Fig 4.

Effects of the members of the PKC family on the regulation of MMP-2, Bcl-2, and STAT3 mediated by HCV NS4B. (A to C) Huh7 cells were transfected with plasmids expressing si-PKCδ, si-PKCα, si-PKCβ, si-PKCε, si-PKCζ, or si-Ctrl. A similar vector (pSilencer 2.0) containing an irrelevant sequence that does not have significant homology to any human gene was provided by Ambion, Inc., and used as a negative control. The relative levels of MMP-2 and Bcl-2 mRNA were measured by real-time PCR at 48 h posttransfection, and the levels of p-STAT3, MMP-2, Bcl-2, and β-actin proteins were determined by Western blot analysis. GAPDH was used as an internal reference. MMP-2 and Bcl-2 levels in each sample were normalized by dividing by the GAPDH quantity. Data are expressed as the mean ± SD of three independent experiments. (D) Huh7 cells were transfected with plasmids expressing si-PKCα, si-PKCβ, si-PKCε, si-PKCδ, si-PKCζ, or si-Ctrl. The levels of SOCS3, p-ERK, ERK, p-JNK, JNK, and β-actin proteins were determined by Western blot analysis at 48 h posttransfection. (E) Huh7 cells were transfected with pCMV-NS4B and treated with si-ERK or si-Ctrl at the indicated concentrations, and the levels of the ERK, SOCS3, p-JNK, JNK, and β-actin proteins were determined by Western blot analysis. (F) Huh7 cells were transfected with pCMV-NS4B and treated with si-JNK or si-Ctrl at different concentrations, as indicated. The levels of the JNK, SOCS3, p-ERK, ERK, and β-actin proteins were determined by Western blot analysis. (G) Huh7 cells were transfected with pCMV-NS4B and treated with si-ERK (80 nmol), si-JNK (80 nmol), or si-ERK (40 nmol) and si-JNK (40 nmol). The protein levels of p-STAT3, STAT3, MMP-2, Bcl-2, and β-actin were determined by Western blot analysis.