with 1 μg/mL of Doxycycline for 48 hours. Cells were then infected with HCV-2a J6/JFH-1(p7-rLuc-2A) virus at an MOI of 1. At 24 hpi, cells were transfected with increasing amounts of plasmids expressing Flag-tagged PTEN with wild-type or mutant 3′UTR, or empty vector. Luciferase assay was performed 48 hours after transfection (left panel). In the right panel, the protein levels of PTEN and β-actin at 36 hours after transfection were determined by Western blotting using anti-PTEN and anti-β-actin antibodies, respectively. Relative PTEN band intensities against β-actin were given underneath each sample. This experiment was performed twice. (e) Cell viability was determined by MTT assay in parallel with luciferase assay. This experiment was performed twice. (f) Huh-7 cells were co-transfected with plasmids expressing non-silencing control shRNA or shRNA targeting the 3′UTR of PTEN, together with bicistronic RNA interference reporter plasmids with (PTEN-3′UTR WT) or without (PTEN-3′UTR Mut) the shRNA target sequence. Dual luciferase assay was performed 48 hours after transfection. The renilla luciferase (rLuc) activity representing the knockdown efficiency was normalized against firefly luciferase activity driven by a constitutive promoter on the reporter plasmid. Luciferase activities were expressed as fold changes relative to vector control. The statistical differences between samples were demonstrated as * if p ≤ 0.05, ** if p ≤ 0.01, or NS for not significant. This experiment was performed three times.