Skip to main content
. 2017 Mar 8;8(24):38239–38250. doi: 10.18632/oncotarget.16013

Figure 3. Expression of exogenous wild-type L1 ORF1/2 is necessary and sufficient to confer sensitivity to GCV.

Figure 3

A. Immunoblots showing recombinant ORF1p expression in HEK293-T, HeLa and primary WI-38 cells transfected with a control GFP expression vector or expression vector expressing wild-type ORF1/2p (pJM101L1.3) or wild type ORF1p and a mutated version of ORF2p (D702A) (pJM105L1.3) [45]. Ponceau staining is used as a loading control. B. All indicated cell lines were seeded into a 24-well plate transfected with 0.8 μg per well of A5b vector, 24 hours later cells were transfected by triplicate with 0.8 μg/well of GFP, pJM105 and pJM101vectors, 24 hours later GCV (Sigma-Aldrich) were added at 10 μg/ml. Transfection was carried out using FuGENE® (Promega) (DNA/FuGENE 1/6) for HEK293-T cells, X-tremeGENE9® (Sigma-Aldrich) (DNA/X-tremeGENE9 1/4) and Lipofectamine 3000® (Invitrogen) (DNA/lipofectamine 1/3) for WI-38. Living cells were counted as described above. All experiments were performed in triplicate and the data are expressed as mean ± s.d. with a pvalue determined by Student's t-test.