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. Author manuscript; available in PMC: 2009 Jul 27.
Published in final edited form as: Virology. 2008 Mar 26;375(2):611–623. doi: 10.1016/j.virol.2008.02.025

Fig. 2. siRNA knockdown of E6 and E7 expression in E6E7 transduced keratinocytes.

Fig. 2

(A) RT-PCR detection of E6 and E7 mRNA (upper and middle panels) and GAPDH mRNA (bottom panel) in keratinocytes expressing the combined E6/E7 genes. The PCR signal for E6 mRNA was reduced in cells treated with siRNA for either E6 or E7. E7 mRNA was decreased in cells treated with siRNA for E7 only. Myc and control siRNA duplexes did not affect the expression of either E6 or E7. The expression of GAPDH mRNA was unaffected by any of these treatments. (B) Western blot detection of p53 protein (upper panel) and β-actin (bottom panel) in HFKs treated with siRNAs. Keratinocytes were treated identically with siRNAs for E6, E7, or Myc as described in (A) but were then analyzed for p53 protein expression by Western blot. p53 protein was increased in cells treated with either E6 or E7 siRNAs, consistent with their postulated inhibition of E6 protein expression. Cells treated with Myc or control siRNA showed lower levels of p53, consistent with continued E6 expression. β-actin protein expression was not altered by any siRNA treatment. (C) RT-PCR detection of E6 and E7 mRNA (upper and middle panels) and GAPDH mRNA (bottom panel) in HFKs that express E6 and E7 from separate vectors. In contrast to experiments with cells expressing the combined E6/E7 genes (panels A-B), cells expressing the E6 and E7 genes from separate vectors showed a specific inhibition by respective siRNAs. E6 mRNA was reduced only when treated with E6 siRNA and E7 mRNA was decreased only in cells treated with E7 siRNA. Myc and control siRNA duplexes had no effect on E6 or E7 expression. The expression of GAPDH mRNA was unaffected by these treatments. (D) Western blot detection of p53 protein (upper panel) and β-actin (bottom panel) in HFKs expressing the E6 and E7 genes from separate vectors. p53 was increased only in cells that were treated with E6 siRNA, indicating specific knockdown of E6 in these cells. These results also verify the results in panel C demonstrating that the E7 siRNA is not inhibiting E6 expression.