FIG 3.

HPV-18 E6- and E7-specific RGNs induce tumor suppressor gene expression in HeLa cells. HeLa cells were transfected with an S. pyogenes Cas9 expression vector and HPV-18-specific sgRNAs, as indicated, and were processed for Western blotting. These data are representative of data from 3 biological replicates. (A) The lysate was probed for p53 and p21 expression, with endogenous β-actin being used as a loading control. The Cas9 protein was detected by an antibody specific for the Flag epitope tag. (B) Similar to panel A except that the lysate was probed for Rb. (C) Sequence of a mutant E6 expression construct designed to be resistant to cleavage by S. pyogenes Cas9 in the presence of E6 sgRNA1. The mutations are in lowercase type, and the PAM is underlined. (D) Expression of the cleavage-resistant E6 gene shown in panel C reveals trans-complementation of p53 protein repression in the presence of S. pyogenes Cas9 and E6 sgRNA1. N.S., nonspecific.